EP0298094A1 - Method of identifying salmonella - Google Patents

Abstract

Direct test for the identification of bacterial organisms using a specific bacteriophage which combines with the target organisms. The marking of the bacteriophage makes it possible to really identify the species or the genus of the bacterium. The markers can be radioactive, fluorescent or enzymatic. Biphenol can be used as a substrate for markers using peroxidase.

Description

METHOD OF IDENTIFYING UNKNOWN ORGANISMS

Background and Summary of the Invention:

This application is a continuation-in-art of U.S. patent application Serial No. 936,515, filed December 1, 1986.

Various tests procedures are known for detecting the presence of infectious organisms. Indirect methods typically test for the presence of antibodies produced by a host in response to the presence of infectious organisms. Direct testing methods may also be used, for example, specimens may be cultured to grow an organism and the organism can then be identified. Other test procedures have also been used in the art. Applicants are aware of the following U.S. patents. u .S. Patent No. 3,654,090 U.S. Patent No. 4,256,834 ϋ. .s. Patent No. 3,791,932 U.S. Patent No. 4,281,061 u .s. Patent No. 3,826,613 U.S. Patent No. 4,287,300 u, .s. Patent No. 3,867,517 U.S. Patent No. 4,308,348 ϋ, .s. Patent No. 4,016,043 U.S. Patent No. 4,347,311 u, .s. Patent No. 4,098,876 U.S. Patent No. 4,374,925 u, .s. Patent No. 4,104,126 U.S. Patent No. 4,376,110 u, .s. Patent No. 4,244,940. U.S. Patent No. 4,469,786

The disclosures of the above patents are incorporated by reference herein.

In the main, both indirect and direct testing procedures have been cumbersome and time consuming. Direct testing methods are often slow. For example, it may take several days, for an organism to be cultured successively, before a certain identification can be made. Other test procedures have other short comings, in particular, many test procedures have a high incidence of false positives and false negatives.

Applicants have discovered and perfected a new test method for directly testing for the presence of unknown organisms. Applicants method is fast acting and produces a test result which has a high degree of certainty. Applicants test procedure provides qualitative and quantitative evaluation of unknown organisms. The test may be used to identify a particular species, type or genus. Applicants method uses a specific agent which rapidly attaches to a target test organism. The agent is labeled, for positive identification. Applicant's method may use a variety of conventional labeling or reporting techniques. For example, the agent may be marked or tagged for identification using radioactive or chemical marking, for example, peroxidase labeling, fluorescent labeling, biotin labeling or other identification for the agent. The marked or tagged agent is then brought into contact with a test unknown organism. If the agent attaches to the organism its presence can be determined by detecting the tag or marker to provide a positive identification for that particular organism. Specifically, applicants' agent is a bacteriophage. It is generally accepted that bacteriophages are narrowly restricted in their range of host bacteria. Some bacteriophages are specific for a particular species of bacteria. However, there are also some bacteriophages which are specific to particular genuses.

Applicants have discovered that bacteriophages may be labeled or tagged, as described herein, and can then be used to identify a bacterium, based on the binding or non-binding to that particular bacteria species, type, or genus. Since a bacteriophage is smaller than the organism to which it binds, free bacteriophage can be separated from bacteria by filtration through an appropriately sized medium, for example, inert filter paper. The filter traps the bacteria and permits the unbound smaller bacteriophage to pass through the filter. However, if the bacteria is the one to which the bacteriophage is specific, the bacteriophage will bind to the bacteria within a very short time. If the test specimen is filtered after this time has elapsed, the bacteria will be trapped on the filter with the bacteriophage attached. If the bacteriophage is labeled its presence can be determined by developing the tag or marker and the presence of that specific bacteria to which the particular bacteriophage attaches can then be determined. Of course, the greater the number of the specific bacterial organisms present the greater the number of bacteriophage which will be found attached to the specimen on the filter, yielding a greater signal from the reporter groups.

Consequently, it is an object of applicants' invention to provide a specific test method for determining the presence of unknown organisms.

It is a further object of applicants' invention to provide a test method by which a labeled agent rapidly attaches to a specific microorganism to identify that organism.

It is a further object of applicants' invention to provide a test method for unknown microorganisms which acts rapidly and with a high degree of reliability.

It is a further object of applicants invention to provide a test reagent for determining the presence of organisms by labeling an organism specific agent.

It is a further object of applicants invention to produce an organism specific test reagent.

It is a further object of applicants' invention to provide a specific test for bacteria using labeled bacteriophage.

Applicants' invention may be further understood by reference to the following Description of the Drawings and Description of the Preferred Embodiments. DESCRIPTION OF THE DRAWINGS:

FIG. 1 is a curve showing the rate of biphenol consumption as a function of hydrogen peroxide concentration;

FIG. 2 is a curve showing the rate of biphenol consumption as a function of biphenol concentration;

FIG. 3 illustrates the limit of sensitivity of liquid assays for peroxidase using biphenol/H₂0₂, and

FIG. 4 illustrates the stability of biphenol in the presence of H-0₂ compared to other systems. DESCRIPTION OF THE PREFERRED EMBODIMENTS:

Bacteriophage are easy to acquire. Suitable wild bacteriophage may be harvesteα from nature, isolated very readily by conventional means and grown to substantial quantity on the specific host bacterium. Applicants have harvested and isolated bacteriophage specific for Salmonella, E. coli, Staphylococcus aureus and Pseudomonas, among others. In addition, common bacteriophage, for example, Tl, T2, T3, T4 and lambda, which are specific for E. coli, are readily available, as are other known bacteriophage. ATCC 15693-B1 is specific for Pseudomonas aerugenosa, ATCC 6538P is specific for Staphylococcus aureus, ATCC 6051-B1 is specific for Bacillus subtilus and ATCC 23074-B1 is specific for Listeria monocy ogenes. Naturally available and collected bacteriophage are specific for Ca pylobacte , Mycobacterium tuberculosis and others.

The ready availability and rapid growth of cultures of bacteriophage is of particular advantage in using bacteriophage as a test reagent. In comparison, monoclonal and polyclonal antibodies are difficult to obtain in high yield and high degree of purity.

Bacteriophages rapidly bond to the outside of host bacteria and, though infecting .the bacteria, leave the shell of the bacteriophage present on the surface of the host. The properties of bacteriophages in binding to a host are noted in Reco binant DNA, A Short Course, Watson et al. pages 14, 15, 23 and 24, W.H. Freeman & Company, New York 1983 and have been noted in "The Mechanism of Virus Attachment to Host Cells. IV. Physiochemical Studies on Virus and Cell Surface Groups," Arch. Biochem. Biophys. Vol. 51 (1954), Puck et al. , pages 229 through 245.

Bacteriophage are particularly advantageous as a test reagent in that relative to its host bacterium, and to other carriers such as antibodies or gene probes, the bacteriophage is relatively large, enabling the tester to bind sizeable quantities of tags or marker elements, such as, enzyme markers, fluorescent markers, or radioactive markers, to the individual bacteriophage. The protein shell of the bacteriophage remains outside and attached to the bacterium after infecting the host bacterium so that the presence of the bacteriophage can be readily identified. Though being relatively large, the bacteriophage is also sufficiently smaller than the host bacteria to permit the unattached bacteriophage to be readily separated from the bacteria in a test specimen by filtration.

Labeling bacteriophages to provide a marker that identifies the presence of the bacteriophage or the bacteriophage bound to a host can be readily accomplished. The shell of bacteriophage is proteinaceous and a variety of markers can be bound to the bacteriophage surface by using a protein linker. For example, the enzyme horseradish peroxidase (HRP) may be linked to a the protein on the shell of a bacteriophage with a bifunctional cross linking reagent, dimethyl suberimidate 2HC1, an imidoester. The general reaction scheme is as follows:

H -f- KIN-Λ/tf^■.-_. —- c-

^~~ \ \ Hrx _ IZ.=- ^^r_rw) c

generating a' bacteriophage-linker-peroxidase complex. Other enzymes may also be used, for example, alkaline phosphatase and beta-galactosidase may also be used as well as the use of other bifunctional cross linkers such as other amino reacting cross linkers and thiol reacting cross linkers. Fluorescent labels may be attached by using a fluorescent label-fluorescene isothiocyanate through the epsilon-amino groups of lysine found in the bacteriophage capsomeres. Other fluorescent molecules such as rhodamine may also be used in a similar manner. Labeling the bacteriophages with biotin type reporter groups using a succini ide ester group may also be used. Other conventional secondary labeling substances may also be incorporated. Radioactive labeling of the bacteriophages with various radioactive compounds could also be used, for example, radioactive iodine 125 may be linked to the protein of the bacteriophage shell by a conventional reaction.

A modified Nakane method of binding horseradish peroxidase may be used to label bacterophage. The Nakane method is described in Standard Biochemical Methods. The procedure is as follows: Horseradish peroxidase is reacted with 0.032M formaldehyde in the presence of a 0.30M NaHCO- buffer, pH 8.1 to form HRP=CH₂. The HRP=CH₂ is reduced with 1 g NaBH./mg enzyme to form HRP-CH-. (methylated horseradish peroxidase). The HRP-CH-. is buffered with 0.30M NaHCO- and separated by chromotography on a 0.5m exclusion column. The HRP-CH₃ is then oxidized with 0.04M NalO, to form HRP-CHO (horseradish peroxidase aldehyde). The HRP-CHO is buffered to pH 9.0 with a a₂C0₃ buffer and separated on a 0.5m exclusion column to produce the activated HRP enzyme. The bacteriophage is grown on a host bacterium culture, for example S. typhimurlum. The lysed bacteriophage is filtered through a 0.45 micron filter and is separated from the culture material. The activated enzyme is added to the bacteriophage and reacted for 4 to 6 hours at room temperature. The enzyme bonds to the protein coating of the bacteriophage, probably by attaching to lysine (R-NH₂) present in the protein coating. The tagged bacteriophage is buffered to pH 7.0 with a 50mM PO. buffer and separated on a Biogel A 0.5m exclusion column. The separated tagged bacteriophage is then assayed and adjusted for titer. EXAMPLE I, Salmonella Species Type Test: Salmonella specific bacteriophage was harvested from nature and isolated on a host culture of Salmonella typhimurium. (A culture of this bacteriophage, designated ATCC Number 40282, has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852 and is available to the public from the permanent collection.) After isolation and purification on a Salmonella typhimurium host culture, the bacteriophage was labeled with horseradish peroxidase using the Nakane method described. The labeled bacteriophage was then tested against representative Salmonella species and the serologically closely related species of, Citrobacter, as shown in Table I. The Salmonella and Citrobacter organisms were graciously provided by Alma Murlin fr.om her collection at the National Center for Disease Control, Atlanta, Georgia and by the USDA Veterinary Services Center, Ames, Iowa. The Citrobacter, Salmonella type, was provided by the Center for Disease Control, Atlanta, Georgia.

Table I

Organism Colori etric Test

Salmonella, Group 1 worthington Positive anatu Positive cholerae var. suis Positive newington Positive paratyphi B Positive montevideo Positive typhi Positive heidelberg Positive typhimurium Positive

Portsmouth Positive

Johannesburg Positive laardt Positive poona Positive berta Positive meleagridis Positive infantis Positive enteritidis Positive pomona Positive auiana Positive paratyphi A Positive newport Positive agoha Positive cereo Positive brithday Positive luciana Positive london Positive westerstead Positive tennessee Positive πewbrunswick Positive gaminara) Positive florida Positive alachua Positive krefold Positive paratyphi A Positive dublin Positive seftenberg Positive paratyphi C Positive drypool Positive

Inverness Positive barrilly Positive minnesota Positive newington Positive paratyphi A, japan Positive cereo Positive

Salmonella, Group 2 phoenix Positive neb-M23037 Positive

Salmonella, Group 3

3a Positive

3b Positive

Salmonella, Group 4 flint Positive marina Positive Salmonella, Group 5 brookfield Positive bongar Positive

Citrobacter

Citrobacter spp., Salmonella type Negative freundii Negative amalonaticus Negative

The test procedure for each organism listed in the Table I was as follows: A culture of the organism was slurried to a concentration of 10 organisms per ml. A 100 ul sample of the organism slurry was combined with 50 ul of the labeled bacteriophage reagent. The reagent was standardized to a titer

Q of 10 p.f.u. The reagent and Salmonella sample (and Citrobacter) were incubated for 20 minutes. After incubation the mixture was filtered through a 0.45 micron filter with vacuum. The filtered mixture was washed twice using a phosphate buffered saline solution containing 0.05% TWEEN 20(TM). The last wash was vacuumed to dryness. A 100 ul saturated solution of para-para-biphenol, buffered to pH 7.0 by a 50m phosphate buffer, (substrate) was added to the filter and incubated for 15 minutes. A characteristic brown color indicated a positive test in all cases (presence of labeled bacteriophage on the filtered bacteria) . All reactions were at room temperature. Example II, Reliability:

A test for reliability of the labeled bacteriophage was conducted on the array of bacteria shown in Table II and Table III. The bacteriophage used were obtained from the American Type Culture Collection, with the exception of applicants' Salumonella bacteriophage. The bacteriophage were specific for the particular organism, as noted herein. Each bacteriophage used was labeled with horseradish peroxidase using the modified Nakane method described. The tests were conducted as described in Example I and developed using para-para-biphenol. Table II

Labeled Test Number of Number of Number of Percen Bacteriophage Organism True False Tests False Positive Negative Negativ

ATCC 40282 Citrobacter, 0 16 0.00 Salmonella type

T4 E. coli 162 15 177 8.47

ATCC 15692-Bl Pseudomonas 273 9 282 3.19 aeruginosa

ATCC 40282 Salmonella 792 795 0.38 typhimurium

ATCC 6538P Staph 181 17 198 8.59 aureus Total 44 1468 3.00

Table III

Labeled Test Number of Number of Number of_ Percent

Bacteriophage Organism True False Tests ^* False

Negative Positive Positiv

ATCC 40282 Citrobacter, 15 1 16 6.25

Salmonella type present

T4 E. coli 2730 0 2730 0.00 not present

Pseudomonas 2616 9 2625 0.34 aeruginosa not present

ATCC 40282 Salmonella 2313 2316 0.13 typhimurium not present

ATCC 6538P Staph 2709 2709 0.00 aureus not present

Total 13 10396 0.13 Applicants have found that the tests described above may give variable results from batch to batch of. the horseradish peroxidase used, requiring standardization of each batch of coupled HRP and bacterophage against the host bacterium. Applicants have since found that the HRP from commercial sources, for example, Sigma Chemical, type I, contains variable amounts of HRP monomer and polymers. Applicants have found that the polymer content determines the strength of the signal produced by the coupled HRP-bacteriophage system. HRP containing mostly monomeric peroxidase produces a weak signal when coupled with bacteriophage and an increasing level of polymerization produces increasing signal strength when coupled with bacteriophage. Applicants have further found that they can produce and isolate a horseradish peroxidase having a high level of polymerization and which is readily coupled with bacteriophage. Applicants' coupled enzyme and bacteriophage may have an effective ratio of enzyme to bacteriophage of above about 200 enzyme units per bacteriophage and preferably above about 1000 enzyme units per bacteriophage. Using applicants' coupled enzyme-bacteriophage, particularly with a para-para-biphenol substrate, the assay described herein has a sensitivity of detecting fewer than 10,000 bacteria per assay test well, including as few as 100 bacteria per assay well.

Applicants have also found that periodate oxidation is particularly useful for coupling peroxidase, especially when used with methods, such as column chromotography, for obtaining polymeric enzyme activity from the HRP. The basic periodate oxidation is described in "Practice and Theory of Enzyme Immunoassays," by P. Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology, v. 15, R.H. Burdon and P.H. van Krippenberg (eds) Elsevier, N.Y. 1985. A suitable active polymeric fraction of HRP may be obtained by column chromatography over BioGel A 0.5m. The preparations that yield the best activity are those fractions between the void and the leading edge of the major enzyme containing peak. Other methods of increasing the enzyme count per bacteriophage may be used. These methods include but are not limited to the addition of compounds such as bifunctional cross-linking agents, divalent or multivalent amlne contaning compounds, divalent or multivalent aldehyde containing compounds, lecithins, and anti-enzyme antibodies. Those skilled in the art may also appreciate that the sensitivity of the enzyme tagged bacteriophage assay depends on the number of active enzymes per phage, and that the requisite ratio of enzymes per bacteriophage may be generated by methods not requiring enzyme polymers. These methods include but are not limited to the successive or in situ chemical layering of monomeric enzymes onto the bacteriophage surface, or by prior coupling of a polymeric or particulate matrix that can accommodate the requisite number of enzymes per bacteriophage.

Various methods may be used to separate bacteria bound reagent from free reagent. One such method practiced by the applicants is centrifugation and washing of the pellet in buffer. One or two washes are sufficient to reduce nonspecific background to an acceptable level. The preferred . method for separating free enzyme-tagged bacteriophage from bacteria bound enzyme-tagged bacteriophage is by filtration on a filter matrix that retains bacteria but passes enzyme-tagged bacteriophage, followed by washing with a detergent containing buffer. Low protein binding filters used for bacterial sterilization of fluids having pores of 0.45um give satisfactory results. The filter-trapped bacteria/enzyme-tagged bacteriphage complex is then contacted with a solution of chromogenic substrate. The substrate is capable of undergoing enzymatic transformation upon contacting the enzyme-tagged bacteriophage to a chromophoric product. Although soluble chromophoric products may serve as positive indicators of target bacteria, the applicants have found that a chromophoric product that binds to or otherwise is trapped by the filter matrix upon filtration of the reaction mixture gives increased sensitivity. For bacteriophage tagged with HRP, the preferred chromogenic substrate is para-para-biphenol, as described herein. A filter based assay of this type has a sensitivity of detecting less than 10,000 bacteria per assay well, and with reagents having greater than 2,000 enzymes per bacteriophage, less than 100 bacteria per assay well. Example III, Generation of Periodate Oxidized HRP

React horseradish peroxidase (Sigma, Type I) with 0.032M formaldehyde in 0.3M NaHC0₃ buffer, pH 8.1 for 30 minutes. Reduce the Schiff's base residues with either lmg NaBH,/mg protein or 0.15 mg NaCNBH,/mg protein. Incubate at 4°C for 16 to 24 hours. Chromatograph the reaction mixture on a BioGel A 0.5m size exclusion column that had been equilibrated in 0.3 M bicarbonate buffer pH 8.1. Pool all fractions excluding aggregates in the void volume and low molecular weight reactants in the totally included peak. Oxidize the pooled fractions with 0.04M NalO, for 30 minutes at room temperature. Stop the reaction by making the reaction mixture 0.1M in propylene glycol. Chromatograph ' the oxidized reaction mixture over a fresh BioGel A 0.5m column of the same dimensions that had previously been equilibrated in 0.5M carbonate buffer pH 9.0. Pool all fractions after the voided peak, if present, up to the leading edge of the main 206nm absorbing peak to 1/3 its peak height. Pooling the voided peak, if present, results in nonspecific signal, while pooling excess monomeric HRP resulsts in weak signal in the bacteria detection test. Concentrate this pool approximately 7 to 10 fold using an Amicon stirred cell with a PM 10 membrane. Filter sterilize through a 0.45 micron membrane and store at 4 C in a sterile glass tube. The typical pooled concentrate has an absorbance at 280nm of between 0.5 to 0.8, a 403 nm/280 nm absorbance ratio of between 0.3 to 0.8, and contains between 0.44 to 2.6 uM HRP by the pyrogallol assay. Example IV, Coupling Activated HRP to Salmonella Specific Bacteriophage

A Salmonella specific bacteriophage was harvested from raw sewage and isolated on a host culture of Salmonella typhimurium. (A culture of this bacteriophage, designated ATCC

Number 40282, has been deposited with the American Type Culture

Collection at 12301 Par lawn Drive, Rockville, Maryland 20852)

Propagate the bacteriophage on its wild type S. Typhimurium host. After the bacteriophage has lysed its host bacterium culture, separate the bacteriophage from bacteria and debris by low speed centrif gation, followed by filtration through a 0.45 micron filter. Separate bacteriophage from low molecular weight contaminants by chromatography over a BioGel A 0.5m column, the bacteriophage being collected in the void volume peak. Adjust the pH of the reaction mixture to between 9.0 to

9.5 with 0.2M sodium carbonate. The final concentration of phage in the reaction is between 10 8 to 109 pfu/ml, and the final concentration of enzyme is 0.11 to 0.6 uM. Incubate the reactants for 4. to 6 hours at room temperature (or 16 to 24 hours at 4°C) . The enzyme readily binds to the protein coating of the bacteriophage, probably by attaching to lysine residues present in the protein coating. Purify the enzyme conjugated bacteriophage equilibrated on a BioGel A 0.5m column with 50mM phosphate buffer pH 7.0. Collect the tagged bacteriophage in the void volume peak. After filtering through a 0.45 micron filter, assay the enzymatic activity and lytic titer of the tagged bacteriophage.

Determine the peroxidase enzymatic activity as described in the Sigma package insert for horseradish peroxidase. The number of HRP molecules in a given sample of tagged bacteriophage is calculated by multiplying the initial rate

(absorbance change at 399 nm/min) by 3.57 X 10¹² molecules

HRP. The number of HRP enzymes/bacteriophage is 2193 as calculated by dividing the number of HRP molecules/ml in the tagged bacteriophage preparation by its lytic titer on the propagation host. Example V, Salmonella Species Type Test

Test the enzyme tagged bacteriophage of Example 2 against representative Salmonella species and serologically closely related species of Citrobacter as shown in Table II. The test procedure for each organism listed in Table II was as follows: Slurry a culture of test organism to a concentration of 10 organisms per ml. Add- a- 100 ul sample of each organism slurry to a filter well of a BioRad microfiltration apparatus having a 0.45 micron HT Tuffryn filter. Remove the solution by vacuum filtration in the apparatus. Add a 50ul aliquot of

Q bacteriophage reagent (10 pfu/ml) to each well. After a 20 minute incubation at room temperature, separate free reagent from bound by vacuum filtration followed by two cycles of washing and evacuation using phosphate buffered saline containing 0.05% TWEEN

Table II

Organism Colorimetric Test

Salmonella, Group 1 worthington Positive anatum Positive cholerae var . suis Positive newington Positive paratyphi B Positive montevideo Positive typhi Positive heidelberg Positive typhimurium Positive

Portsmouth Positive

Johannesburg Positive laardt Positive poona Positive berta Positive meleagridis Positive infantis Positive enteritidis Positive pomona Positive jauiana Positive paratyphi A Positive newport Positive agoha Positive cereo Positive brithday Positive luciana Positive london Positive westerstead Positive tennessee Positive newbrunswick Positive ga inara) Positive florida Positive alachua Positive krefold Positive paratyphi A Positive dublin Positive seftenberg Positive paratyphi C Positive drypool Positive

Inverness Positive barrilly Positive minnesota Positive newington Positive paratyphi A, japan Positive cereo Positive

Salmonella, Group 2 phoenix Positive neb-M23037 Positive

Salmonella, Group 3

3a Positive

3b Positive Salmonella, Group 4 flint Positive marina Positive

Salmonella, Group 5 brookfield Positive bongar Positive

Citrobacter

Citrobacter spp.. Salmonella type Negative freundii Negative amalonaticus Negative

20 (TM) . Add 100 ul of a sterile filtered saturated solution of para-para-biphenol -0.03% H₂0₂ in 50 mM phosphate buffer pH 7.0 to the retentate of each well and incubated for 15 minutes at room temperature. Vacuum filter all wells to dryness. All wells containing Salmonnella species develop a characteristic brown dot on the filter indicating the presence of HRP labeled bacteriophage on the retained bacteria. The filter at the bottom of wells containing Citrobacter is colorless. Example VI, Sensitivity of the Salmonella Species Type Test

Determine the sensitivity of the Samonella species test by the method of Example V using 2 fold serial dilutions of a

5 Salmonella suspension containing 10 organisms/ml. Assay lOOul aliquots of each dilution in triplicate. Figure 2 shows a plot of % reflectance versus Salmonella dilution. Maximum reduction in % reflectance indicating a uniform deposition of brown products from the enzymatic reaction occurred in all wells containing 250 organisms or more. Statistically significant reduction to 80% reflectance occurred at 10 organisms/ml. At a reading of 80% reflectance, a uniform tan colored deposit was clearly discernable by eye. Thus the applicants conservatively estimate that the sensitivity of the

Salmonella species type test to be 100 organisms/ml.

Applicants have found that a particularly useful substrate for detecting the presence of enzyme coupled peroxidase is one incorporating para-para-biphenol. A substrate of this type is use in Examples I and V above, for example. This substrate has been found to be highly sensitive for colorimetric analysis and is highly stable.

The problem in detecting peroxidase activity is that high sensitivity is linked to high background. In a filter based system, the problem is further complicated by the need for soluble substrate that becomes insoluble after being acted upon by the enzyme. The ideal substrate should be (1) rapid, (2) soluble to the extent that it saturates the enzyme, (3) stable to oxidation by hydrogen peroxide in the absence of enzyme, (4) converted to a colored, highly insoluble product by the action of peroxidase.

4,4'-biphenol fullfills the four criteria enumerated above. (1) It exhibits a rate constant M—1 sec—1) as high or higher₆ than any other substrate known. (2) The overall rate of reaction is independent of biphenol

_5 concentration at ^. 5 x 10 M (a saturated solution of biphenol is 1.6 x 10 -4M) . (3) The addition of hydrogen peroxide to biphenol gives a colorless solution that shows no apparent changes over a period of months at room temperature.

(4) During the course of the enzymatic reaction, the solution becomes yellow (max 400nm) then brown (max 445-450nm) . This brown material is retained by a 0.45uMfliter.

Solid 4, '-biphenol is added (lg/L) to aqueous buffer

(pH6-7) and stirred overnight" to produce a saturated solution.

Solid material is removed by filtration (0.45u filter) and hydrogen peroxide is added (10-lOOul of 30% H₂O₂/100ml biphenol solution) . The solution is ready to use. The substrate is added to the surface of a filter upon whih peroxidase has been deposited. After 5-60 minutes, the solution is filtered. By this means, peroxidase levels as low as 1 fmol (femptomole, 10 -15 moles) can be detected. No color change can be detected in the absence of enzyme. This substrate can be used in filter assays for detecting peroxidase, peroxidase labeled reagents (antibodies, gene probes, bacterial viruses and the like), and coupled enzyme assays where the final enzyme is peroxidase. Example VII, Preparation of Biphenol/H Oo Substrate

A stock solution of Horseradish peroxidase (EC 1.11.1.7),

Sigma type VI, 4 mg/ml, was prepared in 50 mM phosphate buffer, pH7.0 and aliquotes were frozen for future use. For each days

₅ testing, an aliquot was thawed, diluted (H₂0) to 10 fold, and the concentration- of enzyme in the diluted stock determined

(See Example IX). A working solution of hydrogen peroxide (30% stock) was prepared by diluting 0.167 mL of 30% H₂0₂ to 10 mL with H₂0. Determination of the exact concentration of

H₂0₂ in the working solution was an average of determinations made at 240 nm (Hildebrant, A.G. and I. Roots,

Arch. Biochem. Biophys. 171:385 (1975) and at 230nm (George, ^p" Biochem J. 54:267 (1953)).

Saturated solutions of biphenol (4,4 '-Dihydroxybiphenyl) in water or buffer were prepared by adding lg to IL of solution with stirring overnight. The resulting suspension was filtered through a 0.45 u filter and stored at room temperature. The exact concentration of the stock solution was determined by

₄ measuring the absorption at 262.5nm (E=2.16 x 10 as determined experimentally) .

Liquid Assay. A Gilford Response spectrophotometer equiped with a kinetics accessory package was used for all assasys. The change in absorbance at 399 nm was monitored for

2-5 minutes. Initial rates were determined by the spectrophotometer and converted to M sec -1 using 3.47 x 104

M as molar absorptivity of biphenoquinone (Pelizzetti, E.,

E. Mentasti and C. Baiocchi, J. Inorg. Nucl. Chem. 38:557

(1976) .

At room temperature, donor (biphenol), substrate

(H-O-) and enzyme are mixed in the cuvette and the rate measured. The rate dependence of H-0~ and biphenol were determined. The sensitivity of the optimized assay for detecting peroxidase was determined.

Solid Phase Assay. A 96 well dot-blot apparatus (BioRad) with a 0.45um pore size polysulfone membrane (HT-Tuffryn, Gelman) was used to perform reactions and then trap insoluble product. Results were scored visually, based upon the appearance of any brown material apparent after filtration. The effect of pH, ionic strength, hydrogen peroxide concentration, incubation times and test sensitivity were tested.

Substrate and donor were combined in the appropriate buffer. Serial 1:2 dilutions of peroxidase were tested to the "limits of detectability" in each test.

Stability Assay. The stability of biphenol in the presence of hydrogen peroxide was compared to the stability of several other donors under the same conditions. Wavelength scans (375-650 nm) were performed on donor solutions prior to the addition of hydrogen peroxide. After peroxide addition, scans were repeated at 1, 2, 3, and 20 hours. Changes_ in the absorbance at the appropriate wavelengths were determined.

RESULTS: LIQUID ASSAY

I. Effect of substrate (H₂0₂) concentration on rate.

Conditions: Biphenol = 1.484 x 10 —4M

Peroxidase = 5.18 x 10~¹³M

Buffer = 46.9mM phosophate, pH7.0

H-0₂ = 9.69 x 10^"4 to 6.33 x 10~²M

Results: As shown in FIG. 1, optimal concentration is between 0.3 and 15mM hydrogen peroxide. Other experiments indicate a decrease in optimum peroxide concentration, within this range, as a donor concentration descreases. These data are consistent with optimal values determined with other donors (Tyssen, P., D.-M. Su and E. Kurstak, Arch. Virol. 74:277 (1982).

II. Effect of donor (biphenol) concentration on rate.

Conditions: H₂0₂ = 4.61 x 10~³M

Peroxidase = 1.34 x 10~¹²M Buffer = 46.9mM phosophate, ρH7.0 Biphenol = 1.43 x 10~⁶M to 1.43 x 10~⁴M

Results: As shown in FIG. 2, at high concentration of biphenol the rate is independent of biphenol (zero order). At intermediate biphenol concentration the rate is more complex and at low biphenol concentration the rate is apparently pseudo first order. Given the general rate expression.

Rate = -.k₄ [Peroxidase! rH202> k₁[H₂0₂] + k₄[Biphenol]

were k-, and k₄ are the rate constants associated with the substrate and first donor steps respectively, the values of these constants may be determined.

8 —1 These calculations yield k_Λ 2.5 x 10 (Msec) and k, 1.2 x 10 6 (Msec)—1. These results are in good agreement with previsously determined values. (Chance, B.,

Arch; Biochem. 22:224;24:11 (1949) and Chance, B., Advan.

Enzymol. 12:171 (1951).

III. Limits of Sensitivity for liquid assay.

Conditons: Biphenol = 1.48 x 10 -4M

^H2°2 ^{= 4*84 X 10}~^3M

Buffer = 46.9mM phophate, pH 7.0

Results: As shown in FIG. 3, the apparent limit seems to be 30-35 x 10 -14M peroxidase in a 3.2 mL reaction mixture ( 1 x 10^~ moles of peroxidase) . This is as sensitive as any other system (Ngo, T.T. and H.M. Lenhoff. Anal. Biochem.

105:389 (1980).

RESULTS: SOLID PHASE ASSAY

IV. Effect of substrate (^H _? ⁰2 concentration on filter assay sensitivity.

_₅ Condition: Biphenol = 9.5 x 10 M

Buffer = 37.5mM phophate, pH 7.0

H-O- = 4.04 x 10^_5M to 8.26 x 10^_2M Peroxidase = 8.9 x 10 -17 moles/assay to 1.14 x 10 -14 moles/assay

Volume/well = 200 uL

Results: Maximum sensitivity for the assay lies between 0.7 and 5.0 mM hydrogen peroxide. Less than 1 fmole of enzyme can be detected under these conditions. Effect of pH on filter assay sensitivity.

Conditions: H- 2O2- = 10~⁴M Biphenol = 1.27 x 10~⁴M

Buffer = 0.0375 M buffer (pH) =

Citrate (5.0), Citrate (5.5),

Phosphate (6.0), ADA* (6.0),

Phosphate (6.5), ADA (6.5),

Phosphate (7.0), ADA (7.0),

Phosphate (7.5), Tricine (7.5),

Trincine (8.0), Tricine (8.5) Peroxidase = 8.9 x 10 -17 moles/assay to 1.14 x 10 -14 moles/assay

*ADA - N-(2-acetamido) i inodiacetic acid

Results: Less than 1 fmole of peroxidase can be detected at pH 6.5-7.0 with ADA or phosphate buffer. Reductions in sensitivity can be observed outside this pH range.

VI. Effect of ionic strength on filter assay sensitivity.

Conditions: H₂0₂ = 9 x 10^~4M

Biphenol = 1.27 x lθ^"4M

Buffer = 0.0375M Phosphate, pH 7.0

Salt = 0-0.75 M Sodium Chloride Peroxidase = 8.9 x 10 —17 moles/assay to 1.14 x 10 -14 moles/assay

Results: A small (2-4 fold) decrease in sensitivity was observed as the salt concentration increased between 0.5 and 0.75M. RESULTS: STABILITY ASSAY

VII. Stability of biphenol

Conditions: ^H2°2 ^{= *}^* ^mM

Buffer = 0.1 MNaH₂PO4,pH6.0

Donors:

ABTS (2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid)); 0.4mg/mL

MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloric acid salt); 0.14mg/mL plus (3-dimethylamino benzoic acid); 5mg/mL

PYROGALLOL (1,2,3-trihydroxy benzene); 50mg/mL

DAB (3,3'-diaminobenzidine) ; 0.5mg/mL

H-Y Hanker-Yates reagent (Polysciences; a combination of p-phenylenediamine and pyrocatechol); 0.5mg/mL BIPHENOL a saturated solution, 1.5 x 10 -4M.

Results: The absorbance at the appropriate wavelengths is shown in FIG. 4 (ABTS, 414nm; MBTH, 590nm; PYROGAL, 420nm; DAB, 460nm; H-Y, 430nm; BIPHENOL, 400nM) . Clearly, with the exception of biphenol, substrate solutions must be made fresh on a daily basis. We have not seen color or precipitation of a biphenol/H₂0₂ solution in our experience. Solutions stored at room temperature over a period of months show no discernable changes, yet still react upon the addition of peroxidase. Example VIII, Biphenol/H Q-. Sample Assay

Blot diluton assay (P.aeruginosa^' phage reagent) 1. Prepare a series of microorganism concentrations of the host microorganism used to propagate the phage. The solutions should be a series of 11 1:2 dilutions in 0.45%

5 sodium chloride, beginning at approximately 10 organisms/ml. Procedure a. Block filter (HT-Tuffryn; 0.45u) with lOOul Trypticase Soy Broth b. Add 10 ul of microorganisms, filter. c. Add 50 ul phage reagent, incubate 20 min., filter. d. Wash 2x2min with 200ul (ea.) PBS-tween 20. e. Add lOOul substrate, incubate 20 min., filter. f. Dry and measure reflectance values. Results

Org./blot % Reflectance Org./blot % Reflectance

10,000 76.2 156 86, .5

5,000 82 78 87, .9

2,500 73 39 77, .9

1,250 78 20 76, .5

625 87 10 79. .5

312 85 91.3+ 0 .94 (n= =25)

Example IX, Determination of peroxidase concentration in an unknown sample.

Assay Conditions

Described in the package insert provided with peroxidase purchase from Sigma Chemical Company, St. Louis, Missouri 63178

Calculations

Units/mg = A420nm/20 sec

12) (mg/3mL Reaction Mix)

therefore,

Units = A420nm/min/mL

12

Since I g pure peroxidase gives 330 units of activity and 1 mg equals approximately 1.357E16 enzyme molecules,

3960 = A420/min/mL for 1 mg. So, when A420/min = 1.0, the concentration of peroxidase in the sample is 3.57E12 molecules/sample volume tested.

It will be appreciated by those skilled in the art that various modifications may be made to the invention disclosed herein. The invention is not to be limited to the specific embodiments given herein for purposes of illustration, but is limited only by the scope of the appended claims and their equivalents.

Claims

1. A method of identifying specific organisms comprising selecting a binding agent for the specific organism, labeling the binding agent, contacting the labeled binding agent with a specimen of an unknown organism, binding the binding agent to the specific organism present in the specimen, separating the bound organism and binding agent from the specimen and testing to determine the presence of the label on the bound organisms.

2. The method of claim 1 wherein the specific organism is a bacterium.

3. The method of claim 1 wherein the binding agent is a bacteriophage.

4. The method of claim 3 wherein the label is bound to the protein coating of the bacteriophage.

5. The method of claim 3 wherein the label is a peroxidase colorimetric indicator.

6. The method of claim 1 wherein the organism and bound binding agent are separated from the specimen by filtration.

7. The method of claim 6 wherein the binding agent is a bacteriophage and the label is a colorimetric indicator bound to the protein coating of the bacteriophage, the indicator being developed on the filter.

8. The method of claim 7 wherein the label is horseradish peroxidase.

9. The method of claim 1 wherein the binding agent is species specific.

10. The method of claim 1 wherein the binding agent is genus specific.

11. A specific test reagent for directly identifying salmonella type bacterial organisms comprising a specific bacteriophage capable of ^' binding to the salmonella type bacteria, the bacteriophage having an identifiable label thereon.

12. The test reagent of claim 11 wherein the label is a peroxidase colorimetric indicator.

13. The test reagent of claim 11 wherein the label is attached to'the protein shell of the bacteriophage.

14. The test reagent of claim 11 wherein the label is an activated peroxidase colorimetric indicator, the peroxidase being Nakane activated.

15. The test reagent of claim 11 wherein the label is an activated peroxidase colorimetric indicator, the peroxidase being periodate coupled.

16. A method of identifying specific organisms comprising selecting a virus binding agent for the specific organism, labeling the virus with greater than 200 enzymes per viral particle, of contacting the labeled virus with a specimen of an unk^'nown organism at a multiplicity of greater than 100, binding the enzyme-tagged virus to the specific organisms present in the specimen, separating free virus from those that are bound to the specific organisms, detecting the enzymatic activity carried by the bound virus at a sensitivity of less than 10,000 specific organisms per test.

17. The method of claim 16 wherein the specific organism is a bacterium.

18. The method of claim 16 wherein the binding agent is a bacteriophage.

19. The method of claim 16 wherein f ee bacteriophage is separated from those that are bound to the specific organisms by filtration.

20. The method of claim 19 wherein the label is horseradish peroxidase.

21. The method of claim 20 wherein the detecting agent is a peroxidase chromogenic substrate.

22. The method of claim 21 wherein the poduct of the enzymatic reaction is trapped on a filter prior to detection.

23. The method of claim 22 wherein the substrate for the detection of enzymatic activity is para-para-biphenol.

24. The method of claim 18 wherein the binding agent is species specific.

25. The method of claim 18 wherein the binding agent is genus specific.

26. The method of claim 18 wherein the binding agent is specific for Salmonella type bacteria.

27. The method of Claim 20 wherein the horseradish peroxidase enzyme is activated for chemical coupling by periodate oxidation.

28. The method of claim 16 wherein the ratio of enzymes per virus particle is greater than 2000.

29. The method of claim 16 wherein the sensitivity of the test is less than 1000 specific organisms per test.

30. A method of testing for the presence of a peroxidase enzyme comprising preparing a stable premix solution of hydrogen peroxide and para-para-biphenol, subsequently reacting the premix with a sample containing an unknown quantity of a peroxidase enzyme and reading the reaction product to determine the presence of peroxidase enzyme in the sample.

31. The method of claim 30 wherein the reaction forms a colored solid reactant and the reactant is trapped on a filter medium.

32. The method of Claim 31 wherein the presence of color on the filter medium is read as a positive for the peroxidase enzyme.

33. The method of claim 31 wherein the absence of color on the filter medium is read as a negative for the peroxidase enzyme.

34 . The method of claim 31 wherein the test is effective to detect the presence of one f mole/ml of enzyme in the sample.

35 . The method of claim 31 wherein the method is automated and the premix is stored in the reservoir of an automated testing apparatus.

36. The method of claim 35 wherein the test is conducted in an assay well of the automated testing apparatus.

37. The method of claim 35 wherein the filter medium is a 0.45 urn filter.

38. A method of testing for the presence of a specific organism comprising selecting a virus binding agent for the specific organism comprising selecting a virus binding agent for the specific organism, labeling the virus with a peroxidase enzyme, preparing a stable premix of peroxide and para-para-biphenol, contacting the' labeled virus with the specific organism and binding at least part of the labeled virus to the specific organism, separating the bound virus and organism from unbaound labeled virus, contacting the bound virus and organism from unbound labeled virus, contacting the bound labeled virus with the premis and reacting the peroxidase enzyme label on the vound virus with the premix to form a colored solid reactant, trapping the colored solid reactant on a filter medium and reading the colored solid reactant to determine the presence of the specific organism.

39. The method of claim 38 wherein the test is effective to determine the presence of one f mole/ml of labeled enzyme in the sample.

40. The method of claim 38 wherein the virus is a bacteriophage.

41. The method of claim 38 wherein the test is conducted in an assay well of an automated testing apparatus and wherein the filter medium is a 0.45 um filter.