DE3824109A1 - ANALYZING METHOD FOR DETERMINING A DETECTING SUBSTANCE AND A DEVICE FOR CARRYING OUT THIS METHOD - Google Patents

Abstract

In an assay method for determining quantitatively the presence of an analyte in a sample by means of specific binding, the sample is contacted with a solid phase carrier material on which is immobilised a first binding reagent having specificity for the analyte, and with a labelled specific binding reagent which can participate in either a "sandwich" or a "competition" reaction with the first reagent in the presence of the analyte, the label participates in a chemiluminescent reaction the light output from which is used as a means for determining the extent (if any) to which the labelled specific binding reagent has become bound to the solid phase, and the light is derived from a point source and is recorded on a light-sensitive film in such a manner that the size of the image formed on the film provides a measure of the quantity of the analyte in the sample. Light recording apparatus, incorporating a multi-well micro-analytical tray and wherein the point source for each well is defined by an aperture in a perforated plate which can be situated directly beneath the tray and spaced appropriately from the film, is also described. <IMAGE>

Description

The present invention relates to an analytical method, i. i.e., assays, for the determination of a detection substance in a sample by specific Binding using a solid phase support on which a immobilized first binding reagent with specificity for the detection substance and a labeled binding reagent that participates in a sandwich reaction or a competitive reaction with the first Binding reagent can participate in the presence of the detection substance, the label participating in a chemiluminescent reaction, their amount of light to determine the extent (if any) in which the labeled binding reagent has been bound to the solid phase is used, and particularly relates to immunoassays and a device that records the results of such assays can be.

Solid phase immunoassay systems, d. i.e., systems based on particles, are already known. These include the qualitative and / or quantitative Determination of immunogenic species in a test sample by a immunological binding reaction between the immunogenic species (Analyte) and one or more specific binding partners for this a, of which at least one of the binding partners to an insoluble Carrier material in contact with a liquid medium containing the test sample contains, is bound. The fact that the resulting immunological Complex bonded to the carrier material can be advantageous during the subsequent determination of the extent to which the Binding reaction has taken place. The determination is usually marked using a specific Binding partner made. The binding reaction can be either be a "competitive" reaction or a "sandwich" reaction, both Reactions in the prior art are widely used.  

In a typical competitive reaction, the assay medium that is in the is generally aqueous, brought into contact with the solid phase support, on which a specific binding reagent (e.g. a monoclonal Antibodies) with specificity for the detection substance to be determined is immobilized. The assay medium contains a known amount of Detection substance (or an analog substance thereof, the identical Epitope) has a label and a known amount a sample that is believed to be the detection substance contains. In the absence of the detection substance in the sample, it binds the marked detection substance or an analogue with the specific one Binding reagent immobilized on the solid phase support. The The extent of this binding is determined by standard experiments. If the sample contains the detection substance, then it occurs with the marked detection substance or its analog substance for the specific Binding reagent in competition. This reduces the extent in which the specific binding reagent with the labeled component is combined. By comparison with the standard situation (i.e., the Result that can be obtained in the absence of the detection substance) a statement about the concentration of the detection substance in the sample be made.

A typical sandwich reaction is a first binding reagent immobilized on the solid phase support with specificity for the detection substance. The assay medium, which is generally aqueous, also contains a second binding reagent with specificity for the detection substance and carries a mark. In the absence of the detection substance can be between the immobilized reagent and the marked reagent no coupling. If the detection substance then it acts as a bridge between the immobilized and labeled specific binding reagents and leads to an indirect coupling of the labeled reagent to the solid phase. The extent of this coupling is a measure of the concentration of the detection substance in the rehearsal. These specificities of the immobilized Reagent and the labeled reagent for the detection substance can be different. However, if the molecule of the detection substance has more than one identical epitope, it is possible that  same specific binding reagent both in the immobilized form as well as to use in the freely marked form.

European patent 01 49 565 (Amersham International PLC) describes assays such as immunoassays and immunometric "2-site" assays, which are carried out using a marked Reagent and another reagent that is magnetically attractable Particle is bound which is suspendable but in a liquid Assay medium are insoluble in which after distribution of the labeled Reagent between the liquid phase and the particles in Ratios that depend on the concentration of the detection substance in the Depending on the sample, the liquid phase is removed. The particles are in suspended in another liquid medium. Then the Concentration of the mark found. The described Analysis method is especially for fluorescent and chemiluminescent Marking systems suitable and can be conveniently in Plates with multiple wells are made, using the wells are optically shielded from each other and the observations above or below the wells. On page 5 of EP 01 49 565 found that centrifuging the microtiter plates was inappropriate and it is also difficult to take any useful measurements make optical signals from the pellet of solid material generated on the sedimentation of a particulate Reagent from a suspension. Because of this, stay the authors of EP 01 49 565 that the use is magnetic attractable particles that appear before the signal is detected in the Resuspended liquid, the widespread use of Microtiter plates allowed for immunoassays.

EP 01 49 565 relates to quantitative assays in which the fluorescent or chemiluminescent signal through instruments is displayed numerically. It may well be that such advertisements more easily dispersed from a particulate solid phase Condition.

In contrast to this prior art, however, the We found quantitative assay results using  of a signal-generating luminescent system and a solid phase can be obtained that are localized rather than through the assay medium is dispersed when that generated by the localized solid phase Signal recorded using a photosensitive film becomes. The need for an expensive optical recording device is avoided. The developed film can be easily stored Provide a permanent record of the analytical result. This is Especially useful when it comes to collecting samples in Hospitals, clinics and medical laboratories on a large scale acts. The present invention offers special techniques with which robust and simple assays in which a photosensitive film as Recording medium is used can be carried out.

Essentially, the present invention provides an assay for a Detection substance in a sample, with a luminescent signal at the determination of the assay result and the signal on is recorded on a photosensitive film. The recorded Light derived from a point source in such a way that the size of the image formed on the film is a measure of the Represents the amount of detection substance in the sample.

The invention particularly relates to an analytical method, i. i.e., a Assay procedure for the determination of a detection substance in a sample by specific binding using a solid phase support material, on which a first binding reagent with specificity for the Detection substance is immobilized, and a labeled binding reagent, in a sandwich reaction or in a competitive one Reaction with the first reagent in the presence of the detection substance can participate, the marking on a Chemiluminescence reaction takes part, the amount of light for determination the extent (if any) that the labeled binding reagent has been bound to the solid phase is used by this is characterized in that the light on a photosensitive film recorded and derived from a point source in such a manner becomes that the size of the image formed on the film is a measure for the amount of detection substance in the sample.  

A point source will probably achieve that the area of the sensitized solid phase aligned with the photosensitive film is significantly smaller than the area of the photosensitive film, which is exposed by the light source. In practice, the area is the solid phase, which is local to the film, preferably not larger than about 50% of the exposed film area and very special preferably no greater than about 25%. This is explained below an example will be explained in more detail.

In one embodiment of the invention, the solid phase defines itself Point source. This is preferably accomplished by using a particulate Solid phase support attached to the light emitting body or the light emitting substance is bound in a small area of a container is located. For example, if the assay is in a small cells, e.g. B. in a series of wells in a typical transparent plate with several wells, at the end of the assay procedure then the particulate solid phase localized in a narrow area in the bottom of the well will. The small mass of accumulated solid phase then acts as Point source. This can easily be done in practice using Cups are achieved that have a much narrower cross section in the lower one than have in the upper part. Typical examples are wells that are sharply conical or are conical or rounded in the bottom leak. The inner shape of the cup therefore forces the small one Amount of the localized particulate solid phase into a narrowly defined Measure into the bottom of the cup. At this Design was found to be surprisingly accurate quantitative Results can be obtained by using the well or the row of cells directly in the vicinity of a photosensitive Film that can record light from the point source defined by the particulate carrier has been. Preferably the photosensitive film is direct Contact to the bottom of the cell.

In another embodiment of the invention, the point source is through defines an opening that is immediately adjacent to the exterior of the container in which the analysis is carried out. At  In this embodiment, the solid phase carrier does not define the point source itself and can therefore, if desired, be a much larger one Mass of material available. In fact, the carrier material can be a particulate solid phase contained in dispersed form if there is is appropriate. Alternatives to this are a localized particulate solid phase, a larger single particle of a solid phase, such as a Beads, or a permanently localized solid phase, such as a sensitized one Inner wall of the container in which the analysis is carried out. In all of these configurations, the outer opening defines the point source from a larger area of the light emitting material. Around to achieve the necessary quantitative nature of the assay result with this configuration, it is necessary to ensure that the photosensitive Film is placed away from the opening rather than with it is in direct contact. The distance between the film and the The opening should not be so big that if there is any light, the passes through the opening, reaches the film, the intensity of which increases is low to produce any usable image on the film to let. In a typical practical situation where a regular Arrangement of cells in a conventional transparent plate with multiple cups is used as a container, in which individual Analyzes can be carried out through an opening perforated plate can be created directly below the well is arranged. Every opening should be at least approximately at the central vertical axis of the associated cell. With Opening plate should be in direct contact with the bottom the cups are standing. The film should be below the one with the opening provided plate, but this is in a critical Distance should be done to make it possible for a quantitative Result is achievable. In a typical practical situation under Use of conventional cups with an inner diameter in the upper one Part of about 8 to 10 mm, while the openings have a maximum Dimension, d. i.e., have a diameter of at least about 0.5 mm should. The opening should preferably be no larger than about 3 mm. The openings should particularly preferably have a maximum Have dimensions of about 1 to 2 mm. The distance between the Underside of the plate having openings and the photosensitive Film should be at least about 0.5 mm. Generally need  not to be exceeded here about 5 mm. Generally is the optimal distance 1 to 3 mm. It is apparent to the person skilled in the art that simple experiments were carried out with every practical arrangement should be specified before the optimal values of these parameters can be.

Preferably, a perforated spacer between the Apertured plate and film provided to the to maintain the necessary distance between the opening and the film exactly.

One advantage of this second embodiment compared to the first is in that the opening defines the point source and thus the point source regardless of the actual body of the light-generating is solid phase. This will provide greater operational flexibility reached. In addition, if desired, the solid phase be selected in such a way that it acts as a screen to prevent extraneous light e.g. B. by unbound marking also present in the medium is generated to keep entering the opening. If, for example the solid phase is a single large opaque bead, then that can Filling the bottom of the container will cause only the light from the underside of the beads creates access to the opening can reach.

If the localized solid phase is in the lower part of a pipe is located and the film is located directly below the tube, then the light on the film will create a point in its area according to the amount of light emitted by the marker varies, which has been bound to the carrier. A variety of assay containers can be checked in this way at the same time by a single piece of film is used. The relative amount of light from each Container can be estimated by comparing the points of the film.

Accordingly, the invention creates another embodiment Analytical method in which, after a suitable incubation with a Sample and a soluble or dispersible labeled specific Binding reagent is a particulate solid phase support material that a  immobilized specific binding agent carries, within a liquid Medium is located in the vicinity of a light-sensitive film, so that the light of a point source can be detected, the Label can participate in a chemical reaction leading to Generation of chemiluminescent light leads, and being the liquid Medium contains such additional reagents that may be necessary to run the reaction.

In another embodiment, the invention extends to a method for determining a detection substance, this method comprising the following measures:
(a) Preparation of an aqueous assay medium containing:

• (I) a particulate solid phase support material on which a specific Binding reagent for the detection substance to be determined is immobilized, the particulate solid phase support in the aqueous assay medium can be suspended, but can be readily localized therein is
• (II) a sample which is believed to be the one to be determined Detection substance contains, and
• (III) a labeled labeled soluble or dispersible in the assay medium Reagent that is either a second specific binding reagent that with the detection substance and the first specific binding reagent in a sandwich reaction can participate, or the detection substance itself or is an analog of the detection substance and the label is a chemiluminescent Reactant is

(b) incubation of the assay medium so that the labeled reagent can be bound to the particulate solid phase support,
(c) locating the particulate solid phase support within the assay medium,
(d) replacing or diluting the assay medium using a liquid medium containing such additional reactants that may be necessary for the chemiluminescent reaction to occur or replacing and diluting together with such additional reactants, and
(e) measuring chemiluminescent light on a photosensitive film emitted from a point source that has a label attached to the particulate solid phase support in the localized state.

When the above procedures are modified, the incubation is divided into two Stages performed an initial incubation of the particulate Solid phase support material and the sample in the presence of the labeled Reagent comprises, at least the bulk of the sample thereon removed and replaced with a liquid medium that contains the soluble or contains dispersible labeled reagent, and a second Incubation is carried out, after which measures (c) to (e) of the above sequence. A localization measure should be carried out before the bulk of the sample between the two Incubation measures are removed. Another or alternative modification can be the addition of one or more or actually all the reagents necessary for the appearance of the chemiluminescent Reaction are necessary at an earlier stage in the process take place, e.g. B. during procedural measure (a) or if the labeled reagent is added during the two-stage incubation becomes.

The particulate solid phase support preferably contains magnetically attractable particles.

The use of "camera" devices to record the Results of the luminescence reaction in analytical methods already known. Such devices are used for. B. in the European Patent application 00 71 859 and in the British patent 21 69 704. The latter document describes one Recording device having a holder for a variety of Reaction containers, this holder being a plate with a hole arrangement  for accommodating an arrangement of reaction containers, a housing for receiving the holder, the housing so is designed so that the entry of stray light is prevented and a too opening cover includes means for holding a photographic Films adjacent to the bottom of the plate and a removable one Closure for insertion between film and plate comprises, the removable closure between the narrowest position and the open Position is movable and the film in the latter position at the Use is exposed. Said plate is arranged so that it (a) can be stored on and carried by said closure, when the latter is in its closed position, and (b) open the photographic film falls when the shutter is in the open Position is moved. Such an apparatus was not used in the past been used to record quantitative analytical results by To deliver light emitted by point sources.

The present invention now provides an improved device for Determination of the result of a specific bond analysis under Using the amount of light from a chemiluminescent reaction for quantitative Determination of a detection substance in a sample, the Device comprises a holder for several cells, the Bracket a first plate with an arrangement of arranged therein Holes for receiving an array of cups and a housing for receiving the bracket, the housing being able is to prevent stray light from entering and one to open Cover comprises, further comprising a device to a photographic Hold film near below the plate, and further a removable one Closure is provided between the film and the plate can be arranged, the closure between a closed position and an open position is movable, and in the latter the film is exposed, and further between the base plate the holder and the closure provided a second perforated plate whose individual openings are each one opening below create a single opening of the bracket, each opening one in a substantially smaller cross-sectional area than that of the assigned one Opening the holder together with the device for maintaining the holder a predetermined distance between the film and the  perforated plate when the closure is in the open Position.

Preferably, those that set or maintain the distance include Mean a perforated third phase in which the holes are essential the openings are larger than the second plate, each hole in the third plate arranged directly below an opening in the second plate is. The triple combination of the plates is preferably so arranged that it is stored on the closure and carried by it when it is in its closed position, and on lower the photographic film when the shutter is in its open Position is moved.

The photosensitive film should be extremely "fast" to ensure that small amounts of light are recorded and differentiated can be used to convey quantitative information. Films, currently available commercially have e.g. B. ASA values of 20,000. These are very useful.

A "Polaroid" film is very suitable and convenient. The Camera setup on a standard commercially available camera "Polaroid" camera "back" based. However, the normal paper film size, which is obtained in this way is not sufficient to total area of a microtiter plate of the standard laboratory format (96 cells / 8 × 12 cells) to cover. It is therefore recommended To use plates with a smaller number of cells, e.g. B. by the use of standard plates that are reduced to a Size have been reduced.

If desired, then any risk of interference from Light any unbound mark in the bulk of the Sample remains, thereby minimized by that Assay medium a damper is incorporated, the light blocked or absorbed.

According to a further embodiment, the invention creates a particle based immunoassay method that uses a specific binding partner  uses that is marked such that the marking continuously a chemiluminescent signal in the test medium generated, this signal masked, deleted or otherwise suppressed is, while the marked binding partner evenly within the Test medium is dispersed, and in this method the Binding reaction following a local concentration of the particulate Carrier material is set in the test medium such that the local concentration of the complex-labeled binding partners, which by the particles are carried, provides a local luminous flux that is sufficient to local signal suppression capability to the test medium fix and thus a detectable chemiluminescent signal deliver, the strength of which depends on the extent of the bond that is between the immunogenic species to be tested and the specific one Binding partner that is carried by the particles. At this embodiment is therefore an immunoassay method according to the Invention characterized by the following features:

• a) The marker continuously generates light while it is in the Test medium is located,
• b) while the label is dispersed in the test medium, independently whether the label is free or on a complex on the support the light is masked, extinguished or otherwise suppressed,
• c) complex formation of the labeled binding partner with the other Binding partner itself changes the light generated by the marking not significant and
• d) a detectable light signal is only generated when the carrier material, to which the complexed label is bound, in a comparatively narrow zone localized within the test medium becomes.

The solid phase, e.g. B. the particulate carrier initially has access to the bulk of the test medium and can therefore be influenced any light suppressing activity that is the main medium  owns. The solid phase is then transferred to a situation in who has and becomes relative freedom from the bulk sample less affected by the light suppressing effect. The movement The fixed phase is the only measure whose implementation is necessary to get a readable result. The constant signal generation the marking itself eliminates the need add more reagents or other stimulating measures to seize or add funds after particle localization has taken place, and creates maximum detection sensitivity with a minimum of complexity or the number of measures. The simple process of physically localizing the carrier particles is all that is required to get a detectable light signal produce. The suppression of the binding partner marked by the generated light when dispersed in the test medium is a highly significant feature of this embodiment of the invention.

The invention uses a label that is chemiluminescent or those involved in a chemiluminescent reaction with other components participates in the medium. If the medium is opaque or at least insufficient is permeable to the light to be determined and if the labeled specific binding partners evenly dispersed in the medium then no optical signal is detectable. Alternatively, you can The medium contains a chemical species that contains an ingredient of the light-generating system interacts and education extinguishes or inhibits optical signals. The subsequent localization of the particulate carrier, on which a complex, the labeled contains specific binding partner, is bound, can give rise to a give localized optical signal, its intensity for detection is sufficient.

Such a system can e.g. B. using horseradish peroxidase as a label, with luminol and a peroxide (such as H₂O₂) in be created in the medium. The medium can be a dye included, which is chemically sufficiently inert to the peroxide reaction not to compromise the medium's optical density appropriate wavelength that is sufficiently high (preferably O. D. of at least about 2) to the light that is dispersed by the  Marker is generated to make it insignificant compared to the that is perceptible at a localized concentration of the marker is. The use of dyes (dampers) that light at wavelengths absorb, including that of emissive luminescence in Immunoassays, including luminescence reactions as Marking system is described in EP 01 65 072. Alternatively the test medium may contain a chemical reagent that is compatible with the Generation of luminescence interferes.

Another way by which the chemiluminescent signal in the Main test sample can be reduced or excluded in introducing an extinguisher into the sample. If e.g. B. the peroxide enzyme of horseradish is used as a marker, with Peroxide or luminol is reacted in solution to give visible light then deliver the reaction by incorporating a competitive Reagent such as gallic acid or thioglycolic acid inhibited in the sample will.

It can be advantageous to use a fluorescent system incorporate that absorb the chemiluminescent light and at different wavelength can emit. Such a system can help to exclude the effect of the light generated in the main sample, especially when the light emitted by a suitable filter is considered. So z. B. blue light from Luminol can be absorbed with coumarin, which emits yellow / green light. The fluorescent system can be on or near the signal sensor be localized. Alternatively, the fluorescent system can be used as Labeling agents dispersed in the main sample can be used, so that only localized generation of chemiluminescence is perceptible is.

Although luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is a special one preferred chemiluminescent agent for use in connection with the invention is a range of alternative compounds and reactions are used or used. For example, you can other derivatives of 2,3-dihydro-1,4-phthalazinedione, such as that 6-amino derivative (isoluminol) can be used. Other examples are  Luciferin and acridinium esters. Such systems are generally e.g. B. in GB 15 52 607, GB 21 12 779 A and 20 95 830 B described. In general, the chemiluminescent reactions tend to be short-lived to be, causing time constraints on the assay procedure can lead. It is therefore useful to consider light emission in this regard improve, e.g. B. by the addition of reagents that control the emission extend or postpone. Improving chemiluminescent Reactions using compounds such as substituted 6-hydroxybenzothiazoles, p-iodophenol, 4-phenylphenol and / or 2-chloro-4-phenylphenol and aromatic amines, is described in EP 87 959, EP 1 16 454 and GB 21 62 946 A are described. The use of p-iodophenol is very particularly preferred.

Another embodiment of the invention is a particle-based one Assay method comprising:

• (a) a first population of localizable particles (e.g. "magnetic" particles) that have a specific binding partner for carry chemical species under test conditions,
• (b) a non-particle specific binding partner, which is marked in such a way that the specific binding partner is continuous a precursor for chemiluminescent within the test medium Reaction forms
• (c) a consumable reagent dispersed in the test medium, that is effective for the prepress and then the education inhibited by chemiluminescence, while the labeled specific Binding partner remains dispersed in the test medium, and
• (d) a second population of localizable particles containing a reagent wear or contain that with the precursor for generating chemiluminescence can react, and

with this method a common localization of the two Particle populations in the vicinity of a photosensitive film the perception of chemiluminescence enables that in a lot  is generated, which depends on the extent to which the specific Binding partner, carried by the first population of particles, bound to the species under the test conditions.

In the previous embodiment, the marking z. B. glucose oxidase be the one with glucose and oxygen in the test medium reacted to produce hydrogen peroxide as a precursor, the can be the consuming substance of the catalase enzyme, and the bound Reagent can be horseradish peroxidase mixed with luminol in the Test medium interacts. If desired, it can bound reagent can be carried from the surface or within the particles of the second population.

The sequential addition of ingredients to the test medium can be advantageous to e.g. B. a suitable time ("incubation") for the to allow immunological binding reaction (s) to occur prior to the Addition of a substrate take place to produce a chemiluminescent Signal is required. It is therefore estimated that the continuous generation of light only takes place when the necessary substrates have been added. This encore is always carried out before the solid phase again in the detection zone or in the detection environment is localized. The continuity of light generation must be available for at least the period of time required to localize the solid phase is selected, and sufficient for the film to produce To demonstrate light.

The incubation period required for the specific binding reactions What is required for this to take place depends on many factors, so on the nature of the reagents and the detection substance, their relative and absolute concentrations and physical factors like that Temperature. These parameters are known to the person skilled in the art. In general the incubation period does not exceed about 1 hour. Both In most applications, it is in the range of 30 minutes to 1 hour.

The location of the support material can be done in a variety of ways respectively. As stated earlier, the sensitized firm  Phase part (or indeed all) of the inner wall of the container form in which the analysis is completed. Alternatively, a single large portion of the sensitized solid phase, e.g. B. in the form a pellet or pencil in which there are containers and in the neighborhood to the photosensitive film at the recording stage be. For example, plastic (e.g., polystyrene) beads in general used as fixed phases. Also "plastic coated" Steel balls with a diameter of 2 to 5 mm, d. that is, a coating from a polymer, like from a polycarbonate that can be applied from solution used. These have the advantage that they can be used externally Magnets can be positioned. In a preferred one Embodiment of the invention, however, the solid phase comprises a large one Number of small particles.

It is possible to remove the solid phase particles under the influence of To let gravity settle, although this requires that a uniform suspension of the particles in the test medium is maintained, e.g. B. by stirring during the binding reaction expires. Centrifugation can be chosen as an alternative to gravitation will. Alternatively, particles can be localized by a porous membrane or filter through the mass of the test medium is carried out, whereby the particles are carried away into a smaller volume will. Another alternative is to use a particulate carrier material that is influenced by magnetism or other externally imposed forces in a particular local Location within the volume of the test medium. The Particles can pass through the test medium by ultrasound generation drifting stationary shaft.

A particulate solid phase can be made from any suitable carrier material getting produced. So-called "magnetic" (i.e., particles, which can be stimulated to move through a liquid under the Influence of a magnetic field to move) particles of a variety suitable sizes, the z. B. between less than about 1 micron up to more than about 300 µ are commercially available. For gravity separation particle sizes of at least about 50 microns and not  more than about 300 µ preferred, depending on the density of the material depends. Commercially available ethylene oxide acrylic beads are very suitable, especially for the assays involving labeled haptens. Measures to raise awareness and / or to couple such Solid phase carriers on binding reagents, such as immunoglobulins, are good known standard measures and do not form part of the present Invention.

The field of detection substances within the scope of the invention can be used is extensive. The following detection substances, which are only intended to be examples, can be mentioned : Steroid hormones, such as those that occur during pregnancy Play a role or have an impact on fertility, such as Progesterone and estrogens, urinary metabolites such as estrone-3-glucuronide and Pregnandiol-3-glucoronid, peptide hormones such as human chorionic gonadotrophin, luteinizing hormone and follicle stimulating hormone, Proteins such as urnalbumin, beta-2 microglobulins and retinol binding proteins, Lipoprotein high and low density, the detection of which in the cardiovascular observation is valuable, and cardiac active troponin, infectious pathogens, such as antigens from Chlamydia, Neisseria, Herpes, Candida and HIV₁ (AIDS) and antibodies from Treponema (Syphilis), Rubella and Toxoplasma, and immunoglobulins such as monoclonal Antibody.

The test medium can thus come from many sources, in particular of body fluids such as urine, serum and plasma. The invention is especially applicable for the separation of cell culture media for Immunoglobulins, i.e. i.e., for the production of monoclonal antibodies.

The specific binding partners can be polyclonal or monoclonal Antibodies or antigens, lectins or hormones or their receptors be. Such materials are well known and many of them are in the Available commercially. Their precise nature does not form part of the present Invention.

If the sample is believed to be the detection substance contains, is of a variable nature (e.g. serum or urine) or constituents  contains that with the specific binding reactions or with the formation of the perceptible signal could interfere, then it can be beneficial be to dilute the sample, e.g. B. with water or a buffer solution, before performing an analytical method of the invention to: reduce the likelihood of such interference.

In some circumstances it is possible to measure the sensitivity of an analytical To improve the process by at least the main mass the test sample before observing the signal from the localized one Solid phase carrier is removed. This is beneficial as many natural samples, such as blood serum, contain materials associated with the interfering signal-generating reaction (so to a misleading low or negative observation result) or can initiate the signal-generating reaction (and thus to a wrong one positive observation result). This problem is already good known. Many conventional assays therefore acquire a series of "Washing measures", which are intended to complete the solid phase to separate from the rest of the sample.

In another embodiment of the present invention therefore includes the analytical procedure the following measures:

• a) Localize the solid phase support material after a suitable one Incubation period in the test sample,
• b) removing at least the bulk of the liquid test sample,
• c) adding an amount of a liquid medium, the other such Contains reagents as required to complete the light emission reaction to occur, and
• d) relocating (if necessary) the particulate solid phase support material and recording any light emitted by a Point source is broadcast.

A modification of the one in the immediately preceding section The procedure described includes the measures:  

• a) Localize the solid phase support material after a suitable one Incubation time in a first liquid medium containing the test sample Absence of the labeled reagent contains or includes
• b) removing at least the bulk of the liquid test sample,
• c) adding an amount of a second liquid medium that the contains labeled reagents,
• d) localizing the solid phase support material again after a suitable incubation period,
• e) removing at least the bulk of the second liquid Medium,
• f) adding an amount of a liquid medium, such further Contains reagents that are required to stop the light emitting Reaction, and
• g) relocating (if necessary) the particulate solid phase support material and recording all the light emitted by one Point source is broadcast.

If this last embodiment is simplified, the second and third liquid medium and the same, so that only one only fluid removal and replacement measure performed too are needed. Alternatively, the third liquid medium at the end of the second incubation measure without removing any second liquid medium are added to the container.

Furthermore, it was found in connection with the invention that it is not necessary to wash the solid phase so carefully that all remaining test sample and / or all unbound marked reagent is removed. It is indeed an advantage of the present invention that with this configuration an analytical Process can be created that is the bulk of the liquid  Medium removed using a minimal number of measures, those with the achievement of a reliable analytical result To be in harmony. It is the goal to have a substantial part of the liquid Remove the medium from the container. Generally this part at least about 50% and usually at least about 90% of the total volume make up the liquid in the container.

Optionally, the supernatant liquid can be decanted, after which the localization of the particulate solid phase support follows. The Container in which the analytical reaction is carried out is then refilled with another liquid medium. If it If necessary, this procedure can be repeated to create a to take intermediate washing measures (which require a renewed Localize the particulate support prior to a second Decanting and refilling the container included). However, if it is is possible, this should be avoided to reduce the number of measures the total required for the analysis on a Reduce minimum. In any case, what ultimately needs to be replenished Contain those reagents that are required to prepare the medium Initiate or accelerate chemiluminescent reaction.

As an alternative to decanting, the liquid medium can be extracted from the Containers removed by pipetting or suction with a siphon will.

All separation measures that have just been described can be closed lead to the problem of removing the liquid coming from the Container has been removed. In a particularly preferred embodiment In the invention, it is preferred to use a wick material use to remove the bulk of the supernatant from the Remove the reaction container by capillary action. The expression "Wick material" is intended to express any porous material that is in the container can be immersed to get the liquid out of it absorb, this material then removed from the container can be located during the localized particulate solid phase support remains essentially unaffected in the container. To the removal of the wicking material that is the absorbed liquid  contains, the container can be refilled with additional liquid medium will. If necessary, an intermediary Wash stage can be made, and second wick material can be used to rinse the washing liquid with subsequent Remove localization of the particulate support material.

The wicking material can be any porous or fibrous Material is made for the rapid absorption of liquid is capable. The porosity of the material can be expressed in one direction (i.e. there are pores that are complete or predominant run parallel to the longitudinal axis of the wick material) or in different Directions (in all directions, so that the Wick material has an amorphous, sponge-like structure). A porous plastic material, such as polypropylene, polyethylene (preferably with very high molecular weight), polyvinylidene fluoride, ethylene vinyl acetate, Acrylonitrile and polytetrafluoroethylene, can be used will. The wicking material may also be paper or other cellulosic Materials such as nitrocellulose are made. Materials currently in the writing tips of so-called fiber pens are particularly suitable. Indeed found within the scope of the invention nib units currently are commercially available for the production of such writing instruments, and provide the ideal wicking materials for practicing the invention. Have the wells of microtiter plates with multiple wells typically a capacity of 300 to 400 microliters. A Tip unit that is about 5 mm in diameter and one 3 cm long, provides an ideal wick material for rapid removal of a volume of up to e.g. B. about 400 microliters a liquid medium from such a well.

The speed at which the liquid from the container into the Wick material pulled should be selected so that it is neither is too fast or too slow. Preferably the wick material should be be able to contain the bulk of the liquid medium in the container Absorbing courses of seconds should still have the capacity have to absorb more liquid. In practice it can be a significant one Liquid film adhere to the walls of the container and thus  stay behind when the wick material is almost instantly liquid is absorbed and then immediately removed from the container. If the absorption rate is slightly slower, or if that Wick material is left in the container for a shorter period of time, then all residual liquid film can be on the bottom of the container run off and are absorbed by the wick material. Vice versa this can unnecessarily extend the operating time if the wick material the liquid only slowly (e.g. in minutes instead of seconds) absorbed, which should preferably be avoided.

Because the wick material is used to make the liquid medium a container containing particulate carrier material absorb, it is preferred to in the particulate carrier material localize short distance without contact with the wick material, to prevent the particulate carrier material from adhering to it or actually drawn into the wick material by the flow of liquid becomes.

Conventional multi-well microtiter plates contain one Variety of wells arranged regularly. A corresponding Arrangement of wick units according to the invention can be used to be able to simultaneously fluid from each element throughout this Remove arrangement of wells if desired.

A particular advantage of the invention is that the liquid the analytical container with no risk of pouring or without need, remove the removed liquid in a free-flowing form, can be removed. The liquid remains in the wick material and can be eliminated much sooner. A significant advantage of a wick material according to the invention is that if it is the liquid Has absorbed medium, removed from the container and without risk of Draining the liquid medium can be handled. This is particularly so important when performing analytical tests that with such samples used, of which it is believed that they contain infectious disease organisms. The risk of aerosol formation is also reduced to a minimum, which is an important point of view where liquid media,  that contain infectious organisms.

The dimensions and overall shape of the wick materials can be so be selected so that the optimal volume of liquid from the Analytical container is removed while the solid phase support (and thus any sensitive material attached to it) as far as possible remains undisturbed.

Embodiments of the invention are intended to be exemplary only below described with reference to the subsequent drawings will.

Recorder

A "camera" recorder that can be used in connection with the invention is shown in Figures 1 and 2 of the accompanying drawings. Fig. 1 shows a perspective view of the entire device or the device, the cover covering the camera being shown in the open position. Fig. 2 shows a partially sectioned view of the device, seen along the line AA in Fig. 1, with the lid of the camera closed.

The recording device shown in Figs. 1 and 2 operates as described in GB-PS 21 69 704.

The apparatus shown in Fig. 1 comprises a conventional instant camera 100 having a hinged portion 101 which can be opened to receive a film (not shown) and a slide fastener 102 which can be pulled out from the rear side 103 around the film to expose. A flat rectangular body 105 is attached to the top 104 of the back of the camera and has a central rectangular opening 100 through which the film can be exposed when the shutter 102 is pulled out to open it. The opening 106 receives an easily replaceable plate 107 which has a plurality of vertically arranged holes 108 which are arranged at right angles to receive cells of a commercially available plate for several cells (not shown). A small button 108 is located near one end of the plate 107 on the surface 109 of the plate so that the plate can be gripped and lifted out of the body 105 . The depth of the body 105 is greater than the thickness 107 . The button 108 does not extend over the upper side 110 formed by the body 105 . A closable cover 111 is arranged on the rear side 112 of the device and can be tilted downwards to close the body 105 . A light-tight seal is provided by means of a narrow continuous bead 113 which extends around the lower surface 114 of the closable lid 112 near the edge 115 of the lid and which engages with a corresponding continuous slot 118 which extends around the top 110 of the body 105 runs.

The view of the device shown in FIG. 2 shows how the device is mounted on the surface 200 by means of feet 201 attached to the bottom 202 of the rear side 100 of the camera. The structural details of the rear of the camera are not shown since these are generally known and do not directly relate to the invention. The lid 111 is shown in the closed position, the flanging 113 lying in the slot 116 in the top side 110 of the body 105 . The shutter 102 is shown in the closed position. The perforated plate 107 rests on the surface 203 of the closure. When the shutter 102 is opened, the perforated plate 107 falls down and rests on a film (not shown) which is placed immediately under the shutter. FIG. 2 further shows a transparent multiple specimen bottom 204 which is mounted in the perforated plate 107 , the individual cups 205 equipped with a round bottom extending into the holes 108 in the perforated plate. The thickness of the plate is such that each well extends to the bottom of each opening so that it is supported with the plate on the film after the shutter is opened so that each well is in direct contact with the film. One of the wells is shown to have a liquid sample 206 that has a particulate solid phase 207 located at the bottom of the body. The amount of solid phase 207 is greatly exaggerated for reasons of drawing technology, since in practice the volume occupied by the particulate solid phase, when it is localized, is very small.

At the end of an analysis carried out in a microtiter plate in a perforated plate can be a particulate solid Phase localized at the bottom of each well (e.g. by magnetic influence) will. The perforated plate can be inside the recess of the Arranged camera body and the lid can be closed. The Shutter is then opened to expose the film and allows that chemiluminescent light is recorded on the film, which in a chemical reaction by a component of the localized solid phase is generated.

For example, various point sources are shown in FIGS. 3 through 6.

FIG. 3 shows a single transparent well 300 (which forms part of an arrangement of such wells in multiple well plates) in cross section, as is arranged in the opaque fastening plate 301 . The diameter of the well decreases noticeably in the direction of the bottom 302 and forms a narrow conical zone at the bottom of the well. The mounting plate 301 sits directly on top of a photosensitive film 303 and the well 300 extends to the bottom 304 of the mounting plate so that the tip 302 of the well communicates directly with the film. The vertical sides 305 of the opening 306 in the mounting plate 301 , which receives the cells 300 , forms an exposure zone 307 of the film and has a diameter which is substantially larger than that of the cell at its lower end. The test specimen contains a liquid reagent volume 308 and a small amount 309 of a finely divided particulate solid phase which, e.g. B. is localized by magnetic action at the bottom of the well. Due to the small amount of the particulate solid phase and the internal dimensions of the well, the particulate solid phase is concentrated in a small volume at the bottom of the well, so that when the solid phase contains a reagent, it is able to undergo a chemiluminescent reaction To emit light, the light collects and is emitted in a zone that is much smaller than the area of the exposed film and thus acts as a point source. Although the accumulated solid phase is separated only by the film due to the thickness of the material of the wall forming the well, it is nevertheless sufficient for the light spot formed on the film to have a relatively larger or smaller diameter depending on the amount of light generated in the solid phase having.

The use of an opening to create a point light source is shown in FIGS. 4, 5 and 6. FIG. 4 shows a single beveled, transparent cup 400 with a round base in cross section in the opaque fastening plate 401 . Immediately below the bottom 402 of the plate 401 in contact with the bottom 403 of the cup is a thin perforated opaque plate 404 with a small opening 405 just below the tip of the cup. The width of the perforation 405 is significantly smaller than the diameter of the opening 406 in the fastening plate which receives the cup. A photosensitive film 407 is arranged below the perforated plate 404 at a small distance X therefrom. The well contains a volume 408 of a liquid reagent and a single large opaque bed 409 as a solid phase which rests on the bottom of the well and substantially fills the lower portion of the well. The diameter of the bed 409 is significantly larger than the size of the opening 405 . If the surface of the bed carries a bound reagent that can emit light due to a chemiluminescent reaction, the light from the surface of the bed that is immediately adjacent to the opening can pass through it and get onto film. The diameter of the point created on the film changes depending on the amount of light that has passed through the opening and the size of the distance X.

A further embodiment is shown in FIG. 5, in which the well 500 has a flat bottom and the inner surface 501 of the sample body is sensitized at its lower region with a reagent and acts as a permanently arranged solid phase. If chemiluminescent light is emitted from the arranged reagents which are bound to this sensitized surface, the entire lower zone will emit light, but only the light passing through the opening 502 will strike the film 503 arranged below. As with the embodiment described above, the diameter of the light spot created on the film depends on the amount of light passing through the opening.

A further embodiment is shown in FIG. 6, in which the well 600 has a round bottom, but is relatively wide at its base and contains a substantial mass 601 of the particulate solid phase, which occupies most of the bottom 602 of the well. The opening 603 forms a smaller part through which light passes and thus serves as a point source.

In Fig. 7, a pair of wells 700 and 701 are arranged in a row in a clear multi-well plate, which is a sectional view and the wells can be seen in the opaque mounting plate 702 . The general arrangement corresponds to that described with reference to FIG. 4. In this case, however, the opaque perforated plate 703 , which forms the opening 704 under each well, is separated from the film 705 below by another thick opaque perforated plate 706 , in which openings 707 are formed with a substantially larger diameter than that of openings 704 and which, as is actually shown in the drawing, have the same diameter as the openings 707 of the fastening plate 702 which receive the cells. Each opening of the partition plate 706 forms a zone 708 of the film which can be exposed to any light passing through the opening. Since the partition plate is opaque, each limited zone of the film is separated from the zones associated with the surrounding wells so that the risk of light hitting the opening from an opening in an adjacent zone is minimal.

Fig. 8 shows components for a holder of a Mehrfachnäpfchenbodens in an exploded view. For reasons of simplification, the transparent base 800 is shown as a 12-well plate with a 4 × 3 arrangement, although in practice the number of cells can be considerably larger. The microtiter plate can be received in an opaque perforated mounting plate 801 with an opening 802 for receiving each well. The thickness 803 of the mounting plate is identical to that of the outer depth of each well. An opaque apertured plate 804 is attached below the attachment plate 801 and has a corresponding number of small openings 805 , each of which lies in the vertical axis of an opening of the attachment plate 801 . Arranged under the apertured plate 804 is an opaque spacer plate 805 , which has the same number of openings, which in this case have the same diameter as the openings of the fastening plate 801 . The thickness 807 of the spacer plate 806 forms the distance that separates each opening 805 from a photosensitive film (not shown), which can be arranged directly below the spacer plate.

Example 1

A "camera" recorder, which is basically the corresponds to that just described, was in the following hCG assay used.

materials a) Solid phase

Commercial macroporous polystyrene particles (Dyno: Dynospheres XP-6006), average size 2.6 microns, in which a uniform Distribution of iron oxide is present. These even, spherical Particles have a hydroxyethyl methacrylate based one outer casing, derivatized with lateral tosyl groups. The Tosyl moieties can easily be replaced by nucleophilic substitution Amino groups are subject to, as in the outer surface of a Proteins are present, which leads to immobilization. These were with Mouse anti-hCG monoclonal antibody sensitized by what the following procedure took place.

To an aliquot of a suspension, the Dynospheres XP-6006 (10 mg) contained, a boric acid buffer (0.005 M, pH 9.5, 0.5 ml) was added. The antibody solution (200 µg in 1 ml PBS, pH 7.2) was then added  given, after which the addition of the boric acid buffer (0.5M, stopped to pH 9.5 with NaOH, 0.5 ml) followed.

This was used to couple at room temperature overnight on a Vibrating shaker left. The beads were then removed using of a magnet sedimented. The protein solution was aspirated. they was washed once with PBS (5 ml). The coupling measure was repeated, this time using BSA (20 mg) was done to couple all unreacted tosyl groups. The Beads were then overnight over a phosphate buffer (0.25M KH₂PO₄, adjusted to pH 8 with NaOH, the 0.5% BSA and 0.1% Tween®20 (5 ml) to any adsorbed antibody from the bead surface. Finally, the Beads in the same buffer (2 × 5 ml) and then in PBS (2 × 5 ml) washed and in a known volume of the PBST buffer, which Contained 0.1% BSA and 0.01% thiomersal preservative at 4 ° C stored.

b) conjugate

The same mouse anti-hCG monoclonal antibody was used with horseradish peroxidase enzyme using a conventional method conjugated in a PBST buffer.

c) Detection substance

A series of urine samples showing known concentrations of hCG of 0 contained up to 450 mIU (immunization units) / ml.

d) Luminol cocktail

A solution in 0.1M Tris / HCl pH 8.5 buffer of 1mM luminol, 1mM p-iodophenol and 10 mM urea peroxide.

e) camera film

Polaroid type 612 with high contrast black / white 20,000 ASA film.  

method

The following were pipetted into each of the series of wells with a round bottom in a conventional transparent plastic-cast plate (with several wells) (“microtiter”):
50 µl of a detection sample from the range of the above concentrations,
50 µl conjugate and
50 µl of the solid phase (0.05% solid, resulting in 25 µg solid phase per well)
(Position of each sample in the arrangement of the wells, which are noted on the dish).

The microtiter plate was covered with a plastic film and opened a laboratory shaker for one hour at room temperature shaken. The plate was then placed on top of an arrangement placed small magnets, which the arrangement of the cups in the Plate corresponded to the particulate solid phase on the bottom to locate each well. After about 2 minutes the plastic film removed and (with the arrangement of the magnets below the Plate held) decanted the supernatant liquid from each well and the plate dried ("blotted"). The record was made by the Removed magnets. 200 µl of the PBST buffer was added to each well given. The plate was left for 1 hour without shaking. The solid phase was then renewed at the bottom of each well localized. This was followed by the decanting and washing measures repeated. The plate was then removed from the arrangement of Removed magnets. 100 µl luminol cocktail was added to each well given. After 1 minute, the plate was placed on the plate again for 2 minutes Arrangement of the magnets set and carefully removed and in the perforated plate of the camera is set and the complete unit in the camera is inserted and the lid is closed. The clasp was opened and the film exposed for 5 minutes.  

FIGS. 9 and 10, the subsequent drawings illustrate the typical results obtained. A typical photograph is shown in Fig. 9 with two parallel arrays of wells that were used in the experiment to obtain duplicate results. The increasing diameter of the spots of the exposed film correlates with the increasing concentration of the detection substance in the samples. The graph in Figure 10, plotting the spot diameter (X) versus hCG concentration (Y) expressed in MIU / ml, shows that within the experimental measurement error there was a fairly good linear correlation between the log concentration and the spot diameter.

Example 2

This example illustrates the separation of the supernatant from monoclonal Antibody cell line cultures and the partial removal of the Sample between two incubation measures.

material and methods Fixed phase

Dynospheres XP-6006 beads as described in Example 1 were used as the particulate solid phase. These were sensitized with Dakopatt's Z₂₅₉ rabbit anti-mouse immunoglobulins.

Rabbit anti-mouse / biotin

The Dakopatts Z₂₅₉ (rabbit anti-mouse immunoglobulins) reagent was treated with biotin-N-hydroxysuccinimide (BNHS) at pH 7.5 marked biotin. The unreacted BNHS was expanded by Dialysis removed.

Avidin-HRP conjugate

Conventional Sigma No. A₃₁₅₁ horseradish peroxidase labeled Avidin conjugate, as recommended by manufacturers, was  used.

Wicks

Two types of "wicks" were used to make sample liquids to remove during the two part incubation. These were commercially available Pen tip units running out of fibers:

• a) Polyethylene writing units that are porous in all directions are more cylindrical Shape with a regularly tapered spout, manufactured by Porex Technologies, Fairburn, Georgia, USA. These were able to add 200 µl water in about 14 seconds absorb.
• b) predominantly non-directional fibrous "chisel-shaped" tip units, manufactured by Stylex SA, Lamone-Lugano, Switzerland. They were able to drink 200 µl of water in less than 1 second absorb.

HS medium

The "HS medium" represents a 5% horse serum in RPMI 1640, which consists:

• 1) 500 ml of commercial RPMI 1640 medium (Flow laboratories)
• 2) 25 ml horse serum
• 3) 25 ml penicillin streptomycin (penicillin 5000 IU / ml and streptomycin 5000 mg / ml)
• 4) 4 ml sodium pyruvate, 100 mM
• 5) 5 ml L-glutamine, 200 mM

Assay procedure

1. 5 ul of the 0.625% dynosphere suspension were mixed with 15 ul Antibody sample, diluted in 5% HS medium, in wells of round bottom a conventional transparent plastic cast "microtiter" dish 30 minutes at room temperature on a shaker  incubated. The sample was a solution of a mouse monclonal antibody the concentrations from 1 ng / ml to 500 µg / ml, in the HS medium, or contained a blind HS medium as a control.

2. After incubation, the Dynosphere beads were coated with a Magnets isolated. The excess of antibody solution was removed Aspirated using either a Stylex or Porex wick.

3. 30 µl of prepared avidin-HRP-Biotin (RaM) complex were added given to each well. The Dynosphere beads were again suspended. (The complex consisted of equal volumes of 1/500 rabbit anti-mouse / biotin and 1/2000 avidin-HRP conjugate mixed together and left for at least 30 minutes.) The Dynosphere beads were added to the complex for 30 minutes Incubated room temperature on a shaker.

4. 150 ul of a luminescent reagent was added. The Dynosphere beads were placed on a magnet for 2 minutes localized on the ground. The reagents were passed

10 mM luminol, 5 mM p-iodophenol,
100 mM urea peroxide (volume ratio = 1: 1: 1).

15 ul of a conventional black ink per 1 ml of Add the mixture before placing it in the wells.

5. After the magnetic localization of the Dynosphere beads the wells were placed on a 20,000 for about 1 to 2 minutes ASA polaroid paper film in a camera as in Example 1 above described, exposed. The film was developed for 1 minute. To At that time, every positive result was a blank spot on one marked black paper.

Results

The analysis was able to detect antibodies in concentrations from 1 ng / ml to 500 µg / ml, while the blank sample of the HS medium did not produce a stain. Within certain limits, the spot size was directly related to the concentration of the detection substance. Beyond this limit, however, there was a plateau effect, since the spot size then tended to decrease with a very high concentration of the detection substance. A typical print is shown in Figure 11 of the accompanying drawings. These are the results of a typical test, which is shown graphically in FIG .

Example 3

The "camera" recorder of Examples 1 and 2 was used with a minor change using an assay for mouse immunoglobulin G (1gG). A cut-down flexible transparent plate with multiple wells ("microtiter") was placed on a movable plate which was adapted to an orifice plate and a spacer plate as shown in Figs. 4, 7 and 8 of the following drawings had been. With this arrangement, each well was placed over an opening 2 mm in diameter, either 3 mm or 1.8 mm from the surface of the film, depending on the spacer plate attached. Since this holder was movable, it was interchangeable with the standard holder used in Examples 1 and 2. The holes in the spacer plate were circular and 8 mm in diameter so that they defined a nominal maximum spot size of 8 mm in diameter.

materials a) Solid phase

Plastic-coated steel ball bearings (3 mm diameter), below referred to as "beads" were with Dakopatt's Z₂₅₉ rabbit anti-mouse immunoglobulin sensitized according to the following procedure.

The immunoglobulin was added to 47 µg / ml in a 0.05M carbonate / bicarbonate buffer,  pH 9.6, diluted. This solution was in a lot of 50 µl per bead (e.g. 5 ml for 100 beads) to the beads given. This sensitizing solution was with the beads for 16 hours left in contact for long (overnight) at room temperature, after which it was decanted. The beads were in twice distilled water.

Conjugate

A commercial conjugate consisting of Peroxidase-conjugated rabbit immunoglobulin to mouse immunoglobulin (Dako, code P 161) existed. Dilutions were made with PBST a content of 0.5% BSA.

Detection substance

A series of samples were made by diluting a stock solution purified monoclonal mouse IgG in PBST containing 0.1% BSA produced to deliver the following concentrations:

100ng / ml; 10ng / ml; 1ng / ml; 0.1 ng / ml; 0.01 ng / ml.

Luminol cocktail

Luminescent reaction mixture, which consists of a solution of 1 mM Luminol, 1mM p-iodophenol and 10mM sodium perborate.

Camera film

The "camera" recorder was made with a high contrast black and white film, 20,000 ASA of the Polaroid type 612.

Action

Cups of the reduced plate were filled with a volume of 150 µl Sample solutions, 3 for each concentration, are loaded. An additional one Set of 3 wells was filled with PBST / 0.5% BSA diluent  negative control, loaded. A sensitized bead became too given to each well.

The plate (plus samples and beads) was placed on a plate at 37 ° C Shaker placed in an incubator. The incubation lasted 30 minutes. The samples were then subjected to suction. To do this, the plate was placed on a block with an arrangement of small magnets attached so that each bead is on the side of his well, which was done sufficiently offset to to be able to insert a narrow tube at the bottom of the cell. Each Cups were filled with PBST, which was placed on them in the same way was aspirated to give minimal rinsing.

Then 150 ul volume of the conjugate (diluted 1/100) to given to each well and bead. The floor was under again Shake incubated at 37 ° C for 30 minutes. After this incubation measure the conjugate was aspirated as before. Every bead was rinsed seven times with PBST.

Each rinsed bead was then treated with 150 µl luminol cocktail covered. The plate was then placed in a "camera" device Standard plate carrier used. The film was made for 30 seconds Wells (containing beads + cocktail) exposed for exposure. The plate was placed on a modified support with 2mm openings and a 3 mm spacer plate placed in the camera and 2 minutes long exposed to film. This was with another Spacer plate of 1.8 mm repeated and a fourth exposure with the the latter arrangement carried out, but this time 1 minute. The different Recording conditions are described in the following Table 1 compiled.  

Table 1

conditions

Results

Typical deductions are shown in Fig. 13 of the following drawings.

The 30 second exposure (trigger A) with an opening restriction showed that the 100 ng / ml samples produced the most light during as another extreme, the zero control an almost undetectable amount of light generated. In the absence of an opening restriction, the light yields provided a blurry overlapping image. At 1 ng / ml a sharp full stain, but the stains were below this value angular and poorly defined.

Print B gave a clearer picture. The 1ng / ml, 10ng / ml and 100ng / ml samples gave identical full spots during the 0.1ng / ml samples weak, blurry spots. The 0.01ng / ml samples gave hardly any detectable stains.

If the space between the opening and the film is reduced to 1.8 mm (trigger C) greater resolution was observed to the extent that the 1 ng / ml samples reduced spot size and the 0.1 ng / ml samples delivered an even smaller size while the 0.01ng / ml samples with very small spots. Zero control provided barely noticeable faint background stains  lower intensity than the 0.01 ng / ml samples.

By reducing the exposure time from 120 seconds to 60 seconds (Deduction D) sensitivity and area were slightly different in the 101 ng / ml samples changed, which alone gave full-surface stains, while the spots for everyone else as the diameter progresses were significantly reduced.

Claims (27)

1. Analysis method for determining a detection substance in a sample by specific binding using a solid phase support material on which a first binding reagent with specificity for the detection substance is immobilized and a labeled binding reagent which is involved in a sandwich reaction or in a competitive reaction with the first Binding reagent can participate in the presence of the detection substance, the label participating in a chemiluminescent reaction, the amount of light of which is used to determine the extent (if any) in which the labeled binding reagent has been bound to the solid phase, characterized in that the light is on recorded on a photosensitive film and derived from a point source in such a manner that the size of the image formed on the film provides a measure of the amount of detection substance in the sample. 2. Analysis method according to claim 1, characterized in that the Area of the solid phase that is locally presented to the photosensitive film is not larger than about 50% of the area of the light source exposed film. 3. Analysis method according to claim 2, characterized in that the Area of the solid phase that is locally presented to the photosensitive film is not larger than about 25% of the area of the light source exposed film. 4. Analysis method according to claim 1, characterized in that the fixed phase itself determines the point source.   5. Analysis method according to claim 4, characterized in that the fixed Phase is particulate and the point source is created by the particulate solid phase in a small area of a container, in the analysis is being carried out. 6. Analysis method according to claim 5, characterized in that the formation the point source is achieved in that the particulate solid phase on Bottom of a well that has a much narrower cross section in the lower part than in the upper part. 7. Analysis method according to one of claims 4 to 6, characterized in that that it is in one or more wells of a conventional microtiter plate is performed and the photosensitive film for recording of the luminescence signal in direct contact with the bottom of the cell Plate is arranged. 8. Analysis method according to one of claims 1 to 3, characterized in that that the point source is defined by an opening that in in the immediate vicinity of the outside of the container which the analysis is carried out and the photosensitive film from the Opening is placed away. 9. Analysis method according to claim 8, characterized in that a perforated spacer is attached between the opening and the film keep the distance between the opening and the film exactly. 10. Analysis method according to claim 8 or 9, characterized in that the Analysis in one or more cells of a conventional microtiter plate performed and the opening is formed by a perforated plate which can be arranged directly below the microtiter plate, so that an opening is directly below each well. 11. Analysis method according to one of claims 4 to 10, characterized in that that each opening has a maximum dimension of about 1 to 2 mm and the distance between the opening and the photosensitive film is at least about 0.5 mm, but not more than about 5 mm. 12. Analysis method according to claim 11, characterized in that the removal between the opening and the film is 1 and 3 mm.   13. Analysis method according to one of the preceding claims, characterized characterized in that after a suitable incubation with the sample and a solution or a dispersible labeled specific binding reagent a particulate solid phase support material that immobilizes carries specific binding reagent, within a liquid Medium located in the vicinity of a light-sensitive film so that light from a point source can be detected, whereby the marker is able to participate in a chemical reaction, which leads to the production of chemiluminescent light, and the liquid Medium containing those additional reagents that are necessary to get the Letting the reaction occur contains. 14. Analysis method according to one of claims 1 to 12, characterized in that it comprises the following measures:
(a) Preparation of an aqueous assay medium containing:

• (I) a particulate solid phase support material on which a specific binding reagent for the detection substance to be determined is immobilized, the particulate solid phase support being suspendable in the aqueous assay medium but readily located therein,
• (II) a sample suspected of containing the detection substance to be determined, and
• (III) a labeled reagent soluble or dispersible in the assay medium, which is either a second specific binding reagent that can participate in a sandwich reaction with the detection substance and the first specific binding reagent, or detection substance itself or an analogue of the detection substance and the label is a chemiluminescent reactant,

(b) incubation of the assay medium so that the labeled reagent can be bound to the particulate solid phase support,
(c) locating the particulate solid phase support within the assay medium,
(d) replacing or diluting the assay medium using a liquid medium containing such additional reactants that may be necessary for the chemiluminescent reaction to occur, or replacing and diluting together with such additional reactants, and
(e) measuring chemiluminescent light on a photosensitive film emitted from a point source that has a label attached to the particulate solid phase support in the localized state. 15. Analysis method according to claim 14, characterized in that it modified by performing a two-step incubation which is an initial incubation of the particulate solid phase support material and the sample in the absence of the labeled reagent, wherein at least the bulk of the sample is removed thereon and by a liquid medium is replaced, which is the dissolved or dispersible contains labeled reagent, and also a second incubation is carried out, after which measures (c) to (e) of the above sequence be performed. 16. Analysis method according to one of claims 1 to 12, characterized in that it uses a specific attachment partner who is such is marked that the marking in the test medium is a continuous Chemiluminescence signal generated, this signal masked, extinguished or otherwise suppressed while the labeled binding partner is even is dispersed within the test medium, and wherein after the Binding reaction a local concentration of the particulate carrier material is set in the test medium such that the local Concentration through the complexed, labeled binding partner carrier the particle provides a local light flux that is sufficient to match the local To overcome signal suppression ability of the test medium and thus a deliver detectable chemiluminescent signal, the strength of which Extent of binding depends on the immunogenic to be tested Species and the specific binding partner carrier through the particles has occurred.   17. Analysis method according to one of the preceding claims, characterized characterized in that the chemiluminescent agent luminol or a is similar derivative of 2,3-dihydro-1,4-phthalazinedione. 18. Analysis method according to claim 17, characterized in that the Chemiluminescence reaction enhanced by the presence of p-iodophenol becomes. 19. Analysis method according to one of the preceding claims, characterized characterized that it is an immunoassay. 20. Analysis method according to one of the preceding claims, characterized characterized in that it is in one or more wells of a conventional transparent micro-analytical plate with many cells is carried out. 21. Device for determining the result of a specific binding analysis using the amount of light from a chemiluminescence reaction for the quantitative determination of a detection substance in a sample, characterized in that the device has a holder ( 301 , 401 , 501 , 601 ) for a plurality of cells ( 300 , 400 , 500 , 600 ), the holder having a first plate with an arrangement of holes arranged therein for receiving an arrangement of cups ( 300 , 400 , 500 , 600 ) and a housing for receiving the holder, the housing being able to to prevent stray light from entering and an openable lid ( 111 ) further includes means for holding a photographic film near below the plate and a removable shutter ( 102 ) provided between the film and the plate can be arranged so that the closure ( 102 ) between a closed position and an open position ng is movable, and in the latter the film is exposed, and that between the base plate of the holder and the closure ( 102 ) a second perforated plate is provided, the individual openings of which each create an opening below a single opening of the holder, each opening having a has a substantially smaller cross-sectional area than that of the associated opening of the holder together with the means for maintaining a predetermined distance between the film and the perforated plate when the closure is in the open position. 22. The apparatus according to claim 21, characterized in that the Spacer comprises a perforated third plate in which the perforation are substantially larger than the openings in the second plate, each Perforation in the third plate just below the opening of the second Plate is arranged. 23. The device according to claim 22, characterized in that a Triple combination of plates is arranged so that (a) they on the Closure is stored when it is in the closed position and (b) falls on the photographic film when the shutter is in its open position is moved. 24. Device according to one of claims 21 to 23, characterized in that each opening has a maximum diameter of at least has about 0.5 mm. 25. The device according to claim 24, characterized in that each Opening has a maximum diameter of about 1 to 2 mm and the distance between the opening and the photosensitive film is at least about 0.5 mm, but not more than about 5 mm. 26. The apparatus according to claim 25, characterized in that the distance between the opening and the film is 1 to 3 mm.