JP4438455B2 - Method for measuring free human immunoglobulin light chain and kit - Google Patents

Description

  The present invention relates to an immunological measurement method for a free human immunoglobulin light chain (free human human immunoglobulin light chain: FLC) and a kit used therefor.

  Free immunoglobulin light chain kappa (FLCκ) or λ type (free immunoglobulin light chain lambda: FLCλ) has been used in clinical studies so far as immunological disorders such as multiple myeloma and amyloidosis. It is shown to be useful for diagnosis and is expected as a marker for clinical examination.

  In order to measure this FLC, antibodies specific to FLC, particularly polyclonal antibodies, have been widely used so far. However, human-derived components, particularly serum, contain intact immunoglobulins several thousand times the concentration of FLC, and the specificity of polyclonal antibodies is insufficient to avoid the influence of this intact immunoglobulin, It was difficult to say that the measurement method was satisfactory. In patients with proteinuria, since intact immunoglobulin in the blood passes through the glomeruli and is excreted in a large amount in the urine, there is a concern about the influence of intact immunoglobulin in the urine sample.

  The present inventors have developed a measurement system using an immobilized anti-FLC monoclonal antibody and a monoclonal antibody specific for the light chain of human immunoglobulin (this antibody reacts with the light chain of intact immunoglobulin and FLC). (Journal of Immunological Methods, 275 (2003), 9-17), which was superior to the conventional measurement system using polyclonal antibodies. It cannot be avoided completely.

Journal of Immunological Methods, 275 (2003), 9-17 Japanese Patent Laid-Open No. 53-44622 Japanese Patent Publication No. 2-39747

  In an immunological measurement method, as one means for improving measurement specificity, a monoclonal antibody is used as an antibody for trapping an antigen to be measured (see JP-A-53-44622), or A method using a monoclonal antibody for both antibodies to be detected (Japanese Patent Publication No. 2-39747) is common knowledge.

  The present inventors also examined these methods. However, the FLC measurement is not always effective, and the development of a measurement system with higher specificity has been desired.

  The present inventors have repeated trial and error to avoid the influence of intact immunoglobulin and to develop a highly specific FLC measurement method. As a result, the present inventors have been able to capture (trap) free human immunoglobulin light chain in a sample. By using a polyclonal antibody specific for FLC as an antibody and a monoclonal antibody that specifically reacts with FLC as an antibody for detecting trapped FLC, the FLC present in the sample is specifically and highly expressed. We found that it can be measured with sensitivity. This was completely unexpected from the common sense for improving specificity in the immunological measurement described above.

  Therefore, the present invention is a method for specifically measuring FLC present in a sample by immunoassay using two types of antibodies that react specifically with FLC, and capturing FLC in the sample. A FLC-specific polyclonal antibody is used as an antibody for trapping, and a monoclonal antibody that specifically reacts with FLC is used as an antibody for detecting trapped FLC. It relates to an immunoassay.

  In addition, a kit for specifically measuring FLC present in a sample by using an immunoassay using two types of antibodies that react specifically with FLC, which captures (traps) FLC in the sample. Immunology of FLC, characterized in that a polyclonal antibody specific for FLC is used as the antibody to detect) and a monoclonal antibody specifically reactive with FLC is used as the antibody for detecting trapped FLC It is related with the kit for static measurement.

A special antibody that uses a polyclonal antibody specific for FLC as an antibody for capturing (trapping) FLC in a sample, and a monoclonal antibody that specifically reacts with FLC as an antibody for detecting trapped FLC By combining these antibodies, it was possible to avoid the influence of intact immunoglobulin in the serum sample and to measure FLC with high sensitivity.
In patients with proteinuria, the influence of intact immunoglobulin is also a concern in urine samples, but in the present invention, it is possible to avoid the influence of intact immunoglobulin, so that monoclonal FLC in urine can be avoided. It is also applicable to the measurement of Bence Jones protein (BJP).

  The sample used in the present invention is not particularly limited as long as it contains FLC, and specifically, blood itself or blood fraction such as serum, plasma, or urine can be used as a sample.

The antibodies used in the present invention are two types of antibodies, a monoclonal antibody that reacts specifically with FLC and a polyclonal antibody that reacts specifically with FLC. Such an antibody may be the antibody itself or an active fragment thereof [F (ab ′) ₂ , Fab ′, etc.].

  Here, an antibody that specifically reacts with FLC is a light chain that is extremely weak or not recognizable at all for a light chain bound to a heavy chain present in intact immunoglobulin and is not bound to a heavy chain. Means an antibody that reacts strongly.

  Such monoclonal antibodies and polyclonal antibodies that specifically react with FLC can be prepared by a conventional method. For example, a monoclonal antibody is prepared using FLC as an antigen, and an antibody that specifically binds to FLC without binding to intact immunoglobulin may be selected from the obtained antibodies.

  In addition, a polyclonal antibody that specifically reacts with FLC can be prepared by a conventional method. For example, a polyclonal antibody may be prepared using FLC as an antigen, and antibodies that bind to intact immunoglobulin may be absorbed and removed from the obtained antibody components. Furthermore, such antibodies are already commercially available and are available. However, it is necessary to confirm the specificity for FLC.

  The present invention uses a polyclonal antibody specific to FLC as an antibody for capturing (trapping) FLC in a sample when attempting to quantify FLC in the sample by an immunoassay using the antibody as described above. A monoclonal antibody that specifically reacts with FLC is used as an antibody for detecting trapped FLC. The immunoassay method itself is not changed in any way, such as a conventional method and procedure.

As an antibody for capturing (trapping) FLC in a sample, a solid phase antibody bound to a carrier can be exemplified.
Carriers used in preparing the solid phase antibody include those conventionally used, such as polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol. , Synthetic organic polymer compounds such as polyacrylamide, polyacrylonitrile, polypropylene, polymethylene methacrylate, dextran derivatives (such as Sephadex), polysaccharides such as agarose gel (such as Sepharose, biogel), cellulose (such as paper disc, filter paper), Inorganic polymer compounds such as glass, silica gel and silicone can be exemplified, and these may be those into which a functional group such as amino group, carboxyl group, carbonyl group, hydroxyl group or sulfhydryl group has been introduced.

The shape of the carrier is flat (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, membrane, fine particles (latex particles, etc.), capsule, vesicle Any suitable shape may be selected depending on the measurement method.
The carrier and the antibody can be bound by a known method such as a physical adsorption method, an ionic bond method, a covalent bond method, a comprehensive method [for example, “Immobilized enzyme” (edited by Ichiro Chibata, March 20, 1975, Inc.) (See Kodansha issue)), and the physical adsorption method is particularly preferable because it is simple. Moreover, the said coupling | bonding may be performed directly and you may carry out through another substance between both substances.
The solid phase reagent thus obtained may be subjected to a blocking treatment using a normal blocking agent such as gelatin or BSA in order to suppress non-specific binding.

Moreover, as an antibody for detecting the trapped FLC, an antibody labeled with a labeling agent can be exemplified.
Examples of the labeling agent include radioisotopes (eg, ³² P, ³ H, ¹⁴ C, ¹²⁵ I, etc.), enzymes (eg, β-galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, catalase, glucose oxidase, lactic acid Oxidase, alcohol oxidase, monoamine oxidase, etc.), coenzyme / prosthetic group (eg, FAD, FMN, ATP, biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein ovalbamyl, etc.), rhodamine derivatives (eg, For example, tetramethylrhodamine B isothiocyanate, etc.), fluorescent dyes such as umbelliferone and 1-anilino-8-naphthalenesulfonic acid, luminol derivatives [for example, Minoru, isoluminol, N-(6- aminohexyl) -N- ethyl isoluminol, etc.] and the like can be exemplified.
The binding between the antibody and the labeling agent is described in a book [for example, “Second Life Chemistry Laboratory Lecture 5 Immunobiochemical Research Method”, Tokyo Chemical Dojin Co., Ltd., (1986) pages 102-112]. What is necessary is just to select suitably from such a well-known method and to implement.

  As a measurement procedure using such a solid phase antibody and a labeled antibody, a normal immunoassay procedure may be employed as it is. That is, the solid phase antibody and the test sample are reacted, and after separation of BF if necessary, the labeled antibody is further reacted (two step method), or the solid phase antibody, the test sample and the labeled antibody are reacted simultaneously (one step method). ), FLC in the sample can be detected or quantified by a method known per se thereafter.

For details of the immunoassay, the following documents may be referred to, for example.
(1) Hiroshi Irie “Continuing Radioimmunoassay” (Kodansha Co., Ltd., issued on May 1, 1979)
(2) Edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition)
(3) Clinical pathology Special issue No. 53 “Immunoassay for clinical examinations-technology and application” (Clinical Pathology Publications, published in 1983)
(4) “Biotechnology Encyclopedia” (CMC Co., Ltd., issued on October 9, 1986)

(5) "Methods in ENZYMOLOGY Vol.70" (Immunochemical techniques (Part A))
(6) "Methods in ENZYMOLOGY Vol.73" (Immunochemical techniques (Part B))
(7) “Methods in ENZYMOLOGY Vol.74” (Immunochemical techniques (Part C))
(8) “Methods in ENZYMOLOGY Vol.84” (Immunochemical techniques (Part D:
Selected Immunoassay))
(9) “Methods in ENZYMOLOGY Vol.92” (Immunochemical techniques (Part E:
Monoclonal Antibodies and General Immunoassay Methods))
[(5) to (9) are issued by Academic Press]

The kit of the present invention is for carrying out the above-described measurement method, and includes, for example, the following reagents.
1) Solid-phased FLC-specific polyclonal antibody
2) Labeled FLC-specific monoclonal antibody Further, instead of the labeled FLC-specific monoclonal antibody, an unlabeled FLC-specific monoclonal antibody and a secondary antibody may be used, and in this case, the following reagents are used.
1) Solid-phased FLC-specific polyclonal antibody
2) FLC-specific monoclonal antibody
3) Labeled anti-immunoglobulin antibody In addition to the above-mentioned reagents, if necessary, an appropriate reagent according to the measurement method is appropriately selected from each reagent such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent. What is necessary is just to select and to attach to the kit of this invention.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

<Method>
(1) FLC-specific monoclonal antibody FLC-specific monoclonal antibody was prepared by immunizing BALB / c mice with FLC (Bethyl laboratories, Montgomery, TX) (J. Immunol. Methods, 275, 9-17. (2003)). . The F (ab ′) ₂ fragment of the obtained antibody was labeled with horseradish peroxidase (HRP) (J. Histochem. Cytochem., 22, 1084-91. (1974)).
In addition, an anti-total light chain monoclonal antibody (anti-total light chain antibody) that reacts with both free and bound immunoglobulin light chains was similarly prepared and used in this experiment (J. Immunol. Methods, 275, 9 -17. (2003)).

(2) FLC-specific polyclonal antibody FLCκ and FLCλ-specific polyclonal antibody were purchased from Dakopatts (Stockholm, Sweden).
The FLCκ-specific polyclonal antibody was used after removing the antibody that cross-reacted with IgG using a CNBr-Sepharose column to which IgG was bound. Antibodies that react specifically were recovered and used. For these antibodies, F (ab ′) ₂ was prepared and labeled with HRP.

(3) Immunoassay FLCκ measurement method FLCκ-specific antibody 10 μg / ml solution was dispensed into a 96-well microplate (Nunc, Immunoplate I) every 100 μl and reacted at 4 ° C. overnight. The antibody solution was removed by suction, washed with PBS, and blocked (4 ° C., overnight) with 300 μl of PBS containing 0.5% skim milk. After the reaction, the blocking solution was removed by suction, and the plate was dried.
A standard concentration solution, appropriately diluted IgG, and a specimen sample were added every 100 μl to each well of the 96-well microplate, and reacted at room temperature. After washing with 20 mM HEPES solution containing 0.05% Tween 20, 100 μl of HRP-labeled FLC specific antibody or anti-total light chain monoclonal antibody was added and allowed to react at room temperature.
After washing away unreacted HRP-labeled antibody, the HRP activity remaining in the well is detected with tetramethylbenzidine (TMB) as its substrate, and the absorbance of the resulting dye is measured with a microplate reader (TECAN Spectra) did. As assay buffer, 50 mM Tris-HCl, pH 7.5 containing 0.1% Tween 20 and 1% bovine serum albumin was used.

FLCλ measurement method The procedure was the same as the FLCκ measurement method. However, phosphate buffered saline (PBS) containing 0.05% Tween 20 was used as a washing solution, and PBS containing 1% bovine serum albumin was used as an assay buffer.

(4) Serum specimen The serum specimen used in this experiment was obtained from 43 healthy volunteers.

<Result>
(1) Comparison of specificity for FLC in immunoassay using various antibodies In immunoassay for FLC quantification, the following four types of antibodies were examined.
(Measurement method 1)
Solid phase FLC specific polyclonal antibody and HRP labeled FLC specific monoclonal antibody (Measurement method 2)
Solid phase FLC-specific polyclonal antibody and HRP-labeled anti-total light chain monoclonal antibody (Measurement method 3)
Solid phase FLC-specific monoclonal antibody and HRP-labeled FLC-specific polyclonal antibody (Measurement method 4)
Solid phase FLC-specific monoclonal antibody and HRP-labeled FLC-specific monoclonal antibody

FIG. 1 shows the results of measurement methods 1 to 3. In measurement method 2 or 3, the specificity of FLC for IgG is about 100 to 1000 times, whereas measurement method 1 has a high specificity of 100,000 times for FLCκ and 10,000 times for FLCλ. Indicated.
For measurement method 4, 9 types of anti-FLCκ monoclonal antibodies and 8 types of anti-FLCλ monoclonal antibodies obtained were used, and the usefulness of FLCκ or FLCλ measurement was evaluated for all possible combinations. I couldn't find a combination.
Based on these results, the influence of serum intact immunoglobulin was continuously evaluated for Measurement Method 1 and Measurement Method 2 that showed high specificity for FLC. Measurement method 3 was not used in subsequent experiments because of its low specificity and measurement method 4 because there was no detectable combination.

(2) Influence of serum intact immunoglobulin Using measurement methods 1 and 2, the FLC concentration in 43 serum samples was measured (FIG. 2). In addition, in order to confirm the influence of intact immunoglobulin on these measured values, the same measurement was performed on a sample from which serum intact immunoglobulin was absorbed and removed using a subclass-specific antibody column. The concentrations of IgG, IgA and IgM before absorption are 12.0 mg / dl, 2.1 mg / dl and 1.3 mg / dl on average, and 0.02 mg / dl, 0.005 mg / dl, 0. It was 004 mg / dl.

The measured value of the specimen after absorption was corrected by the recovery rate obtained using bovine serum to which FLC was added. As a result of the measurement, the value in measurement method 1 was almost the same before and after absorption, but in measurement method 2, the value was very high in the serum before the absorption of intact immunoglobulin, and the intact immunoglobulin was cross-reacted. I found out.
From these results, it was shown that the combination of the solid-phased FLC-specific polyclonal antibody and the FLC-specific labeled monoclonal antibody is most useful for specifically detecting FLC. In addition, no obvious effect of serum intact immunoglobulin was observed in this combination measurement.

FIG. 1A shows the measurement results of FLC and IgG by measurement method 1 (immunoassay using solid phase FLC-specific polyclonal antibody and HRP-labeled FLC-specific monoclonal antibody). FIG. 1B shows the measurement results of FLC and IgG by measurement method 2 (immunoassay using solid phase FLC-specific polyclonal antibody and HRP-labeled anti-total light chain monoclonal antibody). FIG. 1C shows the measurement results of FLC and IgG by measurement method 3 (immunoassay using solid phase FLC-specific monoclonal antibody and HRP-labeled FLC-specific polyclonal antibody). The left figure shows the measurement result of FLCκ, and the right figure shows the measurement result of FLCλ. FIG. 2 shows the measurement results after absorption of 43 serum samples or their intact immunoglobulins in Measurement Method 1 and Measurement Method 2. A and B show the measurement results of FLCκ and FLCλ, respectively.

Claims (8)

A method for specifically measuring a free immunoglobulin light chain present in a sample by immunoassay, using two types of antibodies that specifically react with the free human immunoglobulin light chain, A polyclonal antibody that is specific for a free human immunoglobulin light chain immobilized as an antibody to capture (trap) a free human immunoglobulin light chain and absorbs and removes an antibody that binds to an intact immunoglobulin. A free human, characterized in that it uses a monoclonal antibody that specifically reacts with a free human immunoglobulin light chain labeled with a labeling agent as an antibody for detecting the trapped free human immunoglobulin light chain Immunoassay for immunoglobulin light chain. The method according to claim 1, wherein a polyclonal antibody specific for a free human immunoglobulin light chain and a monoclonal antibody specifically reactive with a free human immunoglobulin light chain are used in the form of an active fragment. The method according to claim 1, wherein the labeling agent is an enzyme. The measuring method of Claim 1 whose free human immunoglobulin light chain is Bence Jones protein (Bence Jones protein). A kit for specifically measuring a free immunoglobulin light chain present in a sample by immunoassay using two types of antibodies that react specifically with the free human immunoglobulin light chain, Polyclonal that absorbs and removes the antibody that binds to intact immunoglobulin and is specific for the immobilized free human immunoglobulin light chain and is immobilized as an antibody to capture (trap) the free human immunoglobulin light chain An antibody is used, and a monoclonal antibody that specifically reacts with a free human immunoglobulin light chain labeled with a labeling agent is used as an antibody for detecting the trapped free human immunoglobulin light chain. A kit for immunological measurement of free human immunoglobulin light chain. The kit according to claim 5, wherein a polyclonal antibody specific for the free human immunoglobulin light chain and a monoclonal antibody specifically reactive with the free human immunoglobulin light chain are used in the form of an active fragment. The kit according to claim 5, wherein the labeling agent is an enzyme. The kit according to claim 5, wherein the free human immunoglobulin light chain is Bence Jones protein.

Images