DE2919334A1 - Reverse passive haemagglutination test for Neisseria gonorrhoeae - by incubation of cell lysate with particles sensitised with antibodies to Neisseria gonorrhoeae strains - Google Patents

Abstract

Microorganisms are identified as N. gonorrhoeae by incubating a lysate of the microorganism with a suspension of discrete particles sensitised with antibodies to N. gonorrhoeae strains ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407. Agglutination indicates positive identification. The reagent comprising the above sensitised particles in also claimed. Test gives rapid and reproducible identification of any known N. gonorrhoeae strains and is not subject to false +ve reactions by usual interfering organisms.

Description

Method for the detection of Neisseria gonorrhoeae

The invention relates generally to methods and materials for the Detection of N. gonorrhoeae in a microbial growth culture. In more specific Regarding the invention, it relates to a rapid and reproducible reverse passive Hemagglutination (RPHA) test for each of the known strains of the microorganism.

The known methods for identifying N.gonorrhoeae in one Culture medium, e.g. Thayer-Martin agar, include a preliminary analysis based on origin and morphology, of the grief and of an oxidase test. Oxidase positive gram negative Diplococci that were found on Thayer-Martin medium inoculated from a patient's urogenital tract showing a typical colony morphology are initially considered to be gonococci. The microorganism is usually examined to confirm this preliminary analysis result with the help of additional tests specific to the detection of N.gonorrhoeae in contrast to other microorganisms showing similar behavior are. These specific tests include the carbohydrate breakdown analysis for their Complete implementation takes around 48 hours and is sometimes unreliable is because some strains of N.gonorrhoeae either fail to grow or can result in false negative sugar reactions. An alternative common test is based on the use of fluorescent antibodies; during this test relatively is rapidly feasible and specific for all N.gonorrhoeae strains, it requires a relatively expensive one to be operated by a highly qualified professional Fluorescence microscope Evgl. e.g. Health Laboratory Science (1975), Volume 12, No. 3, Pages 215 to 2183.

Although it is known to be used to confirm identification the RPHA test applied to the microorganism faster and more reproducibly than e.g. the carbohydrate breakdown analysis is assumed to be the former test for a selective analysis of N.gonorrhoeae due to the often acute specificity of Antibodies against the various strains of N.gonorrhoeae is unsuitable. For example, it was generally assumed that a large number of analyzes in which one a similar number of specific antibodies is used for the unambiguous characterization of a microorganism as one of the many strains of N.gonorrhoeae is necessary.

There was therefore a need for a rapid, reproducible identification method for N.gonorrhoeae which do not have the cumbersome or costly measures that requires conventional procedures.

The invention creates new methods and materials for a reverse passive hemagglutination test, which is used for the detection of N.gonorrhoeae a microbial growth on Thayer-Martin agar medium or other comparable Media is suitable.

According to the invention, antibodies which are produced by means of eleven strains of N.gonorrhoeae, i.e. ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407, obtained as immunogens, are used to sensitize immunologically inert ones Particles (e.g. stabilized erythrocytes) are used and in an RPHA test for effectively detecting the presence of any one or more of the known N.gonorrhoeae strains used. The test according to the invention does not suffer from false ones positive reactions due to microorganisms that make the antibody-oriented Otherwise test for N.gonorrhoeae usually interferes.

In short, antibodies are raised against any of the aforementioned strains combined and used to sensitize discrete carrier particles. The reagent particles are in turn in a hemagglutination test with a lysate of the suspects cultured cells to detect the presence or absence of N. gonorrhoeae used. The entire system requires minimal equipment and delivers Exact results within 3 hours.

There are numerous aspects out of the detailed description below and advantages of the invention are apparent.

The eleven N.gonorrhoeae strains used according to the invention are subject to each of the following taxonomic description: Order: Eubacteriales Family: Neissericeae Genus: Neisseria Species: Gonorrhoeae Morphology: Gram-negative, globular or bean-shaped diplococci with flattened opposite sides, usually 0.6 x 1.0 pein and more uniform size.

Biochemical and Plant Characteristics: Aerobic, requires optimal growth 4 to 10% CO2 and incubation at 360C.

The cultures grow slowly on Thayer-Martin medium and produce after 24 hours small, barely visible colonies (0.1 mm diameter), while one typical morphology is recognizable in 48 to 72 hour old cultures. The colonies are small (1 mm in diameter), gray-white, transparent, smooth, with a round whole Edge, shiny surface and buttery consistency.

Oxidase +, catalase +, ferments glucose but not maltose, lactose or sucrose (sucrose).

Virulence: All 11 strains were originally derived from patients with symptomatic Gonorrhea isolated.

According to the invention, each of the eleven strains is used as an immunogen to be all Immunization of a test animal used. Then you combine the immune sera, which of the different animals immunized each with a single strain can be obtained by means of a serum combination containing antibodies against all strains to dispose of. This bulk sample of antisera is passed through an immunoadsorbent column which cell walls or extracts used to form the immune sera Contains strains. The antibodies bound to the cell walls are then tested on chemical Paths eluted. Preferably, the antibodies are additionally made with selected strains absorbed by N. meningitidis to eliminate antibodies directed against other Neisseria species than N. gonnorrhoeae are reactive. Optionally, each individual Antiserum over a cell wall or extracts of the particular, used as an immunogen in the Column containing development of the antiserum used, eluted, absorbed against the meningitid nerve and then collected or pooled.

The collected antibodies thus obtained are used for coating or Sensitization of discrete particles made of immunologically inert materials such as stabilized human erythrocytes of type 0 are used. Suspected as N. gonnorrhoeae Microorganisms are taken from their culture medium, and it a suspension of the organisms in water up to a standard turbidity (measured on the optical density) and then for the development of a lysate test material treated. This comes into contact with the antibody-sensitized particles brought; an agglutination reaction confirms the presence of N. gonnorrhoeae.

The following examples relate to aspects of the invention Procedure: a) Isolation of N. gonnorrhoeae cell walls; b) Making an N .gonnorrhoeae cell wall phenolic extract; c) production of inactivated meningitidis nerve; d) production of stabilized erythrocyte carrier particles; e) Manufacture of carrier particles sensitized with N.gonorrhoeae phenol extract; f) Manufacturing an immunoadsorbent column; g) production of N.gonorrhoeae antiserum; h) isolation from N.gonorrhoeae antibodies; i) Absorption of the N.gonorrhoeae antibody preparation with inactivated meningitid nerve; j) Production of antibodies with N. Gonorrhoeae sensitized reagent carrier particles; k) preparation of a bacterial lysate; 1) RPHA test for N.gonorrhoeae; and m) control test of the specificity of the reagent.

The N.gonorrhoeae strains ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406, and 31407 have been assigned by the assignee to American Type Culture Collection, 12301 Parklawn, Rockville, Maryland 20852, and are available there.

Example 1 Isolation of N. gonorrhoeae Cell Walls The following method described is for the preparation of a cell wall preparation for each of the eleven N.gonorrhoeae strains ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407 applied. The strain is based on Thayer-Martin medium cultured and then from the agar by washing with 0.01 M phosphate buffered normal saline pH 7.2 (PBS) removed. The cells are through 40 minutes long centrifugation (16300xg) at 40C and once with normal saline washed. A 20% (w / v) suspension of the cells in distilled is then produced Water. The cells are then placed in a homogenizer cooled with liquid carbon dioxide broken up with glass beads (about 150 to 200 µm). Then the cell walls are removed from the Glass beads separated with the help of a sintered glass funnel and again in distilled Suspended in water. After centrifugation (9750xg) for 18 hours

at −20 ° C. the pellet obtained can be stored at 40 ° C.

B e i s p i e l 2 Manufacture of N. gonorrhoeae cell wall phenol extract Equal amounts by weight of wet-packed cell walls produced according to Example 1 of the eleven strains are combined and a 20% (w / v) suspension is made prepared in distilled water. The suspension is an equal volume of 90% aqueous phenol and 0.1 M sodium acetate buffer (pH 5.0) containing of 0.5% naphthalenedisulphonic acid and 0.5% sodium dodecyl sulphate for 20 min. at 700C extracted. Then the phases are separated and centrifuged for 30 minutes gained at 175 g. The phenolic phase is 3 days at 4 ° C against common exchanged distilled water dialyzed The extract is then through 30 minutes Long centrifugation (16300xg) at 40C cleared through a 0.45µm membrane filter filtered and stored at 40C.

Example 3 Production of inactivated N.raeningitidis N.mengingitidis, Group A, strain MK01A (Naval Medical Research Unit No.4, Great Lakes, Illinois); Group B, strain 3951 (King County Department of Health, Seattle, Washington) and Group B, strain 5066-335 (Naval Medical Research Unit No.4, Great Szkes, Illinois) are in each case in a rotary shaker for 18 hours at 37 ° C. on a Mueller-Hinton nutrient broth cultivar. The microorganisms are separated for 30 min.

Pelleted at 40C and 16,300 x g and centrifuged three times washed with equal volumes of PBS. Then the cells down to a fifth the volume of the original broth (generally 1 liter) is resuspended. The suspension is placed in a Pyrex dish, which is enclosed in a plastic bag, which has a has a cotton stopper closed opening, is sealed. The cells are inactivated with ethylene oxide and stored at 4 0C (the failure of growth there is an indication of inactivation on Mueller-Hinton agar).

Example 4 Preparation of stabilized erythrocyte carrier particles Human erythrocytes are determined according to the method of Hirata et al., Journal of Immunology, Volume 100, No. 3 (1968), pages 641 to 646, U.S. Patents 3,714,345, 3,715,427 and / or 3 925 541 stabilized. The procedure is briefly summarized performed in the following manner. A suspension of potentially fused erythrocytes is in a neutral buffer solution at a concentration of about 10 vol .-% and with a small amount in relation to the volume of the erythrocytes (preferably 1.5 to 5 VolO-nÕ) pyruvinaldehyde during a stabilization of the erythrocytes for sufficient time (preferably 12 to 24 hours). The erythrocytes are then used to remove the excess pyruvinaldehyde and any other materials that may be derived from the serum several times washed. The erythrocytes treated with pyruvinaldehyde are then treated with a Small amount in relation to the erythrocyte volume (preferably 1.5 to 5% by volume) Treated formaldehyde for 12 to 24 hours, after which the excess formaldehyde removed by washing with a buffered solution.

Example 5 Preparation of Sensitized with N.gonorrhoeae Phenol Extract Carrier particles Stabilized erythrocytes are produced according to Example 4 and at 10% (w / v) concentration in 0.11 M phosphate buffer of pH 7.2 (PB) stored. The particles are then coated, for example by Mix 2 ml of the phenol extract from Example 3, 20 ml of 0.1 M sodium acetate buffer (pH 4.0), 1 ml of aqueous chromium (III) chloride (10 mg CrCl3.6 H20 / ml) and the pellet, by centrifuging 2 ml of the aforementioned 10% gene suspension for 3 1/2 minutes was formed by stabilized erythrocytes at 700 x g.

After mixing, the suspension is incubated at 37 ° C. for 30 minutes, it is moved shortly after 15 minutes so that the erythrocytes as little as possible dismount. The sensitized Cells are then done four times Centrifugation for 2 minutes (700 x g) at 25 ° C with ten times the erythrocyte volume washed on PB. Thereafter, the cells are reduced to 1% (w / v) in PB with a content resuspended from 0.2 9s sucrose and 10% bovine serum albumin and stored at -30 ° C.

Example 6 Preparation of Immunoadsorbent Column The following procedure can be used to produce eleven columns, each consisting of one of eleven different, corresponding to the eleven strains of N.gonorrhoeae used Gels are made up (a similar technique can be used to make cell walls of all eleven strains in a single gel to produce a single, large one To include column). 8 ml each of the eleven 20% genes produced according to Example 1 (W / v) cell wall suspensions become 20 ml of a gel of 10% total monomer (Acrylamide and N, N'-methylene-bis-acrylamide (BIS), of which 25% are BIS) together with 40 / ul N, N, N, N-tetramethylethylenediamine and 2 ml of a freshly prepared 40% gene given aqueous ammonium persulfate solution. Each gel is about 30 min. At room temperature left to polymerize and then broken into small pieces by pushing it through an 18 gauge needle attached to a 50 ml syringe. The particles are then mixed with 100 ml of 0.1 M tris (hydroxymethyl) aminoethane hydrochloride buffer (pH 7.5) with a content of 0.15 M NaCl (THS), then with 100 ml 0.1 M glycine hydrochloride buffer (pH 2.3) with a content of 0.15 M NaCl (GHS) and finally with 100 ml THS washed. 2 g of Bio-Gel P-100 (Bio-Rad Laboratories, Richmond, California) are placed in a 2 x 36 AN chromatography column, after which the immunoadsorbent particles to be added. The whole column is then washed again as above with THS, GHS and THS.

Example 7 Preparation of N. gonorrhoeae Antiserum The Colony Types 3 and 4 of the eleven N.gonorrhoeae strains are placed in a candle vessel for 18 hours at 370C cultured on Thayer-Martin agar, and each strain is cultured in that described below Way used to produce an antiserum. The colony becomes from the agar with 0.01 M phosphate buffered saline (PBS) from pH 7.2 removed, through three times 15 minutes centrifugation (3000 x g) washed at 40C and resuspended in PBS. The degree of turbidity is used to provide two concentrations (U1 X "and" 10 Xur) set with corresponding optical densities of 0.52 and 5.2 at 630 nm, and the suspensions obtained are stored at -2O0C.

New Zealand white rabbits are tested in several places with 10 ml of a homogenized inoculum, which has equal volumes of thawed Contains 10X bacterial suspension and Freund's complete adjuvant.

The injected is approximately 7, 10 and 15 days after the first inoculation Rabbits 0.5 ml, 1 ml or 2 ml of thawed 1X bacterin. After that, at intervals made two additional 2 ml injections of the 1X bacterin every approximately 5 days. 4 to 6 days later, entire blood samples are taken and (see below) on the Effectiveness of antibodies tested, using particles according to the example 5 sensitized with a phenol extract from cell walls of the inoculated strain became. The animals that provide samples with the desired efficacy the blood is drawn and the resulting antiserum is collected and saved. Those Animals, their samples have a lower than the desired effectiveness show, are again twice with an interval of 5 days with 2 ml of the 1 X Bacterin inoculated, after which another blood sample is tested for antibody action. When the desired effectiveness is achieved, the animal is blood drawn and kept the serum.

The effectiveness of the antiserum is determined by passive hemagglutination using the phenol extract sensitized particles from Example 5 certainly. A diluent for the antiserum is prepared by adding 0.1 , 0 add gelatine to 0.11 m phosphate buffer pH 7.2 (PB) until the Gelatin heated to 600C and filtered through a 0.45 µm membrane filter. 251um the 0.25% gen (w / v) erythrocyte suspension prepared according to Example 5 in PB / sucrose / albumin become 25 to be obtained from serial two-fold dilutions of the Antiserum placed in plastic microtiter dishes with a V-bottom. The filled bowls are shaken for 10 seconds with the help of a vortex mixer, covered and over Left to stand overnight at room temperature. The endpoint titer of the sample is said to be reciprocal Denotes the value of the highest dilution which shows an aggutination scheme that obviously different from that of the control sample (25 µl of the 0.25% gene erythrocyte suspension and 25 / ul of the diluent) is different.

Table I shows the reciprocal titers for each antiserum of the eleven strains, which are believed to indicate appropriate antibody activity.

TABLE I ATCC strain titer 31397 6400 31398 1600 31399 1600 31400 800 31401 800 31402 1600 31403 1600 31404 12800 31405 12800 31406 1600 31407 12800 Example 8 Isolation of N.gonorrhoeae Antibodies The following method for Isolation of the individual antibodies from each of the eleven produced according to Example 7 Antisera can be used to isolate a "pool" of eleven antibodies by pooling the eleven antisera first, then a single column of immunoadsorbent used, which contains a gel that the cell walls of all eleven N .gonorrhoeae strains includes.

7 ml of the rabbit antiserum from Example 7 are on a according to Example 6 produced column applied, which cell walls for immunization contains used tribal. The serum is about 2 hours with the help of a peristaltic Pump with a flow rate of about 100 to 150 ml / hour.

circulated through the column. Then the pillar is using THS (pH 7.5) until the absorbance at 280 nm is <0.02. The adsorbed antibodies are then eluted with GHS (pH 2.3). Fractions of 2 to 2.5 ml each are collected and, if necessary, immediately with 0.5 M sodium carbonate buffer (pH 9.5) adjusted to a pH of about 6.9 to 7.1. Consecutive Fractions with an absorbance at 280 nm of> 0.1 are collected, overnight dialyzed against normal saline at 49C, by negative pressure dialysis at Concentrated room temperature, redialysed and stored at 40C.

EXAMPLE 9 Absorption of a N.gonorrhoeae antibody preparation with inactivated meningitidis N. A suspension of inactivated meningitidis N. (6 ml group B, strain 5066-355t 6 ml group A, strain MKO1A; and 0.6 ml group B, Strain 3951) is centrifuged for 1 hour at 40 ° C. and 16,300 × g. 6 ml (1 50 / ug / ml) of antibodies prepared according to Example 8 are then added to the pellet, Slurried and moderately for 1 hour at room temperature with the help of a vortex mixer emotional. After centrifuging for 1 hour (16,300 x g) at 40 ° C., the absorbed Antibodies stored at 40C.

3 e i s p i e 1 10 Production of antibodies sensitized with N.gonorrhoeae Reagent carrier particles Stabilized erythrocyte carrier particles produced according to Example 4 can with the combined eleven antibodies prepared according to Examples 8 and 9 sensitized using the following method. An 18 (by weight) suspension of stabilized erythrocytes in 0.01 M sodium acetate buffer (pH 4) with 1 to 10 μm of the combined antibody preparation per ml of suspension. 50 to 500 / µg CrCl3.6H2O (10 mg / ml previously dissolved in 0.01 M sodium acetate buffer of pH 4.0) are added. The suspension is briefly mixed in a vortex mixer and then at 30 ° C. for 30 minutes incubated, with an intermediate mixing carried out after 15 min. to prevent settling to keep the erythrocytes as low as possible. The suspension is then 0.1 m Washed phosphate buffer, in a 1% suspension of 0.11 M phosphate buffer (pH 7.4) resuspended with a content of 296 sucrose and 10% bovine serum albumin and kept. The respective concentration of the combined antibody preparation and chromium (III) chloride, which has the highest titre against homologous bacterial Lysates and giving the lowest titer against heterologous lysates are selected and referred to as optimal coating conditions for the antibody pool. Sensitized Reagent particle suspensions can be lyophilized for 12 to 18 weeks without loss of activity be kept.

B e i s p i e 1 11 Production of bacterial lysates Lysates of bacteria for the RPHA tests carried out within the scope of the invention are prepared as follows. The microorganisms are incubated for 18 hours on suitable media, after which the colonies removed with a cotton ball and distilled in a 20% glycerin solution Suspended in water. The suspension will be vigorous to break up clumps moved and adjusted to an optical density of 0.52 at 630 nm. (So manufactured Suspensions can be frozen and stored at -300C for up to several weeks). The suspension is distilled in a ratio of 1:10. Diluted water and for example with 30 .mu.m in sodium hydroxide solution per ml of suspension.

The suspension is then briefly agitated to intensify the lysis. The received Preparations are ready to use after 15 minutes and can be used for up to 4 hours. to be used after preparation if kept at 2-27 ° C. It is noteworthy that all the strains of N. gonorrhoeae in general suspensions which clear within about 1 minute of alkaline treatment (i.e. are not cloudy compared to a water-blind sample). An unclear one Suspension can generally be excreted as N.gonorrhoeae.

Ex. 1 12 RPHA test on N. gonorrhoeae A reverse passive Hemagglutination test for suspected Gonorrhoeae is performed as follows. A Gelatine / phosphate powder diluent is produced according to Example 8. Of the Test is performed with a standard microtiter V-plate. Be in the gutters 1 drop of diluent and then 5Xul of that prepared according to Example 11 given alkaline lysate sample. One drop at a time of evenly suspended sensitized carrier particles (0.1% (w / v) prepared according to Example 10 Cell suspension) is in every groove containing the lysate and in some exclusively Gutters containing diluent (cell control) are given. You cover the plate and taps its rim repeatedly to thoroughly mix the reactants. The test mixture is placed on a non-vibrating horizontal at room temperature Incubated area and after 3 and up to 24 hours.

examined. A positive test is indicated by the formation of a disperse Settling or sedimentation image of sensitized carrier particles is displayed, while a negative test due to the formation of a compact button of particles (comparable with the cell control) is displayed.

EXAMPLE 13 Confirmatory test of the specificity of the reagent Die Sensitivity of an RPHA test using the reagents according to the invention by the results of a selection of 79 randomly selected from broadly scattered Gonorrhoeae strains from sources of origin verified.

All strains found in the test with fluorescent antibodies (cf. Health Laboratory Science, Vol. 12, No. 3 (1975), pages 215 to 218) as positive for gonorrheal nerves are also found in the RPHA tests according to the example 12 positive results. Table II illustrates the results of the sensitivity test.

TABLE II Origin of the tested An confirmed as positive N.gonorrhoeae strain number fiuores adorn- RPHA de Antibodies Seattle, Wash. 20 20 20 Milwaukee, Wis. 19 19 19 Chicago, 111. 17 17 17 Lake County, 111 8 8 8 Atlanta, Ga. * 7 7 7 Seattle, Wash. * 2 2 2 Europe 6 6 6 * Penicillinase producers The specificity The earth-based RPHA test for N. gonorrhoeae is based on the results of a Confirmed by tests performed on several non-Gonococcal Neisseria species. All species that are tested with fluorescent antibodies and when tested Carbohydrate breakdown tests do not prove to be N.gonorrhoeae, also result in the RPHA test a negative result Table III shows the results of the specificity test.

Table III Species No Confirmation Tested - Fluorescent Carbon - RPHA te number of ornamental hydrate antibodies N.meningitidis (group A) 1 1 1 1 Nmeningitidis (Group B) 2 2 2 2 N.menirlgitidLis (Group C) 1 1 1 1 N. meningitidis (untyped) 4 3 4 4 Lactamica nerve 5 5 5 4 Sicca nerve 13 13 13 13 Perflava nerve 3 3 3 3 B. catarrhalis 1 1 1 1 A strain is not completely lysed and not subjected to the RPHA test.

## A strain is not subjected to the fluorescent antibody test.

The specificity of the RPHA test according to the invention for N.gonorrhoeae is additionally determined by the results of one of several non-Neiss eri a microorganisms Tests carried out confirm all microorganisms result in the RPHA test a negative result. Table IV shows the results of the specificity analysis.

TABLE IV Microorganism Tested Negative Number RPHA Test Acinetobacter sp. 5 5 Candida sp. 1 1 Flavobacterium sp. 2 2 Bacillus sp. 1 1 Lactobacillus sp. 3 3 Moraxella sp 4 as Corynebacterium sp. 7 enterococci 3 3 Proteus sp. 4 4 Staphylococcus epidermis 6 6 Streptococci (viridans) 2 2 Staphylococcus aureus 12 11 Homophilus influenzae 5 5 Streptococci (group A) 4 4 Salmonella sp. 5 5 Shigella sp. 5 5 One strain is not completely lysed and neither is the RPHA test subject.

From the above detailed description, those skilled in the art numerous modifications and other forms of execution opened up, which also While stabilized human lie within the scope of the invention Erythrocytes represent a particulate carrier preferred according to the invention, For example, numerous other carriers, such as polystyrene latex, bentonite, Charcoal, Cholest @@ in, or lecithin particles can be used. Another variation is, for example, that one can use alkaline bacterial lysates as test material preferred, but also by other chemical or physical treatments (e.g. Treatment with sodium dodecyl sulfate, freeze-thawing or sonication) produced Lysates are to be regarded as equally well suited for the invention.

It can also be assumed that there will be new ones from time to time in the future Strain of N.gorLorrhoeae which do not give lysts, which are reactive with the reagent particles of the invention. It is therefore within of the framework according to the invention, that for the formation of the reagents according to the invention "pool" of eleven antibodies used a further one, as opposed to such a new one Add strain specific antibodies.

End of description

Claims (2)

PATENT CLAIMS 1. Method for identifying a microorganism as N.gonorrhoeae, d a d u r c h e k e n n z e i zu c h -n e t that one a) a Produces lysate of the microorganism, b) a sample of the lysate with a suspension discrete particles with antibodies against the N.gonorrhoeae strains ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407 are brought into contact, c) the mixture of lysate and particles is incubated and d) the presence of agglutination of the incubated mixture, which the positive Identifying the microorganism as N.gonorrhoeae indicates. 2. Reagent for use in a reverse passive hemagglutination test to identify a microorganism as N.gonorrhoeae, containing a suspension of discrete particles, of which surface areas with antibodies opposed the N.gonorrhoeae strains ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407 are sensitized.