WO1996012966A1

WO1996012966A1 - Agglutination test

Info

Publication number: WO1996012966A1
Application number: PCT/US1994/011900
Authority: WO
Inventors: Leonard J. Zubrzycki
Original assignee: Temple University - Of The Commonwealth System Of Higher Education
Priority date: 1994-10-19
Filing date: 1994-10-19
Publication date: 1996-05-02
Also published as: AU1040195A

Abstract

The invention provides a method for detecting the presence of an antigen from a cell capable of under-going rapid lysis to release the antigen into solution. The method includes mixing a sample solution with an aqueous diluent having an effective amount of an agglutination enhancer which contains a non-protein water soluble polymer and a water soluble protein. The solution of sample and agglutination enhancer is then contacted with an antibody specific for the antigen. Agglutination indicates the presence of the antigen.

Description

AGGLUTINATION TEST

Field of the Invention This invention relates to an enhanced anti- gen/antibody agglutination test.

Background of the Invention Aggregates that quickly sediment can be formed from mixing an antigen and antibody in the presence of an electrolyte, for example, NaCl. Visible aggregates occur from mixing a suspension of particles with antigens on their surfaces with particles having a corresponding antibody. Such aggregation is termed agglutination.

Tests based upon the agglutination principle are generally technically simplistic. For example, a drop of a suspension of antigen particles and a drop of an antibody, usually serum containing the antibody, are mixed on a slide. The slide is rotated, by hand or by a machine, and in a few minutes agglutination occurs. Observing the slide with either the naked eye or under a microscope determines the presence or absence of agglutination. Agglutination is a positive test result.

Agglutination tests can also be performed in test tubes. The contents can be checked for agglutination while still in the tube or removed and examined more closely. A variation of the agglutination test involves the use of antibody-coated particles. In the tube variation, the antigen can be a soluble antigen, that is, sedimentation of the antigen is not required. The end result of a positive tube test is still seen as an agglutination, because the antibody- coated particles add the mass for the reaction to be seen. Thus, these larger antibody-coated particles result in a more sensitive test. One of the most common clinical applications of the agglutination test has been to determine an indi-vidual' s blood type from their red blood cells. The detection of antigens other than blood type antigens is more difficult and less common. However, since antigen-antibody tests are specific, agglutination tests can be used to identify the species of cells isolated or cultured from clinical material.

In such tests, cells (bacteria, for example) in suspension can be viable or inactivated, as long as the inactivation does not alter the antigens for recog-nition by the antibody. In some cases outer layer com-ponents of the cells must be removed to expose antigens on the cells' surfaces for recognition by the antibody. Examples of bacterial species whose antigens can be detected by agglutination tests are Hemophilus influenzae, Neisseria meningitis, and Streptococcus pneumonia. In these species, the antigens are both intact on the cells' surfaces and free in the clinical fluid. Thus, the total free and intact antigens provide a sufficient concentration of antigens to be detected in an agglutination test using antibody-coated particles. Agglutination tests are also useful in the diagnosis of suspected cases of strep throat. Several types of agglutination test kits for detecting strep throat are commercially available. They all require an extraction step for extracting the antigens (usually via nitric acid) . The extraction step is necessary to expose and release the C poly- saccharide of group A Strepto-coccus in the mucus and pus obtained from the throat of the patient. In these tests, as in those previously described for clinical fluids, the antibody is coated on particles to increase the visibility of the agglutination reaction.

Sometimes agglutination enhancers are used to increase the visibility of the agglutination reaction. Water soluble polymers, dextran and gum acacia, and proteins, gelatin and albumin, are known enhancers of antigen-antibody reactions (Munk-Andersen, Acta. Path. Microbiol. Scand., 38 259 (1956)) . Recently, polyethylene glycol (PEG) has become the preferred water soluble polymer for enhancing antigen-antibody reactions in complex immunoas- says (Siersted, et al . , Methods of Enz mology 74538 (1981) ) . Istrate (PCT/US 92/01121 (1992)) describes an antigen extraction procedure and subsequent agglutination test for detecting an antigen specific for Mvcobacterium tuberculo¬ sis when present in culture and in clinical material. The test uses monoclonal antibody-coated latex particles specific for detecting the extracted and concentrated lipoarabinomann (LAM) antigen. The test also requires the addition of a water soluble polymer to enhance the agglutination test. However, use of the water soluble polymer agglutination enhancer by Istrate did not provide sufficient sensitivity to avoid the need for concentration procedures.

The agglutination test of Istrate for the LAM anti- gen of Mvcobacterium tuberculosis from culture requires a series of extraction and concentration steps. These steps include sonication and acetone precipitation of the extracted antigen-containing preparation. Absent extraction and concentration, the assay of Istrate is not sensitive enough to detect the antigen at clinical sample concentration levels.

As illustrated by Istrate, when cells are present in clinical material and a target antigen is only present in or on the cell, sensitivity of the agglutination test is low. Frequently, sophisticated equipment is required to isolate cells and concentrate an antigen before testing.

Accordingly, there is a need for improved ag¬ glutination test reagents. Also, a practical aggluti-nation test is needed which is sensitive enough to detect antigens at lower clinical concentrations without the need for an antigen concentration step.

Summary of the Invention A method for detecting the presence of a target cell capable of undergoing rapid lysis comprising the follow- ing steps:

(a) obtaining a biological sample to be tested for the presence of the target cell which expresses a selected target antigen; (b) mixing the biological sample with an aqueous diluent ,^•

(c) contacting the diluted sample with an amount of a strong base which is effective to chemically lyse the target cell to form a solution containing antigen from the target cell;

(d) neutralizing the antigen solution with a strong acid to form a neutralized solution;

(e) contacting the neutralized antigen sol- ution with particles whose surfaces contain an antibody which is specific for the selected target antigen under condi¬ tions permitting agglutination of the particles, which agglu¬ tination is visible to the naked eye or under a microscope; and (f) detecting agglutination of the particles as an indication of the presence of the target cell in the bio¬ logical sample.

In another embodiment, the present invention provides a method for detecting the presence of a target cell capable of undergoing rapid lysis, comprising the following steps :

(a) obtaining a biological sample to be tested for the presence of a target cell which expresses a selected target antigen; (b) mixing the biological sample with an aqueous diluent comprising:

(i) from about 1% to about 3% by weight of a non-protein water soluble polymer based upon the weight of the aqueous diluent; and

(ii) from about 0.2% to about 2% by weight of a water soluble protein based upon the weight of the aqueous diluent; (iii) from about 0.1% to about 4.0% by weight of a strong base, the weight being based upon the weight of said aqueous diluent, which amount of strong base is effective to chemically lyse the target cell by disrupting the walls of the cell to form a solution containing antigen from the target cell; (c) neutralizing the antigen solution with a strong acid to form a neutralized solution;

(d) contacting the neutralized solution with particles whose surfaces contain an antibody, which is specific for the selected target antigen, under conditions permitting agglutination of the particles, which agglutination is visible to the naked eye or under a microscope; and

(e) detecting agglutination of the particles as an indication of the presence of the target cell in the biological sample. In a preferred method the aqueous diluent further comprises from about 0.01% to about 1.0% by weight of a chelating agent based upon the weight of the aqueous diluent .

In a further embodiment the present invention provides an aqueous cell lysis reagent composition comprising:

(i) from about 1% to about 3% by weight of of a non-protein water soluble polymer, based upon the total composition weight; (ii) from about 0.2% to about 2% by weight of a water soluble protein, based upon the total composition weight; and (iii) from about 0.1% to about 4.0% by weight of a strong base, based upon the total composi¬ tion weight. A preferred aqueous cell lysis composition further comprises from about 0.01% to about 1.0% by weight of a chelating agent, based upon the total composition weight.

Preferably, in each of the above embodiments of the invention: the non-protein water soluble polymer is selected from the group consisting of polyethylene glycol , dextran, or gum acacia; the water soluble protein is gelatin or albumin; the chelating agent is ethylenediaminetetra-acetic acid (EDTA) ; and the strong base is a NaOH solution. Detailed Description of the Invention

The present invention provides a practical, simple, and sensitive agglutination test which allows for results in as little as a few minutes. The agglutination test is useful when applied to clinical material. The biological samples used within the method according to the present invention comprise bodily fluids or solids, cell cultures, tissues, or the like, which may be tested for the presence of a target cell, which cell is capable of undergoing rapid lysis to re- lease target antigens from the cell into solution. A target antigen is an antigen characteristic of the target cell which, upon lysis of the target cell, allows the target cell to be detected in the biological sample. Any biological sample in which particle agglutination by a cell lysate is detectable is acceptable.

The type of biological sample may depend upon the type of disease suspected or the condition of the individual being tested. Examples of biological samples include serum, whole blood, urine, feces, tissue specimens, (e.g., pus, exudates, and biopsy specimens) , cold abscess drainage, peritoneal ascitic fluid, uterocervicovaginal secretions, cerebro-spinal fluid, pulmonary secretions, (e.g. bronchoal- veolar and gastric lavage, pleural fluid and sputum) . Some samples may require special pretreatment and/or decontamin- ation prior to testing, but such procedures are well-known to the ordinary practitioner in the art.

A preferred type of biological sample for use in the present method is a biological fluid or tissue suspected of containing target cells of the genus Neisseria, particu- larly the species Neisseria gonorrhoeae. Another preferred type of biological sample is a blood sample or a sample derived from blood, which is to be tested for human CD4+ cell concentration levels.

The target cell is described as "a cell which is capable of undergoing rapid lysis." This means cells which will fragment from chemical treatment in less than thirty min¬ utes, without the need for centrifugation or other mechanical lysis procedures. Particularly preferred cells can be lysed under alkaline conditions in less than fifteen minutes. Lysis of target cells is obtained by exposing the cells to a strong base, followed by exposure to a strong acid which neutralizes the base and adjusts the pH. Ideally, lysis can be obtained using an aqueous diluent containing from about 0.2% to 0.5% by weight of NaOH and about 0.005 % phenol red as an indicator. The preferred neutralizing agent is HC1.

Preferably, the method of the invention is applied to any bacteria cells and to other cells, human or otherwise, which can be lysed by NaOH, or similar alkaline agents. Whether rapid lysis is possible can be quickly determined for a particular cell type by routine experimentation.

Cells which are readily lysed may include patho¬ genic bacteria from genera such as Hemophilus, Neisseria, Streptococcus, Staphylococcus. Escherichia, Clostridia, Pseudomonas, Proteus, and the like. Human cells are readily and rapidly chemically lysed.

Upon being chemically lysed the target cells release target antigens. The target antigens to be detected are those which are not altered by the alkaline lysis tech- nique to such a degree as to be no longer capable of recognition of by a specific antibody in the presence of a water soluble polymer-water soluble protein-chelating agent mixture.

Whether a particular antigen is altered too much by the alkaline lysis conditions to be detected is readily determined as follows. Target cells are obtained from a cell culture or tissue culture in sufficient quantity to produce a 1 McFarland concentration in phosphate buffered saline. Mechanical and alkaline chemical lysing procedures are run on pairs of identical concentration test target cell samples to produce solutions which have the target antigen. The test samples are obtained from progressive 1/2 cell-concentration dilutions of the 1 McFarland stock cell solution. These dilutions are obtainedby diluting a volume of solution having a particular concentration of target cells with an equal volume of buffer solution. Thus, the cell concentration per unit volume is halved.

Agglutination assays of the resulting antigen solutions from each of the pairs of solutions are compared for alkaline lysis deactivation of the antigen. A lower or negative agglutination result for the alkaline lysed samples indicates possible alkaline deactivation of the antigen.

The aqueous diluent according to the present invention comprises:

(i) from about 1% to about 3% by weight of a non-protein water-soluble polymer; (ii) from about 0.2% to about 2% by weight of a water-soluble protein; and

(iii) from about 0.01% to about 1.0% by weight of a chelating agent. A preferred aqueous diluent comprises:

(i) from about 1% to about 2% by weight of polyethylene glycol;

(ii) from about 0.25% to about 1% of gelatin; and (iii) from about 0.03% to about 0.40% of ethylenediaminetetraacetic acid. The non-protein water-soluble polymer and the water-soluble protein are agglutination enhancers. Enhancers are additives which generally promote agglutination of antibody-coated particles. The amount of enhancer added to the aqueous diluent should be sufficient to increase the sensitivity of the aggluti- nation reaction, but not enought to result in autoagglutination. The autoagglutination concentration for a particular antibody-coated particle can be determined by routine experimentation. Thus, the enhancer at increas-ingly higher concentrations can be added to a solution containing antibody-coated particles the absence of antigen, to determine the autoagglutination point. The concentration of enhancer is then selected so as to increase the sensitivity of the assay, but not result in autoagglutination. Preferred agglutination enhancing non-protein water-soluble polymers are selected from the group consisting of polyethylene glycol (PEG), dextran, or gum acacia.

Preferred agglutination enhancing water-soluble proteins are gelatin and albumin. Gelatin is a mixture of proteins obtained by hydrolysis of collagen by boiling skin, ligaments, tendons, etc. Gelatin is strongly hydrophilic, absorbing up to ten times its weight in water. Albumin is any one of a group of water-soluble proteins of wide occurrence in such natural products as milk (lactalbumin) , blood serum, and eggs (ovalbumin) .

The combination of a non-protein water-soluble polymer enhancer and a water soluble protein enhancer permits a very sensitive agglutination test. Surpris-ingly, at the enhancer concentrations set forth above for the aqueous diluent, autoagglutination is avoided while providing sufficient enhancement to permit detection of antigens at clinical concentrations. Thus, the need for an antigen concentration step is avoided or minimized. In agglutination tests with lysates made in a non- protein water-soluble polymer and a protein water soluble polymer diluent solution, DNA from the lysates can act as a further agglutination enhancer. Agglutination enhancement due to DNA can be optimized by adding a chelating agent to the lysate prior to testing. An example of an acceptable chelating agent is EDTA, which is known to chelate ions necessary for the action of deoxyribonucleases (DNases) . Thus, EDTA in the lysate can preserve DNA enhancement of the agglutination test by neutralizing the action of DNases. The enhancing effect of DNA can be eliminated by adding and mixing DNase to a solution on an agglutination slide. About five microliters of a 2000 mg/ml solution of DNase per slide is sufficient.

A preferred diluent solution contains from about 1% to 2% by weight of PEG, from about 0.25% to about 1% by weight of gelatin, and from about 0.03% to about 0.40% by weight of EDTA.

PEG having a molecular weight from about 200 to about 15,000 is acceptable for the diluent solution. About 8000 is the preferred molecular weight for the PEG polymer, but PEG 350 and PEG 15,000 can be substituted. PEG having a molecular weight less than 200 may have insufficient viscosity. PEG having a molecular weight more than 15,000 can be more difficult to work with due to excessive viscosity. Dextran T40 and T70 (e.g. , Sigma Chemical Co. , St. Louis, MO) , at about the same concentration as the PEG, or at a concentra¬ tion up to about 3% of the diluent solution, can substitute for PEG as the water-soluble polymer. Although gelatin is the preferred water-soluble protein, albumin at about 0.5% concentration can be substituted for gelatin.

The particles used in the practice of the invention may be any particles capable of agglutination in a detectable manner when coated with the appropriate antibodies specific for the selected target antigen. The particles may be biological or chemical. Synthetic particles typically in a size range of from about O.lμ to about 15μ have been used as agglutination reagents; however, larger and smaller particles may be used. A preferred size particle for the practice of the present invention is from about 0.15μ to about l.Oμ, more pref-erably about 0.2μ. Examples of such particles include red blood cells, non-blood cells with surface antibodies, glass beads, liposomes, pollen spores, metal oxide particles, latex particles, and carbohy¬ drate particles, e.g.. dextran, agarose, or cellulose beads. The pre-ferred particles are latex particles.

The term latex is art recognized and typically refers to particles made of natural or synthetic rubber or plastic. Latex particles are commercially available and are prepared by addition polymerization processes in aqueous media. Monomers used in preparing latex include acrolein, acrylate, methyl acrylate, methacrylate, methyl methacrylate, glycidyl methacrylate, styrene, vinyl toluene, t.-butyl styrene and mixtures of these monomers. The polymers and copolymers optionally may contain cross-linking agents such as divinyl benzene and butadiene .

The particles may also be colored, thereby enhancing the ease of visually detecting agglutination. The color may be selected to provide a contrast between the particles and the background color of a slide, for example. Preferred particles are red styrene latex beads having an average diameter of about 0.2μ. They can be obtained from Rhone Poulenc, France. Such beads provide a desirable contrast against an opaque slide that is white.

The particles are coated with antibodies specific for the selected target antigen, that is, an antigen characteristic of the target cell or organism being detected. The surface of a particle may be coated using known methods capable of directly or indirectly attaching antibodies. The antibodies may be absorbed directly on the surface of the particle or attached to the particle through a spacer molecule, e.g., a molecule capable of bonding to both the surface of the particle and to the antibody. Preferably, the antibodies are directly attached or absorbed to the particle using pas-sive coating techniques well-known to those of ordinary skill in the art. Such coating techniques tend to pre-serve the specificity and activity of the immunological reagent.

The term antibody is intended to include whole polyclonal or monoclonal antibodies, antibody fragments such as Fab fragments, chimeric antibodies containing portions from two different species, and synthetic peptides identical to or functionally analogous to the antibody. The preferred form of antibody is whole, monoclonal antibody. It should be understood that more than one species of monoclonal antibody may be attached to a particle. Examples of monoclonal antibody-producing cell lines include hybridoma cell lines, myeloma cell lines, or viral or ontogenically transformed lymphoid cells. Hybridoma cells which can produce the specific antibodies for use with the present invention may be made by the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256 495 (1975) or similar procedures employing different fusing agents.

The agglutination conditions according to the invention are described generally as follows.

The conditions and reagents are preferred which permit visually detectable agglutination to occur. The naked eye can detect an agglutination clump when it is about 50μ in size. Thus, visually detectable aggluti- nation requires approximately 100 antibody-coated par- tides, with each particle being about 0.2μ in diameter, to be attached together by antigen-antibody bonds. Two anti-body-coated particles which are about 0.2μ in size will not adequately clump when aggitated on an agglutination slide unless about 10 bonds are formed between them. The bonds are a result of antigen linking the antibodies of the two particles. The conditions and reagents are preferably selected such that agglutination is detectable at levels of about 10,000 bacteria/ml of biological fluid, and preferably at levels of about 100 bacteria/ml of biological fluid, without requiring concen- tration of the antigen solution after cell lysis of the bacteria .

Preferably, antibody-coated particles are cap-able of autoagglutinating in an excess of agglutination enhancer such that they are detectable with the naked eye. Thus, the antibody specific for the target antigen is preferably present on the surface of the particle at a density sufficient to cause this autoagglutination.

The slight excess of agglutination enhancer is a preselected concentration of enhancer which provides an autoagglutination standard for particles.

For example, an acceptable autoagglutination standard is the amount of enhancer capable of autoag¬ glutinating a reagent solution of monoclonal antibody-coated gonococcal reagent Wl (Karo-Bio, Sweden; "Phadebact Gonococcal Reagent") , which has been diluted 1:5 by a buffer solution. This is a slight excess beyond the amount of enhancer to be used in the actual agglutination test. Preferably, only par¬ ticles capable of autoagglutinating at this slight excess are used in the invention process. The target cell samples are processed for the agglutination assay via the procedure generally described below. The general procedures include progressive 1/2 cell- concentration dilution steps for cell culture su- spensions which are usually not necessary for clinical samples. Cells are transferred to a tube containing PBS until a cell suspension solution having a density of about a McFarland number one (Manual of Clin. Microbiol., Lennette ed. , Amer. Soc. Microbiol., (1980)) is obtained, as judged by eye. A two-fold dilution of the cell su- spension is made. For example, 0.5 ml of the susi - ≤ion is added to 0.5 ml of PBS and the resulting 1 ml sol. .on is thoroughly mixed to produce a 1:2 dilution solution The 1:2 dilution solution has 1/2 the concentration of cells per volume as compared to the 1 McFarland cell suspension solution. This 1:2 dilution solution can be further progressively diluted two-fold in a similar manner with PBS to produce a series of two-fold dilution solutions, that have cell concentrations of 1/4, l/B, 1/16, etc., as compared to the original 1 McFarland cell suspension solution. This dilution procedure allows one to determine the minimum concentration of cells in solution which, when lysed, will result in a positive agglutination test result .

After forming the progressive dilution solutions, the cells of each solution are lysed with a strong base to produce a lysed cell solution. About 50 μl of a strong base such as IN NaOH solution per 0.5 ml of cellular solution is usually adequate to rapidly lyse the cells. However, the concentration of strong base may be varied for a particular cell type. After lysing the cells the pH of the lysed cell solution should be returned to neutrality. The neutralization process is best controlled by monitoring the pH. An electronic pH meter can be used to determine the neutrality point or a pH indicator can be used to visually indicate it. About 0.005 % by weight of a pH indicator such as phenol red can be added to sample prior to adding a pH neutralizing acid. The phenol red pH indicator provides a color change which readily indicates the neutrality point and avoids the need for an expensive pH meter. The amount of strong acid such as IN HC1 needed to neutralize the alkaline extract can then be judged by the color change. Usually the amount of strong acid required is less than 50 μl per 0.5 ml of cellular solution, the e,act amount in any particular situation can be determined by adding acid to the negative control, which is void of bacteria cells.

For the agglutination reaction, about 50 μl of lysate and about 25 μl of the reagent, which is antibody- coated particles, can be put in the circle of a glass aggluti¬ nation slide, and mixed with an applicator stick. The slide can be rotated by a mechanical slide rotator or by hand, both of which are standard methods of an agglutination test.

Typically the lysate with the greatest concen¬ tration of cells, such as 1:2 and 1:4, agglutinate in a minute or so. Greater dilutions, such as 1:32 or 1:64, may take about 10 to 20 minutes to develop into a visible aggluti¬ nation.

The agglutination slide may be rotated by hand or mechanically rotated. Weak agglutination reactions become stronger if the slide is finally hand-rotated for a minute or two at the conclusion of the mechanical rotation. Usually, the maximum dilution of the 1 McFarland starting cellular sample which will result in visibly detectable agglutination is 1:128. The agglutination of the maximum dilution is referred to as the "end point agglutination" limit for a particular agglutination process and cell type. Thus, with the present method the end point agglutination of most cell lysates in PEG-gelatin-EDTA is 1:128 as seen by the naked eye, and two to four-fold higher (i.e.. 1:256 and 1:512, respect- ively) when viewed under a microscope at low magnification, such as 10X. Minor variations in end points can be expected with different samples of the same type of cell due to dif¬ ferences in the concentration of cells in suspension and the age of the cells. Surprisingly, the method according to invention which utilizes a three-component diluent (non-protein water- soluble polymer:water-solubleproteinpolymer:chelating agent) rather than separate components is more sensitive. Irrespec¬ tive of the method of reading the test, the PBS buffered PEG- gelatin-EDTA diluent results in an end point which is about four-fold more sensitive than the end points with lysates made in the separate components: PEG in PBS, gelatin in PBS and EDTA in PBS.

Further, the PEG-gelatin-EDTA diluent consistently gives greater sensitivity than any diluent containing two components, i.e., PEG-gelatin in PBS, PEG-EDTA in PBS and gelatin-EDTA in PBS.

The sensitivity of the method according to the invention is such that a single colony of bacteria can be identified. Using a wood applicator stick a colony can be touched and transferred to about 0.5 ml of the PEG-gelatin- EDTA mixture and lysed according to the method of this invention. When the lysate is mixed with the specific monoclonal antibody-particles in an agglutination test, a positive test result is obtained.

The following non-limited examples are provided to illustrate the invention.

Examples

EXAMPLE 1 : Agglutination of Cultured N. Gonorrhoeae

A. Preparing the Aqueous Diluent A 2X stock aqueous diluent of polyethylene glycol, gelatin and EDTA in phosphate buffered saline are prepared as follows. The 2X stock is diluted in half with phosphate buffered saline for use in the aggluti-nation test.

Phosphate buffered saline (PBS) was made by adding one part of one molar (1M) sodium phosphate buffer (pH 7) to thirty-nine parts of 0.85% (w/v) saline. Gel-atin was prepared as a 10% solution (w/v) in distilled water, and autoclaved. The gelatin was liquified by heating in a warm water bath. To 60 ml of PBS, was added 0.6 grams of EDTA and 0.8 ml of IN NaOH. Heating in a warm water bath speeded up the incorporation of EDTA in the liquid. Eight ml of the 10% gelatin was then added, followed by 3.2 grams of PEG 8000 (Sigma Chemical Co., St. Louis, MO) . The volume of the solution was adjusted to 80 ml by adding PBS.

B. Monoclonal Antibody-coated Particles

Monoclonal antibody-coated staphylococcus reagent

Wl was obtained from Karo-Bio, Sweden, distributed as

"Phadebact Gonococcal Reagent." An agglutination reagent solution was formed by diluting the Wl reagent 1:5 in PBS.

C. Preparation of Lvsate

N. gonorrhoeae of serogroup Wl, National Reference Laboratory, U.S.A., strain 32779, was grown on solid media using standard culture techniques. Using a cotton tipped applicator stick, colonies were picked and transferred to a dilution tube containing PBS, until a density of about one McFarland (Manual of Clin. Microbiol., Lennette ed., Amer. Soc. Microbiol., (1980)) was obtained, as judged by eye . This 1 McFarland cell suspension solution was designated as a 1:1 cell suspension solution (see Table 1, below) . A two-fold dilution (1:2 McFarland dilution solution; solution with 1/2 the cell concentration) of the 1 McFarland suspension was made by mixing 0.5 ml of the suspension and 0.5 ml of PBS. Then 0.5 ml of the 1:2 cell suspension solution was diluted with 0.5 ml of PBS to produce a further two-fold dilution solution having 1/4 the cell concentration of the 1 McFarland cell sus¬ pension solution. This cell suspension solution was designated as a 1:4 dilution solution. The two-fold dilution process with PBS was continued until a series of serially diluted cell suspension solutions (1:1, 1:2 ... 1:256, 1:512) was produced. A negative control dilution was made by adding 0.5 ml of PBS to an empty tube. To each 0.5 ml sample, and to the negative control, 50 μl of IN NaOH containing 0.005

% phenol red was added to produce an alkaline solution. The alkaline solution was mixed thoroughly to allow lysing of the N. gonorrhoeae cells to occur and produce a lysate solution. Then about 50 μl of IN HC1 was gradually added to until the alkaline lysate solution was neutralized to produce a neutral lysate solution. The neutralization point was judged by the color change of the phenol red pH indicator from alkaline to the neutral range.

D. Agglutination Reaction Procedure

To perform the agglutination reaction, 50 μl of each of the above N. gonorrhoea lysate dilution solutions and 25 μl of the above agglutination reagent solution of B were placed in the circle of a glass ag-glutination slide, and mixed with an applicator stick. The slide was rotated mechanically on a slide rotator. The procedure was then repeated with rotation by hand. E. Agglutination Test Results The results were the essentially the same for the mechanically rotated and hand rotated slides, both of which are standard methods used in agglutination tests. The slides were evaluated for the presence of agglutination both by eye and under a 10X power micro-scope. The results are set forth below in Table 1.

Table 1 N. Gonorrhoeae Culture Agglutination Results

Cell Suspension Agglutination Agglutination Concentration Visible by Eye Visible at 10X

1:1 McFarland + +

1:2 + +

1:4 +

1:8 + +

1:16 + +

1:32 + +

1:64 + +

1:128 + +

1:256 +

1:512 +/-

0 (Control) visible agglutination no visible agglutination

The lysate from the cell solutions with the greatest concentration of N. gonorrhoeae cells, that is, from the 1:1, 1:2, and 1:4 cell solutions, agglutinate in a minute or so. The greater dilutions, 1:32 and 1:64, take 10 to 20 minutes to develop into a visible agglutination.

When a mechanical rotator was used, weak agglu¬ tination reactions become stronger, if the slide was finally hand-rotated for a minute or two. The end point agglutination of the N. gonorrhoeae lysate in PEG-gelatin-EDTA was 1:128 as seen by the naked eye, and about two to four-fold higher as seen under a 10X microscope. EXAMPLE 2 : Agglutination of -rnorrheal Clinical Sample

A. Collecting the Clinical Sample Applicability of the invention to clinical material was demonstrated with five urethral swabs of pus taken from men who had been diagnosed as having gon-orrhea. Each of the swabs was placed into about 0.6 ml of the PEG-gelatin-EDTA solution (aqueous diluent of Example 1A) and maintained at room temperature for about an hour.

B . Lysing the Sample

Each swab having the sample was swirled in the 0.6 ml PEG-gelatin-EDTA solution, and slowly withdrawn while pressing the swab against the wall of the tube to release the clinical material. Sixty μl of IN NaOH with 0.005% phenol red was added to the clinical material in the 0.6 ml solution, followed by an amount of IN HC1 sufficient to neutralize the alkaline lysate, as judged by the change in color of the phenol red indicator. The volume of IN HC1 needed to achieve neutrality was about 57 μl .

C. Agglutination Reaction Procedure

Fifty μl of the neutralized extract and 5 μl of the agglutination reagent of Example IB (containing Wl antibody-coated particles) , were mixed on a slide for the agglutination test. For a positive control, a 1:2 McFarland dilution of N. gonorrhoeae suspension in PEG-gelatin-EDTA

(lysate produced for this control as in Example 1C) was used.

As a negative control of the diluent, PEG-gelatin-EDTA was treated with IN NaOH, phenol red, and IN HC1 as in Example ID. Fifty μl of the diluent negative control and 5 μl of the agglutination reagent of Example IB were mixed on a slide for the agglutination test. As an antibody reagent negative con¬ trol, plain particles diluted 1 :5 as in Example IB were placed on a slide for the agglutination test.

D. Agglutination Test Results

Within one minute, all five urethral specimens were positive by the agglutination test . The positive control was positive within one minute. The diluent negative control and the antibody reagent negative control were still negative after one-half hour. All references cited with respect to synthetic, preparative and analytical procedures are incorporated herein by reference.

The present invention may be embodied in other spe¬ cific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Claims

1. A method for detecting the presence of a selected target antigen expressed by a target cell capable of undergoing rapid lysis comprising the following steps:

(a) obtaining a biological sample to be tested for the presence of the target cell expressing the selected target antigen; (b) mixing said sample with an aqueous diluent to form a diluted sample, the aqueous diluent comprising: (i) from about 1% to about 3% by weight of a non-protein water soluble polymer, based upon the weight of the aqueous diluent; and

(ii) from about 0.2% to about 2% by weight of a water soluble protein based upon the weight of the aqueous diluent;

(c) contacting said diluted sample with an amount of a strong base which is effective to chemically lyse said target cell to form an antigen solution containing antigens from the target cell;

(d) neutralizing said antigen solution with a strong acid to form a neutralized solution; (e) contacting the neutralized solution with particles coated with an antibody specific for said selected target antigen under conditions permitting agglutination visible to the naked eye or under a microscope; and

(f) detecting agglutination of the particles as an indication of the presence of said target antigen in the biological sample.

2. A method according to claim 1, wherein said aqueous diluent comprises a buffered solution containing: (i) from about 1% to about 3% by weight of polyethylene glycol or dextran, based upon the weight of said aqueous diluent; (ii) from about 0.2% to about 2% by weight of gelatin or albumin based upon the weight cf said aqueous diluent; and

(iii) from about 0.01% to about 1.0% by weight of a chelating agent based upon the weight of said aqueous diluent.

3. A method according to claim 2, wherein said buffered solution is a buffered saline solution.

4. A method according to claim 3, wherein said aqueous diluent comprises:

(i) from about 1% to about 3% by weight of polyethylene glycol ; (ii) from about 0.2% to about 2% by weight of gelatin; and (iii) from about 0.03% to about 0.40% of ethylenediaminetetraacetic acid.

5. A method according to claim 4, wherein said aqueous diluent comprises:

(i) from about 1% to about 2% by weight of polyethylene glycol; (ii) from about 0.25% to about 1% by weight of gelatin; and (iii) from about 0.03% to about 0.40% of ethylenediaminetetraacetic acid.

6. A method according to claim 4, wherein said agglutination is visible to the naked eye.

7. A method according to claim 1, wherein the amount of strong base is from about 0.1% to about 4.0% by weight based upon the weight of said aqueous diluent.

8. A method according to claim l, wherein the amount of strong base is from about 0.2% to about 0.5% by weight based upon the weight of said aqueous diluent.

9. A method according to claim 8, wherein said aqueous diluent comprises:

(i) from about 1% to about 2% by weight of polyethylene glycol or dextran based upoπέt weight of said aqueous diluent;

(ii) from about 0.25% to about 1% by weight of gelatin or albumin based upon the weight of said aqueous diluent; (iii) from about 0.03% to about 0.40% by weight of a chelating agent based upon the weight of said aqueous diluent.

10. A method according to claim 1, wherein said strong base is NaOH.

11. A method according to claim 1, wherein wherein said strong base is from about 0.2% to about 4.0% by weight of NaOH.

12. A method according to claim 1, wherein said target cell is a member of the genus Neisseria.

13. A method according to claim 12, wherein said cell is of the species Neisseria gonorrhoeae.

14. A method according to claim 1, wherein said target cell is a human CD4+ cell.

15. A method according to claim 1, wherein said strong acid is HCl .

16. A method according to claim 1, comprising the additional step of adding a pH indicator to the lysed sample to indicate solution neutrality upon addition of sufficient strong acid.

17. A method according to claim 16, wherein said indicator is phenol red.

18. A method according to claim 1, wherein said antigen is a membrane-bound antigen.

19. A method according to claim 1, wherein said process can be completed in about fifteen minutes or less.

20. An aqueous cell lysis reagent composition comprising:

(i) from about 1% to about 3% by weight of of a non-protein water soluble polymer, lsl upon the total composition weight;

(ii) from about 0.2% to about 2% by weight of a water soluble protein, based upon the total composition weight; (iii) from about 0.01% to about 1.0% by weight of a chelating agent, based upon the total composition weight; and (iv) from about 0.1% to about 4.0% by weight of a strong base, based upon the total composition weight.

21. A composition according to claim 20, which comprises a buffered solution containing:

(i) from about 1% to about 3% by weight of polyethylene glycol or dextran, based upon the weight of said composition; (ii) from about 0.2% to about 2% by weight of gelatin or albumin, based upon the weight of said composition; (iii) from about 0.01% to about 1.0% by weight of a chelating agent, based upon the weight of said composition; and (iv) from about 0.1% to about 4.0% by weight of a strong base, based upon the weight of said composition.

22. A composition according to claim 21, wherein said aqueous reagent composition is a buffered saline solution.

23. A composition according to claim 22, wherein said buffered solution is a phosphate buffered saline solution.

24. A composition according to claim 23, wherein said composition comprises:

(i) from about 1% to about 3% by weight of polyethylene glycol; (ii) from about 0.2% to about 2% of gelatin; and

(iii) from about 0.03% to about 0.40% of ethylenediaminetetraacetic acid.

25. A composition according to claim 24, wherein said strong base is NaOH.

26. A composition according to claim 24, wherein said composition comprises:

(i) from about 1% to about 2% by weight of polyethylene glycol;

(ii) from about 0.25% to about 1% of gelatin; and (iii) from about 0.03% to about 0.40% of ethylenediaminetetraacetic acid.

27. A composition according to claim 26, wherein said strong base is from about 0.2% to about 0.5% by weight of NaOH.

28. A composition according to claim 25, further comprising a pH indicator to indicate pH neutrality.

29. A composition according to claim 28, wherein said indicator is phenol red.