WO1990010232A1

WO1990010232A1 - Composition containing labeled streptococcal antibody, test kit and assay using same

Info

Publication number: WO1990010232A1
Application number: PCT/US1990/000761
Authority: WO
Inventors: Chester Harold Warren, Iii; Gary Louis Snodgrass
Original assignee: Eastman Kodak Company
Priority date: 1989-03-01
Filing date: 1990-02-13
Publication date: 1990-09-07
Also published as: FI905350A0; CA2028146A1; EP0413810A1; KR920700402A; JPH03501653A

Abstract

An aqueous immunological composition comprises a detectably labeled antibody to a Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant. This composition provides improved background and spot quality in assays for Streptococcal antigens. Such advantages are particularly evident with assays carried out using filter membranes to capture the immunological product. The composition can be included in a diagnostic test kit for use in the assay.

Description

C0MP0SITI0N CONTAINING LABELED STREPTOCOCCAL ANTIBODY, TEST KIT AND ASSAY USING SAME Field of the Invention

This invention is related to an immunological composition containing a labeled antibody to a Streptococcus organism. It also relates to an immunological diagnostic test kit including the composition. The invention is useful in diagnostic methods for determining Streptococcal organisms.

Background of the Invention

Organisms classified as gram positive bacteria, such as group specific Streptococcus, are known to be pathogenic in humans. Group A organisms are primarily responsible for causing B-hemolytic pneumonia, scarlet fever, rheumatic fever, cardiac sequelae, glomerulonephritis, septic sore throat and puerpueral sepsis. Because of the serious nature of infections potentially caused by Streptococcus A, it is important to diagnose its presence at an early stage so that an appropriate course of treatment can be pursued. In other words, it is highly desirable to have a highly sensitive assay so that infectious agents can be detected at low concentration. The antigen-antibody reaction is the basis for all immunological test methods. Certain proteins known as antibodies are produced by mammals and humans in response to the presence of an antigen, or foreign substance, which can be another protein or a carbohydrate. This normal body response to a foreign substance has led to the development of a number of diagnostic techniques to detect drugs, diseases or other physiological conditions, including the presence of Streptococcal organisms. For example, in an in vitro assay for Streptococcal organisms in a test specimen, the presence of the antigen from the organism is detected by contacting the specimen with its immunological counterpart, that is an antibody. If the antigen is present, the resulting immunological complex will form and can be detected using appropriate techniques and reagents.

Such complexes are detected in a number of ways. Generally, one or more participants in the reaction is detectably labeled. That is, it is either chosen because it is inherently detectable, or a detectable moiety (for example, an enzyme, radioisotope, chromogen or fluorogen) is incorporated therein in some manner. Many assays (for example, that known as ELISA) today utilize enzymes as detectable moieties because of the convenience they provide in requiring minimum equipment and skills needed for the assays, as well as improved sensitivity is some cases.

One significant problem encountered in many analytical procedures which utilize immunological reactions is the occurrence of non-specific binding reactions. For example, immunological reactantε, such as antibodies or antigens, may indiscriminately react with other proteins, carbohydrates or chemical or biological materials for which they are not specific. They may also react with each other and clump together thereby inhibiting specific binding with a molecule for which they have specific reactivity. In addition, if the assays are carried out using solid supports of some^" type (such as membranes, glass tubes, plates, beads or fibers), the specific binding species may also non-specifically bind to such materials because of the nature of chemical groups on the surfaces. This can be particularly troubling if the immunological complex is detected on a solid support in a certain region only, because unwanted reactions are observable as background signal. If this background is too high, the signal due to the desired immunological reaction is obscured or misread. Low antigen concentrations are undetectable, and assay sensitivity is lowered. Polyamide membranes, in particular, are susceptible to nonspecific interactions with antibodies. It is known that non-specific binding to solid surfaces (such as polyamides) can be minimized by coating them with proteins, such as casein or bovine serum albumin. In copending US—A-4,828,980, it is disclosed that non-specific interactions in agglutination assays can be minimized by coating microporous membranes used in those assays with certain low pi proteins or carbohydrates. Moreover, copending U.S.S.N. 206,257 (filed June 13, 1988 by Warren III et al) describes the use of water-soluble proteins or carbohydrates with labeled antibodies in order to lower background and improve assay sensitivity.

While the noted inventions represent important advances in the art, it has been observed that it would be very helpful if background was reduced further in assays for Streptococcal organisms. In particular, in instances where the assay is carried out in a distinct region of a microporous filtration membrane, the area around that region sometimes exhibits clumps of labeled antibody which obscures the assay signal, can lead to false positive results and can hinder assay determinations in the distinct region. Summarv of the Invention

The problems mentioned above have been overcome with the use of an aqueous immunological composition comprising a detectably-labeled antibody to a Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant.

Moreover, an immunological diagnostic test kit useful for the determination of a Streptococcal organism comprises: a. one or more reagents for extracting Streptococcal antigen from a Streptococcal organism, and b. the aqueous immunological composition described above. This invention also provides a method for the detection of a Streptococcal organism comprising the steps of:

A. contacting a specimen suspected of containing an extracted Streptococcal antigen with the aqueous immunological composition described above, to form a detectably-labeled immunological complex,

B. detecting the presence of the complex as an indication of the presence of the antigen in the specimen.

The present invention provides a composition which enables one to have a highly sensitive diagnostic assay for Streptococcal organisms using a detectably labeled antibody. The improved sensitivity and specificity of the assay are accomplished by using the labeled antibody in admixture with one or more water-soluble or water-dispersible nonionic surfactants. It is not understood how these surfactants provide the improvement, but it is clear that background is lowered and the clumps of labeled antibody formed in background regions of a substrate are noticeably reduced. Thus, spot quality is improved when the test is carried out in a "spot" or region of the substrate. Moreover, assay sensitivity is not adversely affected, and may be improved in many instances because lower concentrations of antigen are readily detected. Detailed Description of the Invention The present invention can be used to rapidly detect the presence of a Streptococcal organism in a biological specimen from a human or animal host. Generally, such organisms are detected by determination of the presence of extracted Streptococcal antigens, such as the carbohydrate antigens extracted from Streptococcal A, B, C or G group organisms. Streptococcal A antigen is most preferably detectable with this invention.

Biological samples which can be so assayed include any solution, specimen or sample which is suspected of containing Streptococcal organisms or extracted Str-eptococcal antigens. Generally, such organisms are found in human mucous and tissue cultures from the mouth, throat or nasal passages. The specimens can be collected using suitable procedures. For example, a throat specimen is generally collected on a swab, and can be tested with or without pretreatment (for example, filtration) to remove unwanted debris or interferents. While some analytical procedures may be suitably carried out using whole Streptococcal organisms since some antigenic sites may be external to the bacterial cells, and thus, available for immunological reaction, it is preferred that the assay be carried out with extracted antigen. Streptococcal antigen extraction can be accomplished using a number of known procedures. For example, extraction can be carried out using hot formamide, autoclaving in the presence of HC1, various enzymes (see TJS—A—4,618,576, or by generation of nitrous acid according to TJS-A— ,673,639 using one or more extracting reagents. A preferred extraction technique is described in copending US-A-4,808,524. In that preferred extraction technique, a specimen suspected of containing Streptococcal organisms (such as from a patient) is subjected to the preferred extraction composition. For example, a swab containing a throat culture can be put into a container containing the extraction composition and incubated therein for a suitable period of time. If desired, centrifugation can be carried out to remove extraneous matter or the resulting extract can be neutralized.

Extracted Streptococcal antigen is contacted with the aqueous immunological composition of this invention to form a detectably-labeled immunological complex. This composition comprises a detectably labeled antibody to the antigen. By detectably labeled is meant that the antibody can be directly detected using appropriate detection equipment and procedures, or it can be detected after further reaction of the label with one or more appropriate reagents. The label can be an inherent portion of the antibody molecule, or a separate moiety attached thereto. Useful labels include radioisotopes, chemiluminescent moieties, chromogens or fluorogens, phosphorescent moieties, enzymes, enzyme cofactors, and others known to one skilled in the art. Generally, the labeled antibody is water-soluble or water-dispersible, so the label must not insolubilize the antibody. Another class of labels are what are termed in the art as specific binding materials which will specifically bind with a corresponding receptor material. The resulting complexes are comparable to immunological complexes. Examples of specific binding complexes include avidin-biotin and sugar-lectin. Thus, the Streptococcal antibody can be labeled with avidin, biotin, a sugar, a lectin, or even another antibody or antigenic material not specific for Streptococcal antigens. Detection of the immunological complex is accomplished by reacting it with the receptor corresponding to the label, which receptor has a detectable moiety (for example, a radioisotope, enzyme or cofactor). Radioisotopes and enzymes are preferred labels with enzymes being most preferred. Representative examples include peroxidase, alkaline phosphatase, urease, glucose oxidase, b_e_£a.-glucosidase and others known in the art. Peroxidase and alkaline phosphatase are preferred with peroxidase being most preferred.

Labeled antibodies can be prepared using known procedures. Many are commercially available. Others are not commercially available, but they are prepared using known procedures (see, for example, Methods in Enzvmologv. 22__> Immunochemical Techniques, p.277, Langone and Van Vunakis, Eds., 1983 for preparing radiolabeled antibodies, EP-A-0 201 079 and US-A-4,276,206 for preparing biotinylated antibodies). An enzyme-labeled antibody is generally prepared by derivatizing the enzyme, purifying the enzyme derivative, reacting the derivative with the antibody, and purification and characterization of the resulting conjugate. A number of procedures are described in the following references: Yoshitake, Eur. J. Biochem.. lp_l, 395 (1979), Nakane et al, i_ Histochemistry and Cytochemistry. 22., 1084 (1974) and Avrameas, Bull. Soc. Chim. Biol.. 5_0_, 1169 (1968). The amount of labeled antibody present in the composition of this invention will vary depending upon the target ligand, the dye-forming composition used (where an enzyme label is used), the particular label used and other factors of a particular assay. Generally, however, it is present in an amount of at least about 0.1 μg/ml, and preferably from about 1 to about 20 μg/ml.

The labeled antibody and unlabeled antibody (described below) used in the practice of this invention can be individually or collectively monoclonal or polyclonal. They can be obtained from several commercial sources (such as Biochemed, Immunosearch or Ventrex), or prepared using standard techniques.

The second critical component of the immunological composition is one or more water—soluble or water-dispersible nonionic surfactants. Such surfactants are generally soluble or dispersible in water in an amount of at least about 1 mg/ml. Useful surfactants include, but are not limited to, polyoxyethylene ethers such as those sold as Triton nonionic surfactants (for example, Triton™ X-100 and Triton™ N101), or as Nonidet or Brij nonionic surfactants (such as Nonidet™ P-40 and Brij™ 35), polyoxyethylene- sorbitan derivatives such as those sold as Tween

TM nonionic surfactants (for example Tween 20 or Tween TM 40), and polyglycol ethers such as those

- T sold as Tergitol nonionic surfactants (for example Tergitol NPX and NP-7). Other useful materials would be readily apparent to one skilled in the art, especially after consulting the standard reference for surfactants, McCutchepπ'g F-πmlsifiers and Detergents. 1986 Edition, McCutcheon Division, Publishing Co., Glen Rock, N.J., as well as the Sigma Chemical Company catalog entitled "Biochemicals Organic Compounds for Research and Diagnostic Reagents" for 1989, pp. 333-335.

Particularly useful surfactants are the polyoxyethylenesorbitan derivatives such as the monolaurate, monooleate, monopalmitate, monostearate and other derivatives. The monolaurate derivative is most preferred. This material can be purchased as Tween 20 nonionic surfactant.

The nonionic surfactants are present in the immunological composition of this invention in an amount of at least about 0.05, and preferably from about 0.1 to about 0.5, weight percent (based on total composition weight).

The immunological composition optionally, but preferably, also contains a number of other components which improve assay performance. For example, it can include a water-soluble protein or carbohydrate which has a low pi. The term pi (or isoelectric point) is known as the pH at which there is an equal number of positive and negative charges in a molecule so that the molecule is neutral in charge. The pi can be measured using standard materials and procedures. Such materials include, but are not limited to, casein derivatives or other protein derivatives which are negatively charged (for example succinylated casein, glutarylated casein, succinylated bovine serum albumin and succinylated collagen) and cellulosic derivatives (such as carboxymethyl cellulose and carboxyethyl cellulose). Succinylated casein is preferred. The composition can also include one or more phenols which improve its keeping stability. Useful phenols are described, for example, in US-A-4,828,983, and include rj,p.^»-biphenol, 4*-hydroxyacetanilide, p.-methoxyphenol, chlorphenol red, p_-cresol, B-methoxyphenol, vanillin, 4-chloro-3,5-dimethylaminophenol and ir-hydroxybenzoic acid. The phenols p-methoxyphenol and 4'-hydroxyacetanilide are preferred, with 4^»—hydroxyacetanilide being most preferred. The amount of phenol present in the composition is generally at least about 0.001 weight percent.

One or more buffers are generally included in the immunological composition. Any buffer, in an appropriate amount, which will effectively maintain composition pH in the range of from about 7 to about 9 are useful. Representative buffers include, but are not limited to, 3-(N-morpholino)propanesulfonic acid, N-(2—hydroxyethyl)piperazine-N'—2—ethane- sulfonic acid and N-tris(hydroxymethyl)methyl-2- aminoethanesulfonic acid.

The complex formed between Streptococcal antigen and detectably labeled antibody can be detected using suitable procedures and equipment depending upon the type of label used.

Where the label is an enzyme, the complex is contacted with suitable substrates and one or more dye-forming reagents. In some instances, the substrate itself provides a dye. Such substrates include benzidine or derivatives thereof,

2,2'—azino—di-(3-ethyl-benzthiazolone-6-sulfonic acid), phenol red, fi-phenylenediamine, pyrogallol, 4-aminoantipyrine, bromopyrogallol red and others known in the art. Alternatively, a hydrogen donor and an electron acceptor can be combined to provide a detectable species (for^" example, as shown in US-A-4,260,679). Preferably, the label is peroxidase and the dye-providing reagents include a leuco dye which provides a dye in the presence of peroxidase and hydrogen peroxide. Useful leuco dyes include imidazoles, arylmethanes and others known in the art. Particularly useful leuco dyes are triarylimidazoles (as described in US-A-4,089,747) and triarylmethanes (as shown in US-A-4,670,385) . A preferred dye-providing composition is described in copending U.S.S.N. 136,166 (filed December 18, 1987 by McClune et al), incorporated herein by reference.

Once a complex has formed, it is detected to determine if the assay indicates the presence of antigen in the test specimen, which would then be indicative of the presence of Streptococcal organisms. Both positive and negative control tests can be carried out simultaneously using the appropriate reagents to give the desired results.

In a preferred embodiment of this invention, a method for the detection of Streptococcus A comprises the steps of:

A. contacting a specimen suspected of containing Streptococcus A with one or more extraction reagents for extracting antigen from the organism,

B. contacting the extracted Streptococcal A antigen with the aqueous immunological composition described herein having an enzyme labeled antibody, to form an enzyme-labeled immunological complex, C. prior to, simultaneously with or subsequently to step B but after step A, contacting the extracted Streptococcal A antigen with an unlabeled antibody to the antigen, which unlabeled antibody is immobilized or capable of becoming so, to form a complex of antigen and the unlabeled antibody, D. contacting the resulting complex of labeled and unlabeled antibodies with the antigen with one or more reagents which provide a dye in the presence or the enzyme, and E. detecting the presence of the dye as an indication of the presence of Streptococcus A in the specimen.

As noted in Step C of this embodiment, the extracted Streptococcal antigen can also be contacted with a second antibody to the antigen, which second antibody is not labeled with detectable moieties as described above for the first antibody. This second antibody can be used to form a "sandwich" of the antigen between the two antibodies. Generally, the unlabeled antibody is either attached to an insoluble substrate (such as beads, membrane, filter, glass tube or other material known in the art), or is capable of becoming so attached during the assay. In the latter instance, the unlabeled antibody could have a moiety or chemical group which readily reacts with another moiety or chemical group on the substrate. Thus, the second antibody could be attached to a substrate through adsorptive, covalent or specific binding means (for example, avidin—biotin).

In a preferred embodiment, the unlabeled antibody is bound in some manner to a water—insoluble microporous article such as a filtration membrane. This membrane can be composed of a number of materials with polyamides (for example nylons) or modified polyamides being the most useful. It has sufficient porosity to drain uncomplexed materials, but readily captures the immunological complex formed between antigen and the two antibodies. The unlabeled antibody can be readily affixed to the microporous article just described. For example, it can be affixed mechanically by coating, spotting or spraying and held thereon by hydrophobic bonding among molecules, or if the antibodies are attached to particles, the bonding can be between particles. It can also be affixed through chemical means. It is preferred that the antibodies be bound covalently to polymeric particles, which particles are mechanically affixed to a microporous filtration membrane of nylon.

The unlabeled antibody is generally used in the assay in an amount sufficient to bind all available antigen to be detected. The timing of contacting extracted antigen with the unlabeled antibody is not critical in most instances in relation to antigen contact with the labeled antibody. In a preferred embodiment where the unlabeled antibody is immobilized on a solid substrate, the antigen is contacted with it before contact with the labeled antibody is accomplished.

The assay of this invention can include one or more wash steps in which uncomplexed or unused reagents are separated from complexed materials. Separation can be accomplished through standard centrifugation or other mechanical means. Preferably, uncomplexed materials are washed through a microporous membrane, leaving the complex on the membrane. Other steps may be needed for adding dye-forming reagents or reagents for immobilizing the unlabeled antibody. One skilled in the art would readily understand how to incorporate such steps in the assay. A representative assay is shown in Example 3 below. While the assay of this invention can be carried out using standard laboratory equipment and glassware, it is preferred that it be carried out in a test device of some type. Various test devices are known in the art includin those described in

US-A-3,825,410, US-A-3,888,629, US-A-3, 70,429 and US-A- , 46,232. Particularly useful devices are described in EP-A-0 308 231 and US-A-4,870,007.

More specifically, the test device comprises a water—insoluble shell having one or more test wells therein each of which can accommodate a sample of a biological specimen and appropriate reagents.

The shell can be prepared from any useful water—insoluble material such as glass, polymeric materials, ceramics, fibrous materials, cellulosic materials and other materials known in the art.

In a preferred embodiment, the test device has three test wells designed for providing a specimen test result and positive and negative control results. Each test well has a microporous article mounted therein. Another test device is described and claimed in EP-A-0 280 558. Other variations of useful test devices would be within the purview of a worker of ordinary skill in the art. One such test device is currently used for the

TM

Surecell diagnostic assay kits commercially available from Eastman Kodak Company.

A diagnostic kit of this invention includes the immunological composition of this invention as well as one or more other reagents or reagent compositions. In one embodiment, the kit includes the immunological composition and one or more extraction reagents. In another embodiment, a kit includes the immunological composition and one or more reagents for providing a detectable species where the label is an enzyme. In either embodiment (where appropriate), the kit can also include buffers, wash solutions, dye-providing solutions, test devices, extraction devices, unlabeled antibodies, immobilizing reagents (for example avidin-coated beads), mixing containers, pipettes, swabs, instructions and the like.

Preferably, any test kit also includes a disposable test device having immobilized therein an unlabeled antibody to Streptococcal antigen.

The following examples are provided to illustrate, but not limit, the present invention. All percentages are by weight, unless otherwise noted.

Materials A leuco dye solution was prepared with 2- (4—hydroxy-3,5-dimethoxypheny1)-4,5-bis(4-methoxy- phenyl)imidazole as follows:

Solid leuco dye (to make a 0.1% solution) was dissolved in a solution of 20% poly(vinylpyrrolidone) in sodium phosphate buffer (5 mmolar). This solution was then added to a solution containing hydrogen peroxide (10 mmolar), 4'—hydroxyacetanilide (0.7 mmolar) and diethylenetriaminepenta- acetic acid (10 μmolar) in sodium phosphate buffer to produce a final concentration of 1% poly(vinylpyrrolidone and 0.005% leuco dye.

Succinylated casein was prepared by reacting casein with an equal weight of succinic anhydride for four hours at 25"C, then purifying the product by dialysis.

TM

LoProdyne nylon microporous membranes (Pall Corp.) were incorporated into the test wells of disposable test devices and pretreated with Fluorad™ FC 135 surfactant (0.05 g/m²). Anti—Streptococcus A-peroxidase conjugate was prepared using immunopurified rabbit polyclonal antibodies which were obtained commercially and horseradish peroxidase by the method described by Yoshitake et al, Eur. J. Biochem. 101. 395 (1979). Examples 1 £.2: Immunological Compositions

An immunological composition for use in an assay for Streptococcal A antigen was prepared as follows: A solution (1 liter) of succinylated casein (5 g) in nanopure water was prepared. 3-(N-fflorpholino)propanesulfonic acid buffer (20.93 g) was added to the solution, and its pH was adjusted to 7.55 using sodium hydroxide. To portions (500 ml each) of this solution were added a phenol (either 0.756 g 4'—hydroxyacetanilide or 6.2 mg p_-methoxyphenol),

TM

Tween 20 nonionic surfactant (1 g, 0.27.) and thimerosal preservative (0.05 g). The resulting solution was filtered through a filter (0.45 μm) and the peroxidase labeled—antibody to Streptococcus A (3.57 ml of phosphate buffered saline solution containing 1.232 mg/ml antibody) was added thereto. Thus, Example 1 contained 4'—hydroxyacetanilide as a stabilizer, and Example 2 contained p_-methoxyphenol. Example 3: Assay for Streptococcus A

This example demonstrates the use of the compositions of Examples 1 and 2 in the method of this invention to detect Streptococcus A antigen in a biological specimen.

Streptococcus A antigen was obtained from Group A strep cultures using a standard nitrous acid extraction procedure wherein aqueous sodium nitrite was mixed with an acidic coreagent prior to the addition of the cultured organism. The extraction fluid was then neutralized by the addition of excess buffer. The Group A carbohydrate antigen was obtained using acidic ethanol and acetone, discarding the supernatant and resuspending the pellet in 0.85% saline solution. The concentration of rhamnose was determined by the method of Dische and Shattles, J. Biol. Chem. 175. 595-603 (1948). This concentration (20 μl) was resuspended in a neutralized extraction fluid comprising citric acid (10 μl, 1.2 molar), sodium nitrite (120 μl, 8 molar) and 4-morpholinopropane sulfonic acid buffer (120 μl, 2 molar, pH 8) .

A microporous membrane as described above was incorporated into each of the three test wells of two disposable test devices which were similar to that described in EP-A-0 308 231. A dispersion of pol rstyrene-co-m &. p_-(2-chloroethylsulfonylmethyl)- styrene] beads [2 μl of a 1% solid suspension in 0-1 molar glycine (pH 8.5) admixed with poly(acryl- amide) (5%) and 0.0005%, of an optical brightener], was added to the center area of the membrane in the test well identified as the specimen test well. To the beads were covalently bound rabbit polyclonal antibodies to Streptococcus A antigen.

A second test well of each disposable test device, considered the negative control well, contained a dried dispersion (2 μl) of the same polymeric beads to which had been attached rabbit gamma globulin (1%, by weight of beads) admixed with poly(acrylamide) (5% ) and the optical brightener. This dispersion was applied in a center area of the membrane.

A third test well of each test device, considered the positive control well, contained a dried dispersion of the reagent (2 μl) described above in poly(acrylamide) (5%), the optical brightener and 20 ng/ml of the Streptococcus A antigen. This dispersion was applied in a center area of the membrane.

A sample of extracted antigen (200 μl containing about 300 ng/ml of carbohydrate) was added to the specimen test well only. The peroxidase labeled antibody compositions (40 μl each, 9 μg/ml conjugate) from Examples 1 and 2 were added to all test wells of the disposables, followed by incubation for two minutes at room temperature. The membranes in the test wells were then washed twice with a wash solution (240 μl) containing decyl sulfate (18 g/1) with fluid draining immediately.

The leuco dye composition (40 μl) described above was added followed by incubation for five minutes at room temperature.

A Control composition similar to those of

TM Examples 1 and 2, but without Tween 20 nonionic surfactant, was similarly used in a Control assay for Streptococcus A. This Control composition is like the composition described and claimed in U.S.S.N. 206,257 (noted above). The resulting spots of dye in the center of the test wells were observed and graded on a scale of 0 to 10 with zero representing no observable dye and 10 being the highest dye density. Background readings were obtained by observing the presence of any dye in the area outside the center zone where the unlabeled antibodies were immobilized on the membranes.

The results of these tests are shown in the following Tables. It is apparent that the Control assay exhibited unacceptable background in the assay, especially in the negative control well. The assays using the compositions of Examples 1 and 2 exhibit little or no background. This demonstrated significant improvement over the Control composition and assay.

T A B L E I (Control)

(p cograms Area roun Area groun Area' ground

0 0 2.5 0 1 7 1 500 0 1.5 3 0.5 7 0

T A B L E II (Example 1)

(p cograms') Area roun Area roun Area round

0 0 1 0 0.5 7 0 500 0 0 4 0 7 0

T A B L E III (Example 2) e s t W e l l s

Antigen Negative Positive Concen¬ Control Specimen Control tration Center Back- Center Back- Center Back¬ (picograms) Area ground Area ££Jind_ Area ground

0 0 0.5 0 0 7 0 500 0 0.5 4 0 7 0

The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

Claims

We claim:

1. An aqueous immunological composition comprising a detectably labeled antibody to a Streptococcal antigen, and a water—soluble or water-dispersible nonionic surfactant.

2. The composition of claim 1 wherein said antibody is labeled with a radioiεotope or enzyme.

3. The composition of claim 2 wherein said antibody is labeled with an enzyme.

4. The method of claim 1 wherein said antibody is directed to a Streptococcal A antigen and is labeled with peroxidase.

5. The composition of claim 1 wherein said nonionic surfactant is present in an amount of at least about 0.05 weight percent.

6. The composition of claim 1 wherein said nonionic surfactant is selected from the group consisting of polyoxyethylene ethers, polyoxyethylenesorbitan derivatives and polyglycol ethers.

7. The composition of claim 6 wherein said nonionic surfactant is a polyoxyethylenesorbitan derivative.

8. The composition of claim 7 wherein said nonionic surfactant is polyoxyethylenesorbitan monolaurate.

9. The composition of claim 1 further comprising a buffer, a water-soluble protein or carbohydrate and a phenol.

10. An immunological diagnostic test kit useful for the determination of a Streptococcal organism comprising: a. one or more reagents for extracting Streptococcal antigen from a Streptococcal organism, and b. an aqueous immunological composition comprising a detectably labeled antibody to said Streptococcal antigen, and a water—soluble or water-dispersible nonionic surfactant.

11. The test kit of claim 10 wherein said antibody is labeled with an enzyme, and said kit further comprises one or more reagents for providing a detectable species in the presence of said enzyme.

12. An immunological diagnostic test kit useful for the determination of a Streptococcal organism comprising: a. an aqueous immunological composition comprising an enzyme-labeled antibody to a Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant, and b. one or more reagents for providing a detectable species in the presence of said enzyme.

13. The test kit of claim 12 further comprising a disposable test device in which is immobilized an unlabeled antibody to said Streptococcal antigen.

14. The test kit of claim 12 wherein said enzyme is peroxidase, and said one or more reagents includes a triarylimidazole leuco dye which provides a dye in the presence of peroxidase and hydrogen peroxide.

15. A method for the detection of a Streptococcal organism comprising the steps of:

A. contacting a specimen suspected of containing an extracted Streptococcal antigen with an aqueous immunological composition comprising a detectably labeled antibody to said Streptococcal antigen, and a water-soluble or water-dispersible nonionic surfactant, to form a detectably labeled immunological complex, B. detecting the presence of said complex as an indication of the presence of said antigen in said specimen.

16. The method of claim 15 wherein said extracted antigen is contacted with an immobilized unlabeled antibody thereto prior to step A and after extraction.

17. The method of claim 15 wherein said antibody is labeled with an enzyme, and detection of said complex is accomplished by contacting said complex with one or more reagents which provide a detectable species in the presence of said enzyme.

18. A method for the detection of Streptococcus A comprising the steps of: A. contacting a specimen suspected of containing Streptococcus A with one or more extraction reagents for extracting antigen from said organism,

B. contacting said extracted Streptococcal A antigen with an aqueous immunological composition comprising an enzyme-labeled antibody to said Streptococcal A antigen, and a water—soluble or water-dispersible nonionic surfactant, to form an enzyme-labeled immunological complex, C. prior to, simultaneously with or subsequently to step B but after step A, contacting said extracted Streptococcal A antigen with an unlabeled antibody to said antigen, which unlabeled antibody is immobilized or capable of becoming so, to form a complex of antigen and said unlabeled antibody,

D. contacting the resulting complex of labeled and unlabeled antibodies with said antigen with one or more reagents which provide a dye in the presence or said enzyme, and E. detecting the presence of said dye as an indication of the presence of Streptococcus A in said specimen.

19. The method of claim 18 wherein said enzyme is peroxidase, and said dye is formed using a triarylimidazole leuco dye and hydrogen peroxide.

20. The method of claim 18 wherein said nonionic surfactant is present in an amount of at least about 0.05 weight percent, and is selected from the group consisting of polyoxyethylene ethers, polyoxyethylenesorbitan derivatives and polyglycol ethers.