FR2458590A1 - 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

 The present invention relates generally to a method and a reagent for the detection of N. Gonorrhoeae in a microbial culture and, more particularly, to a rapid and reproducible test of reverse passive hemagglutination (RPHA) for any of the known strains of this organism.

Current techniques for the identification of N. gonorrhoeae in a culture medium, for example by Thayer-Martin agar, include a preliminary selection according to the source, the morphology, the gram test and the oxidase. The diplococci giving a positive oxidase test and a gram negative test and having a typical colony morphology on Thayer-Martin medium inoculated from the genitourinary tract of a patient are considered to be presumed gonococci. Confirmation of this preliminary determination conventionally includes examination of the organism in a set of additional tests intended in particular for the detection of
N. gonorrhoeae, unlike other organisms with similar characteristics. These specific tests include the selection by degradation of carbohydrates which takes around 48 h to complete and sometimes is not reliable because some strains of N gonorrhoeae may either not grow or give false negative reactions to sugar. Another commercial selection technique involves the use of fluorescent antibodies and, although the procedure is relatively rapid and specific for all strains of N. gonorrhoeae, the use of a fairly expensive fluorescence microscope by a technician very experienced is necessary for this determination. See for example Health Laboratory Science, volume 12, no. 3, pages 215-218 (1975).

Although it is generally known that the RPHA test for confirmation of microorganisms is faster and more reproducible than, for example, the breakdown analysis of carbohydrates, it was thought that these procedures were not not suitable for screening for N. gonorrhoeae due to the often acute specificity of antibodies to different strains of N. gonorrhoeae. It has generally been stated, for example, that a large number of determinations also involving a large number of specific antibodies might be necessary to surely characterize an organism as one of the many strains of
N. gonorrhoeae.

 There is therefore now a need for a reproducible and rapid method for identifying the presence of N. gonorrhoeae which does not include the tedious or costly operating methods of conventional methods.

 The invention relates to a new method and a new reagent for a reverse passive hemagglutination test in the detection of N. gonorrhoeae from a microbial culture on Thayer-Martin agar medium or other comparable medium.

 According to the invention, antibodies derived from the use of eleven strains of N. gonorrhoeae, ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407 are used as immunogens to immunize particles. immunologically inert (e.g. stabilized erythrocytes) and used in an RPHA test to effectively identify the presence of one or more of the known strains of N. gonorrhoeae. The procedure is not subject to false positive reactions from organisms usually interfering with antibody-oriented detection of N. gonorrhoeae.

 In short, the antibodies of each of the above-mentioned strains are pooled and used to sensitize discrete support particles. The reagent particles, in turn, are used in a hemagglutination assay with a cell lysate from a suspect culture to verify the presence or absence of N. gonorrhoeae. The complete system uses a minimum of equipment and gives precise results in no more than 3 hours.

 Various other aspects and advantages of the invention will appear to those skilled in the art on reading the description which follows.

The eleven strains of N. gpnorrhoeae used in the practice of the invention are each capable of the following taxonomic description.

Order: Eubacteriales
Family: Neissericeae
Genus: Neisseria
Species: Gonorrhoeae
Morphology: spherical or bean-edged gram-negative diplococci
flattened neighbors, usually 0.6 xl, O / u and dimen
more uniform.

Biochemical and cultural characteristics: aerobic, optimal growth
with 4-10% C02 and incubation at 36 "C.

 The cultures grow slowly on Thayer-Martin medium, producing small colonies barely visible after 24 h (0.1 mm in diameter) with a characteristic morphology visible on cultures of 48-72 h. The colonies are small, 1.0 mm in diameter, gray-white, transparent, smooth, with a full round edge, scintillating surface and butyrous consistency.

 Positive oxidase and catalase tests, fermentation of glucose but not of maltose, lactose or sucrose.

 Virulence: the 11 strains were initially isolated from patients with symptomatic gonorrhea.

 According to the invention, each of the eleven strains is used as an immunogen in the serial immunization of an animal. A batch of sera is prepared by combining immune serum from several immunized animals each with a single strain to give a combination of serum containing antibodies against all strains. This batch of sera is passed through an immunoadsorbent column containing cell walls or extracts from the strains used to produce the immune sera. Antibodies attached to the cell walls are then eluted by chemical means. Preferably, the antibodies are still absorbed with selected strains of N. meningitidis to eliminate the reactive antibodies against Neisseria species other than N. gonorrhoeae. Each individual antiserum can also be passed over a column containing cell walls or extracts of the particular strain used as an immunogen in the development of the antiserum, elute, absorb against N. meningitidis and then pool the antisera.

The combined antibodies thus obtained are used to coat or sensitize discrete particles of an immunologically inert material, such as type 0 stabilized human erythrocytes.

Organisms suspected of being separated from their culture medium
N. gonorrhoeae and a suspension of the organisms in water is prepared at a given turbidity (measured by the optical density) and then treated to prepare a test lysate. The product to be tested is brought into contact with the particles sensitized to the antibody and an agglutination reaction confirms the presence of N. gonorrhoeae.

 The nonlimiting examples below illustrate the following aspects of the implementation of the invention: (a) isolation of cell walls of N. gonorrhoeae; (b) preparation of a phenolic extract from cell walls of N. gonorrhoeae; (c) preparation of inactivated N. meningitidis; (d) preparing stabilized erythrocytes as carrier particles; (e) preparation of support particles sensitized by the phenolic extract of N. gonorrhoeae; (f) preparation of an immunoadsorbent column; (g) preparation of antiserum against N. gonorrhoeae; (h) isolation of antibodies against N. gonorrhoeae; (i) absorption of the antibody preparation against N. gonorrhoeae with inactivated N. meningitidis; (j> preparation of reactive support particles sensitized by antibodies against N. gonorrhoeae; (k) preparation of bacterial lysate; (1) RPHA test for N. gonorrhoeae; and (m) test for verification of the specificity of the reagent.

The strains of N. gonorrhoeae ATCC 31397 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407 were deposited by the applicant and are available from the American Type Culture
Collection, 12301 Parklawn, Rockville, Maryland 20852.

EXAMPLE 1
Isolation of N. gonorrhoeae cell walls
The following procedure is used to develop a preparation of cell walls for each of the eleven strains of
N. gonorrhoeae ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407. The strain is cultured on Thayer-Martin medium and separated from the agar by washing with normal physiological saline buffered with 0.01 M phosphate (PBS), pH 7.2. The cells are recovered by centrifugation at 16,300 g for 40 min at 40C, washed once with normal physiological saline and adjusted in distilled water to obtain a suspension at 20% by weight by volume. The cells are ground with beads glass (about 150 to 200p) in a homogenizer cooled by liquid carbon dioxide. The cell walls are separated from the glass beads using a sintered glass funnel and resuspended in distilled water. After centrifugation at 9,750 g for 18 h at -20 C, pellets are obtained which can be stored at 40C.

EXAMPLE 2
Preparation of a phenolic extract from cell walls of N. gonorrhoeae.

 The eleven strains prepared as described in Example 1 are combined with equal parts of packed cell walls in the wet state and are suspended in distilled water to have a concentration of 20% by weight / volume. The suspension is extracted with an equal volume of 90% phenol in water and buffer with 0.1 M sodium acetate, pH 5.0 containing 0.5% of naphthaenedisulfonic acid and 0.55 of dodecylsulfate. sodium at 700C for 20 min. The phases are separated and recovered by centrifugation at 175 g for 30 min. The phenolic phase is dialyzed for 3 days at 40 ° C. against distilled water which is renewed frequently.

The extract is clarified by centrifugation at 16,300 g for 30 min at 40C, it is passed through a 0.451r filtering membrane and it is stored at 40C.

EXAMPLE 3
Preparation of inactivated N. meningitidis.

N. meningitidis, group A, strain MKO1A (Naval Medical Research Unit No. 4, Great Lakes, Illinois) is grown; group B, strain 3951 (King County Department of Health, Seattle, Washington) and group B, strain 5066-335 (Naval Medical Research Unit nO 4, Great Lakes,
Illinois), each on Mueller-Hinton broth at 370C for 18 in a rotary shaker. The organisms are separately transformed into pellets at 16,300 g for 30 min at 40C and washed three times by centrifugation with equal volumes of PBS. The cells are resuspended in one fifth of the initial volume of broth (generally 1 liter), placed in a Pyrex dish and enclosed in a plastic bag with an opening closed with a cotton pad. The cells are inactivated by the ethylene oxide and stored at 4 "C. (The lack of growth on Mueller-Hinton agar is evidence of inactivation).

EXAMPLE 4
Preparation of stabilized erythrocyte.

 Human erythrocytes are stabilized according to the Hirata et al method. (Journal of Immunology, volume 100, no.3, pages 641-646 (1968); U.S. Patents 3,714,345, 3,715,427 and / or 3,925,541). In summary, the process is as follows. A suspension of washed erythrocytes is prepared in a neutral buffer solution at a concentration of about 10% by volume and treated with a small amount of pyruvic aldehyde, preferably 1.5 to 5% by volume relative to the volume of erythrocytes, for a sufficient time to stabilize the erythrocytes, preferably 12 to 24 h. The erythrocytes are then washed several times to remove excess pyruvic aldehyde and other substances which may originate from the serum. Pyruvic aldehyde treated erythrocytes are treated with a small amount of formaldehyde, preferably 1.5 % by volume relative to the erythrocytes, for 12 to 24 h and the excess formaldehyde is eliminated by washing with a buffered solution.

EXAMPLE 5
Preparation of support particles sensitized by the phenolic extract of N. gonorrhoeae.

 Stabilized erythrocytes are prepared according to Example 4 and stored at a concentration of 10% w / v in the 0.11 M phosphate buffer (PB), pH 7.2. The particles are coated for example by mixing: 2 ml of phenolic extract from Example 3; 20 ml of 0.1 M sodium acetate buffer, pH 4.0; 1 ml of aqueous chromic chloride (10 mg of CrC13,6H20 / ml); and a button is formed by centrifugation at 700 g for 3.5 minutes of the 10% suspension of stabilized erythrocytes. above. After mixing, the suspension is incubated at 370C for 30 min with rapid shaking after 15 min to reduce the sedimentation of erythrocytes. The sensitized cells are washed four times by centrifugation 700 g for 2 min at 250C with 10 volumes of PB relative to the volume erythrocytes. The cells are resuspended at 1% w / v in the phosphate buffer containing 0.2% sucrose and 10 "1, bovine serum albumin and stored at -300C.

EXAMPLE 6
Preparation of an immunoadsorbent column.

 The following procedure can be used to prepare eleven columns, each consisting of one of the eleven separate gels corresponding to the eleven strains of N. gonorrhoeae used. (An equivalent procedure can be used to fix the cell walls of the eleven layers in a single gel for the preparation of a single large column).

8 ml of each of the eleven cell wall ions 20% w / v prepared according to Example 1 are added to 20 ml of a gel consisting of 10% of total monomers, acrylamide and N, N'-methylenebis-acrylamide (BIS) including 25% BIS, with 40 μl of N, N, N, N-tetramethylethylenediamine and 2.0 ml of 40% aqueous solution of ammonium persulfate ~ freshly prepared. Each gel is allowed to polymerize at room temperature for about 30 min and then broken into small pieces by passage through a 1.0 mm needle (No. 18) attached to a 50 ml syringe. The particles are washed with 100 ml of 0.1 M tris (hydroxymethyl) aminoethane hydrochloride buffer, pH 7.5 containing 0.15 M NaCl (THS), then with 100 ml of 0.1 glycine hydrochloride buffer M, pH 2.3, containing 0.15 M NaCl (GHS) and then with 100 ml of THS. 2 g of Bio-Gel P-100 from Bio-Rad Laboratories, Richmond, California, swollen overnight in THS are placed in a chromatographic column of 2 x 36 cm, then the immunoadsorbent particles are added. The column is then washed again with THS, GHS and THS, as above.

EXAMPLE 7
Antiserum preparation against N. gonorrhoeae.

The types 3 and 4 colonies of the eleven strains of N. gonorrhoeae are grown on Thayer-M) artin agar for 18 h at 380C in a flask and each strain is used to produce antiserum as follows. The colony is removed from the agar with 0.01 M phosphate buffered saline (PBS), pH 7.2, washed three times by centrifugation at 3000 g for 15 min at 40C and resuspended in PBS. The turbidity is adjusted to obtain two concentrations ("1 X" and "10 X") having respectively optical densities of 0.52 and 5.2 630 nm and they are stored at -20 C. White rabbits are again immunized Zealand in several sites per 10 ml of a homogenized inoculant comprising equal volumes of ten times frozen diluted bacterin suspension and complete Freund's adjuvant. Approximately 7, 10 and 15 days after the initial inoculation, Klapins is injected with 0.5 ml, 1.0 ml and 2.0 ml of bacterin respectively at a dilution of 1 X thawed, followed by two additional injections of 2.0 ml of bacterin at 1 X dilution about 5 days apart. 4 to 6 days later, whole blood samples are taken and the strength of the antibodies is determined therein using the particles according to Example 5 sensitized by a phenolic extract from cell walls of the inoculated strain.

The animals giving samples having the desired power are then bled and the antiserum is collected and stored. The animals giving samples which have a lower potency are again inoculated twice, 5 days apart, with 2.0 ml of the bacterin suspension at 1 × concentration, after which the antibody capacity of another blood sample. The animal is bled if the desired power is observed and the serum is stored.

 The potency of the antiserum is determined by passive hemagglutination using the particles sensitized to the phenolic extract of Example 5. A diluent of the antiserum is prepared by adding 0.1 of gelatin to phosphate buffer 0.11 M, pH 7.2 (PB), then heating at 600C until the gelatin dissolves and filtered through a 0.45P filter membrane. 25 μl of 0.25% erythro cytes suspension (poidstvolume) are added to the PB-sucrose-albumin mixture prepared according to Example 5 to double dilutions of antiserum in microtiter trays with V bottom. shake the filled trays with a vortex mixer for 10 s, cover them and allow them to stand overnight at room temperature. The inverse of the highest dilution having a clearly different agglutination scheme than that of the control is chosen as the end point for the titration of the sample (25 μl of the suspension at 0.2570 erythrocytes and 25 μl of the diluana The table I below indicates the inverses of the titles for each of the antisera of the eleven strains considered to be indicative of a suitable antibody potency.

TABLE I
ATCC strain Title
31 397 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 antibodies against N. gonorrhoeae.

 The following procedure can be used for isolating individual antibodies from each of the eleven antisera prepared according to Example 7 to isolate a batch of eleven antibodies by first combining the eleven antisera and then using a single immunoadsorbent column containing a gel in which the cell walls of the eleven strains of N. gonorrhoeae are coated.

 7 ml of rabbit antiserum are applied, according to Example 7, to a column prepared according to Example 6 containing cell walls of the strain used for immunization. The serum is recycled through the column for approximately 2 h using a peristaltic pump having a flow rate of approximately 100-150 ml / h. The column is then washed with THS h pH 7.5 until the absorption at 280 nm is <0.02. The adsorbed antibodies are then eluted by CHS at pH 2.3, fractions of 2 to 2.5 ml are collected and, if necessary, they are immediately adjusted to approximately pH 6.9-7.1 using a carbonate buffer. 0.5 M sodium at pH 9.5.

The successive fractions having an absorption of at least 0.1 at 280 nm are combined, dialyzed against normal physiological serum at 40 ° C. for 1 h, concentrated under a negative dialysis pressure at room temperature, dialyzed again and we keep at 40C.

EXAMPLE 9
Absorption of an antibody preparation against N. gonorrhoeae with
N. meningitidis inactivated.

 A suspension of inactivated N. meningitidis (6 ml of strain 5066-355 from group B, 6 ml of strain MKOLA from group A and 0.6- ml of strain 3951 from group B) is centrifuged at 16,300 g, for 1 ha. 40C. 6 ml of antibody (150 7 / ml) prepared according to Example 8 are added to the button, diluted and stirred gently in a vortex mixer for 1 h at room temperature. After centrifugation b 16 300 g, for 1 h at 4 ° C., the absorbed antibody b 4 "C. is stored.

EXAMPLE 10
Preparation of reactive support particles sensitized by antibodies against N. gonorrhoeae.

Stabilized erythrocytes prepared according to Example 4 can be sensitized with the eleven antibodies prepared according to Examples 8 and 9, following the following procedure. 1.0 to 10.0 Y of the batch preparation is added to a 1% w / v suspension of erythrocytes stabilized in 0.01 M sodium acetate buffer at pH 4.0 antibodies per milliliter of suspension. Add 50 to 500 Y of
CrC13.6H20 (previously dissolved at 10 mg / ml in 0.01 M sodium acetate buffer at pH 4.0), the suspension is mixed for a short time in a vortex mixer and incubated for 30 min of 300C by mixing after 15 min to reduce the sedimentation of erythrocytes. The suspension is washed in 0.1 M phosphate buffer, resuspended at 1% in 0.11 M phosphate buffer at pH 7.4 containing 2% sucrose and 10% beef serum albumin and we store.

The particular concentrations of the antibody batch preparation and the chromic chloride giving the highest titer against homologous bacterial lysates and the lowest titer against heterologous lysates are chosen and designated as optimal coating conditions for the batch. AntibodyX Suspensions of sensitized reactive particles can be lyophilized and stored for 12-18 weeks without loss of activity.

EXAMPLE 11
Preparation of bacterial lysates.

 The bacteria lysates are prepared in the following manner for use in the RPXA tests according to the invention. The organisms are incubated on suitable media for 18 h, after which the colonies are removed with a cotton ball and suspended in a 20% glycerol solution in distilled water. The suspension is vigorously stirred to break up lumps and is adjusted to an optical density of 0.52 at 630 nm (the suspensions thus formed can be frozen and stored for several weeks at -300C).

The suspension is diluted to 1:10 with distilled water and treated for example with 3Oil of 1N sodium hydroxide per ml of suspension. The treated suspension is stirred for a short time to facilitate lysis. These preparations are ready to use in 15 min and can be used up to 4 h after preparation if kept at 2-27 "C. It should be noted that all strains of N. gonorrhoeae generally give suspensions which s lighten in about 1 min of alkaline treatment (they are not cloudy or milky as in a blank test with water) A suspension which does not lighten can generally be discarded as N. gonorrhoeae.

EXAMPLE 12
RPHA test for the determination of N. gonorrhoeae.

 The RPHA test is carried out as follows to detect N. gonorrhoeae, the presence of which is suspected. A gelatin diluent / phosphate buffer is prepared as indicated in Example 8.

The test is carried out with a conventional V-bottom microtiter plate.

A drop of diluent is added to the wells, then 5 ml of the alkaline lysate of the sample, prepared according to example 11. 1 drop of sensitized support particles is added in uniform suspension (cell suspension at 0.1% by weight / volume prepared according to Example 10), in each well containing the lysate as well as in a few wells containing the diluent alone (cell control). The plate is covered and tapped on its edge several times to vigorously mix the reagents.

The test mixture is incubated at room temperature on a non-vibrating surface and the result is read after 3 h and up to 24 h. A positive test is indicated by the formation of a scattered pattern of sedimentation of the sensitized support particles and a negative test by the formation of a compact button of particles comparable to the cell control.

EXAMPLE 13
Reagent specificity verification test.

 The sensitivity of an RPHA test using the reagents of the invention is verified by the results of the selection of 79 strains of N. gonorrhoeae chosen at random from very variable sources.

All strains that are confirmed positive for N. gonorrhoeae by fluorescent antibody test (see Health
Laboratory Science, volume 12, no. 3, pages 215-218 (1975)) also give positive RPHA tests according to example 12. Table II below indicates the results of the sensitivity search.

TABLE II
Source of strain Number of strains Confirmed positive test for N. gonorrhoeae tested Antibodies RPHA
fluorescent
Seattle, Wash 20 20 20
Milwaukee, Wis. 19 19 19
Chicago, I11. 17 17 17
Lake Country, Ill. 8 8 8 Atlanta, Ga. * 7 7 7
Seattle, Wash. * 2 2 2
Europe 6 6 6 * Manufacturers of penicillinase.

 The specificity of the RPHA test of the invention for the detection of N. gonorrhoeae is verified by the results of tests on numerous species of non-gonococcal Neisseria. All species that do not confirm as N. gonorrhoeae in fluorescent antibody and carbohydrate degradation tests also give a negative RPHA test. Table III below indicates the results of the specificity search.

TABLE III
Number No confirmation
Test species Hydra Antibodies RPHA
EPHA fluorescent carbon species
N. meningitidis 1 1 1 1
(group A)
N. meningitidis 2 2 2 2
(group B)
N. meningitidis 1 1 1 1
(group C)
N. meningitidis 4 3 4 4
(type not determined)
aa
N. lactamica 5 5 5 4a
N. sicca 13 13 13 13
N. perflava 3 3 3 3
B. catarrhalis 1 1 1 1 a A strain does not give complete lysis and is not subjected to
the RPHA test.

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

The specificity of the RPHA test of the invention in the search for N. gonorrhoeae is further verified by the results of the test on numerous organisms of species other than Neisseria.

All organisms give a negative test in the RPHA procedure.

Table IV below indicates the results of the specificity search.

TABLE IV
Organism. Number of tests RPHA test negative
Acinetobacter sp. 5 5
Candida sp. 1 1
Flavobacterium sp. 2 2
Bacillus sp. 1 1
Lactobacillus sp. 3 3
Moraxella sp. -4 4 Gorynebacterium sp. 7 6
TABLE IV (continued)
Organism Number of tests Negative RPHA test
Enterococci 3 3
Proteus sp. 4 4
Staphylococcus epidermis 6 6
Streptococci (viridans) 2 2
Staphylococcus aureus 12 11a
Homophilus influenzae 5 5
Streptococci (Group A) 4 4
Salmonalla sp. 5 5
Shigella sp. 5 5 a A strain does not give complete lysis and the test is not subjected to
RPHA test.

 It is understood that the invention is not limited to the preferred embodiments described above by way of illustration and that a person skilled in the art can make various modifications to it without departing from the scope and the spirit of the invention. For example, while the stabilized human erythrocytes constitute a preferred particulate support for the implementation of the invention, numerous other supports can be used such as particles of polystyrene latex, bentonite, charcoal, cholesterol and lecithin.

Likewise, while alkaline bacterial lysates are preferred as the test material, it is expected that the lysates obtained by other chemical or physical treatment (e.g. treatment with sodium dodecyl sulfate or freeze-thaw, or treatment ultrasound) are also suitable for the implementation of the invention.

 Similarly, the Applicant believes that new strains of N. gonorrhoeae may be isolated from time to time in the future and not provide lysates which are reactive with the reagent particles of the invention. The Applicant therefore believes that an additional antibody specific for this strain can be added to the reserve of eleven antibodies used to produce the reagents according to the invention, without however departing from the scope and spirit of the invention.

Claims (2)

 1 - Method for identifying a microorganism with N. gonorrhoeae, said method being characterized in that a lysate of the microorganism is prepared, a sample of said lysate is brought into contact with a suspension of discrete particles sensitized with antibodies against N. gonorrhoeae strains ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407, this mixture of lysate and particles is incubated and the presence of agglutination of said incubated mixture is determined, indicating that the microorganism is N. gonorrhoeae.  2 - Reagent for use in a search test by reverse passive hemagglutination for the identification of a microorganism with N. gonorrhoeae said reagent being characterized in that it comprises a suspension of discrete particles, including portions of the surface were sensitized by antibodies against the strains of N. gonorrhoeae ATCC 31397, 31398, 31399, 31400, 31401, 31402, 31403, 31404, 31405, 31406 and 31407.