CA1052268A - Serological test for neisseria gonorrhoeae antibodies - Google Patents

Serological test for neisseria gonorrhoeae antibodies

Info

Publication number
CA1052268A
CA1052268A CA180,020A CA180020A CA1052268A CA 1052268 A CA1052268 A CA 1052268A CA 180020 A CA180020 A CA 180020A CA 1052268 A CA1052268 A CA 1052268A
Authority
CA
Canada
Prior art keywords
enzyme
antigen
neisseria gonorrhoeae
culture
produced
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA180,020A
Other languages
French (fr)
Other versions
CA180020S (en
Inventor
Hassan A. Gaafar
Dora D'arcangelis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Corp
Original Assignee
Research Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research Corp filed Critical Research Corp
Application granted granted Critical
Publication of CA1052268A publication Critical patent/CA1052268A/en
Expired legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/095Neisseria
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

ABSTRACT OF THE DISCLOSURE
Serological method for determining presence of Neisseria gonorrhoeae antibodies in human sera and products utilized in such testing.
The method comprises diluting the serum to be tested in physiological saline at a dilution of about 1 : 2 to 1 : 1000, heating the diluted serum at about 56°C to 65°C for from about 15 to 30 minutes and thereafter in-cubating with a composition containing a heat labile antigen produced from a growth culture of Neisseria gonorrhoeae to form an antigen-antibody con-jugate when said antibodies are present; the antigen being characterized as follows: 1. inactivated by heating at about 56°C for about 30 minutes;
2. inactivated by suspension in an aqueous medium At pH values up to 5;
3. stable in an aqueous medium at pH values of 7 and above; 4. stable when incubated with the following enzymes: deoxynucleic acidase (Enzyme No. 3.1.4.5), ribonucleic acidase (Enzyme No. 3.1.4.22), lipase (Enzyme No. 3.1.1.3), dextranase (Enzyme No. 3.2.1.11), neuraminidase (Enzyme No. 3.2.1.18), lysozyme (Enzyme No. 3.2.1.17), and 5. inactivated when incubated with pronase (Enzyme No. 3.4.24.4), trypsin (Enzyme No. 3.4.21.4) or chymotrypsin (Enzyme No. 3.4.21.1).

Description

ios~z~8 This invention relates to methods and products employed for determining the presence of Neisseria gonorrhoeae antibodies in human sera by a serological method.
The present mass screening method for the detection of gonorrhea is a bacteriological method. It requires two to seven days for completion because it necessitates waiting for growth of a colony of gonococcus organisms in an appropriate culture medium and confirmatory biochemical reactions by growth in a fermentation medium, such as shown in Table I. Moreover, the bacteriological method requires that a specimen of the gonorrhea caused dis-charge arrive at the testing laboratory with the fragile gonococcus organismstill viable, a natural time limit of as little as two days. There is real need for improvement.
A serological method which would be capable of detecting antibodies in a blood sample would be highly desirable for use in a mass screening pro-gram to demonstrate that an îndividual may be currently suffering from gonorrhea or had been infected in the past. Individuals reacting positively could then be tested by the bacteriological method to determine if the infection is current. Several serological methods have been reported, but none is completely satisfac~ory. The primary reason for dissatisfaction has been the low sensitivity and the high number of false positive reactions.
A serological method has now been deve}oped which substantially alleviates the difficulties with kno~n serological techniques, and is suitable for use in mass screening programs as an adjunct to the bacteriological method with substantial elimination of false positive reactions.
This invention comprises methods for the detection of gonorrhea produced antibodies and novel products used in the method.
It has now been disooYered that Neisseria gonorrhoeae (N.g.) organisms produce a species specific antigen which is heat labile and reacts with N.g. produced antibodies in human sera to thereby detect the presence of a pa5t or current gonorrhea infection. The antigen does not react with cross reacting antibodies which may also be present in the sera to cause the false positive reactions which have plagued previously employed serological proce-~ ;:

:

iO5'~'Z~3 dures.
According to the invention, there is provided a serological method for determining the presence of Neisseria gonorrhoeae antibodies in human serum which comprises diluting the serum to be tested in physiological saline at a dilution of about 1 : 2 to 1 : 1000, hPating the diluted serum at about 56C to 65C for from about 15 to 30 minutes thereafter incubating with a com-position containing a heat labile antigen produced from a growth culture of Neisseria gonorrhoeae to form an antigen-antibody conjugate when said anti-bodies are present and detecting whether any such antigen-antibody conjugate forms the antigen being characterized as follows: 1. inactivated by heating at about 56C for about 30 minutes; 2. inactivated by suspension in an aqueous medium at pH values up to 5; 3. stable in an aqueous medium at pH
values of 7 and above, 4. stable when incubated with the following enzymes:
deoxynucleic acidase (Enzyme No, 3.1.4.5); ribonucleic acidase (Enzyme No. 3.
3.1.4.22); lipase (Enzyme No. 3.1.1.3); dextranase (Enzyme No. 3.2.1.11);
neuraminidase ~Enzyme No. 3.2.1.18); lysozyme (Enzyme No. 3.2.1.17); and 5.
inactivated when incubated with pronase (Enzyme No. 3.4.24.4); trypsin (Enzyme No. 3.4.21.4); or chymotrypsin (Enzyme No. 3.4.21.1).
The invention also provides an antigen composition comprising a fixed cell suspension from a Neisseria ~onorrhoeae growth culture characte-rized by the presence of a heat labile antigen which will form an antigen-antibody conjugate when incubated wi*h sera containing heat stable Neisseria onorrhoeae antibodies; the antigen being characterized as follows:
1. inactivated by heating at about 56C for about 30 minutes; 2. inacti-vated by suspension in an aqueous medium at pH values up to 5; 3. stable in an aqueous medium at pH values of 7 and above; 4. stable when incubated with the following enzymes: deoxynucleic acidase (Enzyme No. 3.1.4.5); ribonucleic acidase (Enzyme No. 3.1.4.22); lipase (Enzyme No. 3.1.1.3); dextranase (Enzyme No. 3.2.1.11); neuraminidase (Enzyme No. 3.2.1.18); lysozyme (Enzyme No. 3.2.1.
17), and 5. inactivated when incubated with pronase (Enzyme No. 3.4.24.4), . trypsin (Enzyme No. 3.4.21.4) or chymotrypsin (Enzyme No. 3.4.21.1); the anti-gen unit value of the composition ;

E

lOS~Z~68 being at least 100 Ag. u./mg of protein.
In accordance with the presently preferred testing procedure utilizing suspensions of microorganisms, such a suspension containing the antigen is prepared, and is incubated with two separate samples of sera and tested for the production of an antigen-antibody complex. One sample of serum is heated to inactivate any heat labile cross reacting antibodies which may be present. The other sample is not subjected to heat. The key to the test is that antibodies caused by a gonorrhea infection are heat stable whereas the cross reacting antibodies are heat labile. Therefore, a positive reaction with both of the test samples is a clear indication of the presence of N.g. stimulated antibodies. A negative reaction in both specimens is a clear indication of the absence of such antibodies. A negative reaction with the heated sample coupled with a positive reaction with the unheated sample is an indication of the presence of natural antibodies.
The formation of a positive antigen-antibody complex can be detected by any of the presently available methods. Generally, the procedure -~
is to incubate the complex with a labeled anti-human immunoglobulin, prefer-ably immunoglobulin G (IgG). The heavy chain IgG is preferred. The immuno-globulin can be labeled, for example, with a detectable radioactive ele-ment, an enzyme or a chemical which fluoresces when exposed to ultraviolet light.
In the presently preferred method described above, separate samples of the antigen containing suspension are placed on a fluorescent antibody slide and incubated with the sera under test, one sample of which is heated, the other unheated. The thus prepared separate specimens are then incubated with an anti-human IgG labeled with fluorescent material such as fluorescein, rhodamine or auramine. The preferred detecting mate-rial is, for this method, anti-human IgG conjugated with fluorescein through an isothiocyanate. The product is well known and commercially available.
Basically, the test method of this invention comprises the detec-tion of a conjugate formed by reaction between the N. g produoed heat labile antigen and complementary heat stable antibody. Detection is preferably . , .

..

~05;~2~

effected utilizing the reaction with an anti-human IgG labeled with an ele-ment or chemical which is detectable by a chemical or physical method. Alter-natively, the reaction may be detected by permitting it to take place after the antigen is adsorbed on a carrier. Suitable adsorbents include, for example, various polymer latices such as a polystyrene latex, bentonite or charcoal. The particles comprising antigen adsorbed on an adsorbent are then mixed with the sera to be tested and the presence or absence of flocculation or clumping noted. The sensitivity of this procedure can be enhanced by wash-ing the particles to remove unreacted protein followed by the addition of anti-human IgG. Positive reactions give clumps while in negative reactions - the antigen coated particles remain homogeneously dispersed.
As stated above, the preferred detection method with suspensions `
is the fluorescent method. However, the anti-human IgG can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The pre-ferred isotope labels are 4C, 31I, 25I and 5S. The enzyme label can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric or gasometric techniques. The enzyme is conjugated to the anti-human IgG by reaction with bridging molecules, such as carbodi-imides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase (Enzyme No. 1.11.1.7);~-glucuronidase (Enzyme No. 3.2.1.31);
~-D-glucosidase (Enzyme No. 3.2.1.21); ~-D-galactosidase (Enzyme No. 3.2.1.23); urease (Enzyme No. 3.5.1.5); glucose oxidase (Enzyme No. 1.1.3.4) plus peroxidase; galactose oxidase (Enzyme No. 1.1.3.9) plus peroxidase and acid phosphatase (Enzyme No. 3.1.3.2).
The optimum sources of antigen for the process of this invention are _i_ e_ a gon rhoeae B-585, B-370 and B-1094. These organisms have been deposited at American Type Culture Collection and have received the accession numbers 21823, 21824 and 21825 respectively.

~ - 3a -c-~

lOS~;268 TAXONOMIC DESCRIPTION
Order: Eubacteriales Family: Neissericeae .
Genus: Neisseria :
Species: Gonorrhoeae ~ ~

: : .-,-, : 3b ~ Al~
.. . . . .. . . . . .

~ C

105;~ 8 orphology: Gram negative spherical or bean shaped diplococci with adjacent sides flattened usually 0.6 X 1.0 ~ and more uniform in size.
_ochemical and Cultural: Aerobic, optimal growth requires 4 - 10%
C2 and incubation at 36C.
The cultures grow slowly on chocolate agar producing small barely visible colonies after 24 hours (0.1 mm in diameter) with typical morphology seen on 48 72 hours cultures. The colonies are small 1.0 mm in diameter, gray white, transparent, smooth, with round entire edge, glistening surface and butyrous consistency. B-1094 produced slightly larger colonies and grows more rapidly.
Oxidase +, catalase +; ferments glucose but not maltose, lactose or sucrose.
Antigenicity: All three isolates share common antigens which is heat labile "L".
Virulence: All three strains were originally isolated from patients with symptomatic gonorrhea.
In the process of the invention the sera to be tested is diluted in physiological saline solution at a dilution of from about 1:2 to 1:1000, heat^
ing the suspension to about 56C. to 65C. preferably 59C. for a period of from about 15 to 40 minutes preferably 30 minutes to inactivate heat labile natural antibodies and then incubating the heated serum with an antigen pro-duced from a culture of N.g~ The heat labile antigen has not been previously detected or reported For the agglutination test the preferred ratio is at the lower end of the range~ ~or the radioimmunoassay, the preferred ratio is at the higher end of the scale, and for the fluorescence test the preferred ratio is from 1:10 to 1:40.
For the preparation of the antibody slides of this invention fresh isolates of the N.g. organisms are lyophilized. They are reconstituted as needed and subcultured as needed, on chocolate agar with rabbit blood (Table II) medium~ Maintenance cultures in semisolid media (Table III) may be trans-ferred once a month to maintain viability. The cultures are transferred as needed to a suitable growth medium, preferably chocolate agar (rabbit blood) 105;~268 slants, and these are incubated at about 34CC. to 38C., preferably 35C. to 37C. for from about 18 to 24 hours normally in a C02 (4-10%) atmosphere. The organisms do not grow appreciably at temperatures significantly below 35C.
and die above about 38C. At periods appreciably below 18 hours the amount of growth is too scarce to be practical, and there is a gradual decline in available antigen after about 24 hours.
Preferably the suspension is checked for purity and typical morphology by Gram staining and streaking on growth medium.
The growth is suspended in physiological saline at a concentration of from about 105 to 109 organisms per ml. For the fluorescence test, the preferred concentration is from 3 X 106 to 4 X 106. For the agglutination and enzyme tests, the preferred concentration is of the order of 108 organisms per ml.
A standard antigen slide is a slide preparation where one drop tabout 0.05 ml) of a suspension of N.g. organisms in physiological saline at a concentration of 3 - 4 X 106 organisms per ml is placed, dried and fixed.
A small drop of the suspension is placed on each end of a cleaned fluorescent antibody slide and smeared with a loop. The slides are air dried, fixed and then rinsed in distilled water to produce a standard antigen slide.
The slide is stable after 1 to 3% formalin fixation or without fixing for up to six weeks if maintained at about -20QC. They can be lyophilized and will retain their usefulness for as long as 2 to 3 months at room temperature.
The preferred fixative for a refrigerator temperature of about 10C. is one containing 10% formalin in phosphate buffered saline at pH 7.6, 95% ethanol, and glacial ace~ic acid in the ratio of 10:90:5. With this reagent the fixed suspension will remain stable for an extended period of time, even more than three months. At refrigeration temperatures, the stability may be enhanced by the presence of a dessicant such as anhydrous calcium chloride.
When utilized in the fluorescent process described above, the antiboty slides are stained with diluted samples of the serum to be tested.
The serum is diluted at 1:10 to 1:40 with physiological saline. When the serum dilution ratio varies appreciably below this range, the test gives 105.~:Z68 false positives; above the range it gives false negatives. The diluted serum is divided into two aliquots of about 0.5 ml each, and one sample is heated, preferably at 59C. for 30 minutes.
One drop of the heated diluted serum is added to the suspension on one end of the slide, and a drop of the unheated diluted serum is added to the suspension at the other end of the slide. The slide is then incubated for about 15 to 30 minutes in a humid chamber, preferably one that is saturated with water vapor at from about 22C. to 37C.
Upon removal from the chamber, the samples are washed thoroughly in buffered saline to remove serum proteins which are not bound to the antigens or cells. A suitable buffer is the standard phosphate buffer at a pH of 7.5 to 7.7. Washing is preferably accomplished by first dipping several times in ; `
the buffer solution, then holding in fresh buffer solution for about 10 A~ minutes, and finally rinsing in millipore H20. The slide is then dried, suitably by gently blotting with an absorbent paper.
The dried smears are stained with fluorescein isothiocyanate con-jugated anti-human IgG for about 20 minutes. The working dilutions for part-icular conjugate will vary from one lot to the other, and are best determined by titration with known controls. The slides are then mounted, suitably in a glycerol-carbonate-bicarbonate buffer at a pH of from about 8.5 to 9.5, pre-ferably 9Ø Any of a number of mounting fluid compositions can be employed, but they should be selected to have a minimum of autofluorescence.
The degree of fluorescence may be determined with a fluorescent microscope, typically a Leitz fluorescent microscope fitted with an HBO-200 light source, a 3-mm BG-12 exiter filter, a BG-38 red excluding filter, an OG-l ocular filter, a dark field condenser, and a 100 x oil immersion objec-tive.
The fluorescence of the smear stained with the heated serum specimen ~s compared with the smear stained with the unheated portion of the serum, $peci~ens that show 1 to 2~ fluorescence or more with the unheated serum with no or insignificant reduction on the heated specimens are con-sidered positive. The determination of fluorescence is somewhat subjective, _~_ ~ ~r~le ~r k~

`` 105;~Z68 but an experienced operator will have little or no difficulty distinguishing between positives and negatives.
Lyophilized products from suspensions of N.g. growth cultures, especially N.g. B-585, B-370 and B-1094 are especially valuable sources of antigens. They may be prepared by the following procedures:
A. Prepare concentrated suspensions in sterile milk (100 gr. of skim milk -- Baltimore Biological Laboratories -- added to 1 1. of water which was preheated to 50C.; sterilize 113C. to 115qC. for 20 minutes), and then add 5% rabbit or horse blood~
B To prepare milk suspension, wash culture slant down with 0.5 ml. milk using a capillary pipette. Dispense 2 drops culture suspension at the bottom of the prenumbered glass tubes.
C Dispense no more than 0.05 ml. of suspension into the bottom of 6-7 X 100 mm. cotton-plugged tubes.
D. Remove cotton plug and insert tube in rubber sleeve of connection to vacuum pump, making sure that; 1. Every plug is discarded directly into con-tainer for sterilization. 2. Every tube on any one connection contains some culture.
E. Place tubes in bath of dry ice and 95% ethyl alcohol with tilting motion to deposit some of suspension on glass a few millimeters above major part of material to be dried~ -F. Until connected to the manifold of the pump and the pressure is satisfactory for rapid drying, keep cultures in, or so near freezing bath, that no thawing can occur.
The lyophillzed products are characterized by the presence of a species specific, heat labile antigen which will react with a heat stable N.g.
antibody in patients' sera to form an antigen-antibody complex. They are especially valuable because they can be used in any of the variations of the test procedures described herein~ They can, for example, be taken up in physiological saline and the suspension used in the fluorescent procedure described above. They are also valuable in the enzyme and radioactive techniques, as will be readily apparent to those skilled in the art.

.:

i(~S'~Z1~8 The adaptation of the process of this invention to an enzyme test-ing technique with cell suspensions is accomplished by labeling the anti-human IgG with an enzyme such as horse-radish peroxidase. The antigen-anti-body conjugate is prepared from a sample of heated serum and antigen suspen-sion as described above. The sample is incubated with the labeled anti-human IgG and washed to remove extraneous protein. The washed product is mixed with a substrate such as o-dianisidine and hydrogen perioxide. After about 3 to 5 minutes the reaction is stopped by the addition of a few drops of 6N sulfuric acid. The optical density is measured at 400 nm. Reactive sera is indicated by a higher optical density than similarly treated negative controls.
Other acids can be used to arrest the reaction, for example hydro-chloric acid~ In that event, the preferred reading for optical density may be other than 400 mm.
~ n the radioimmunoassay procedure anti-human IgG is labeled with the selected isotope according to standard procedures, reacted with the antigen-antibody complex and the product counted using, for example, an autogamma spectrophotometer.
The methods of this invention are applicable, as described above, to antigenic cell suspensions. With cell suspensions, the preferred pro-cedure is the fluorescence method which, when utilized as described above,depends upon the morphology of the cells, ---- as will be recognized by those skilled in the art.
In another, and more preferred, embodiment of this invention, purified antigens or antigenic compositions are employed. With these materials, all of the general methods described above are also applicable. The use of purified antigens or antigenic compositions is preferred over the use of cell suspensions because the results are generally more reproducible and the pro-cedures are more easily automated. Moreover, the fluorescence procedure, when applied to these materials, is not subjective as in the case of cell suspensions, but rather it is objective since the results are determined by comparisons with controls containing known concentrations of antigens and an~ibodies.

lOS~,Zf~8 In the presently preferred procedure for purification of the anti-gen and for the preparation of antigenic compositions the selected N.g.
strains are grown on a suitable media, preferably Rabbit chocolate agar.
The cell growth is suspended in physiological saline solution and filtered -through sterile gauze. During this and all subsequent manipulations involv-ing the antigen or fractions containing the antigen, unless noted otherwise, the solutions, suspensions, etc. are kept cold at about 5C to 10C.
The residue on the filter is washed with physiological saline and centrifuged at about 10,000 RPM for about 15 minutes. The centrifuged mate-rial is decanted and the supernatant liquid discarded. The sediment is re-suspended in physiological saline, pooled and washed by centrifugation.
The antigen may be extracted with cationic detergents such as sodium lauryl sulfate (SDS), sodium deoxycholate, sodium cholate or similar materials.
The heat labile, species specific antigens of this invention are protein in nature as evidenced by the fact that they are inactivated when -~
incubated with protease enzymes such as pronase (Enzyme No. 3.4.24.4), trypsin (Enzyme No. 3.4.21.4) or chymotrypsin (Enzyme No. 3.4.21.1) in the usual manner. They are also inactivated by suspension in an aqueous medium at pH values up to 5, but they are stable in aqueous mediums at pH values of 7 and above. The antigen is heat labile in aqueous media, and is inacti-vated by heating at about 56C for about 30 minutes. It contains no deoxy-nucleic or ribonucleic acid, as evidenced by its stability when incubated with the corresponding acidases (DNase No. 3.1.4.5 and RNase No. 3.1.4.22).
It is not affected by lipase (Enzyme No. 3.1.1.3) or dextranase (Enzyme No. 3.2.1.11). It is not a neuramic acid, since it is stable to neuraminid-~ ase (Enzyme No. 3.2.1.18). Neither is it a cell wall unit (murine), since i it is not inactivated by lysozyme (Enzyme No. 3.2.1.17).
While the purified antigen can be prepared by the procedures described above, and can be used in the process of this invention, it is not necessary, nor is it preferred to do so. The reason for this is that anti-genic compositions containing even relatively high quantities of impurities ~ _ g _ ' . . .

lQ5'~Z~;8 such as extraneous protein can be used.
The antigenic activity of a particular composition can be defined in terms of antigen units by reference to the standard antigen slide as defined above. The method of determining antigen units will require some _ 9~ _ ' ~
., ~

lOS;~Z68 explanation. It is based on the ability of a particular composition to inhibit the preferred fluorescent tes~ which is used with cell suspensions, and is described in detail above.
As described above, the fluorescent test with cell suspensions depends upon the morphology of the cells. More particularly, it depends upon the morphological localization of the antigen on the intact cell. Optimum conditions for the test prevail when the complete cell is intact. During the course of purification in accordance with this invention, more and more antigen is cleaved from the cells. The compositions thus become less and less suitable for the type of fluorescent testing which depends upon cell -morphology.
In the initial stages of purification, there will be relatively large amounts of protein, for example, cellular debris, which is not antigenic in nature in the compositions. This material does not contribute to the antigen unit values. With increasing purification, the extraneous protein is removed and the antigen unit values increase rapidly.
With careful purification, it is possible to obtain products with antigen unit values of 10 - 20,000 units per mg. of protein, or even higher.
However, products with values of at least 100 can be used in fluorescent test-ing, enzyme testing, radioimmunoassay and agglutination procedures to deter-mine the presence of N.g, antibodies in human sera. It is preferred, however, that the values be at least 1000.
The following definitions will be helpful in understanding this aspect of the invention:
Conjugate Unit (C.U~
This is the highest dilution of fluorescein conjugated anti-human IgG which when added in aliquots of 0.05 ml to a standard antigen slide pre-paration stained with excess specific antibodies will give a 4~ fluorescence.
Antibody Unit (Ab.U~) It is the highest dilution of heat inactivated serum which gives a 4~ fluorescence when used to stain standard antigen slides and counter stained with fluorescein conjugated anti-human IgG appropriately diluted to contain 105A~ ;8
2 C.U./0.05 ml.
Antigen Unit ~A~_U.) One Antigen Unit tAg.U.) is defined as that amount of a whole, partially purified, or pure antigen preparation, expressed as ~g protein, which when allowed to react with heat inactivated serum diluted to contain one Ab.U.~0.05 ml will adsorb this unit of activity and when this adsorbed serum is used to stain ~he standard antigen slide and counter stained with fluorescein conjugated antl-human IgG appropria~ely diluted to contain 2 C.U./
0.05 ml will give fluorescence of shadow to 11. An antigen unit is the minimum amount of material which will absorb one antibody unit.
It will be appreciated that the purity of an antigen proteinaceous preparation is directly proportional to the number of antigen units per mg. of protein.
The antigen compositions of this invention can be used in all of the tests described above in connection with cell or organism suspensions. The procedures will be generally the same. For example, the antigen composition can be reacted with the N.g. antibody in test sera on a slide, and the resulting complex incubated with IgG labeled with a fluorescent chemical.
Alternatively, the IgG may be labeled with any of the enzymes or isotypes mentioned above. The procedure for the agglutination test described above is the same, except that the suspensions are replaced with purified antigens or antigenic compositions.
It has been observed that the antigens or antigenic compositions of this invention are particularly useful when conjugated or adsorbed on part-iculate carriers or substrates. Such materials do no~ affect the antigen-antibody reaction. Neither do they affect the subsequent reaction with the labelled compound. Any of a very wide variety of carriers can be employed, including polymeric materials such as polystyrene, inorganic materials such as glass, silica, bentonite or charcoal, and cellulosic materials such as ,~ ~
Sepharose, Sephadex or cellulose~ Biological carrie~s such as red blood cells may also be employed.

For the conjugation of species specific N.g. antigens to non-* Trdd~ rKs l~)Si~Zf~8 antigenic carriers, equal volumes of the partially purified or pure antigenpreparations containing at least 100 Ag.U./mg of protein and the selected carrier, for example, DEAE-cellulose in 0.1 m phosphate buffer at pH 8 are mixed and refrigerated overnight. The particles are recovered by filtration and washed with additional buffer solution, for example, 0.3 m phosphate buffer at pH 7.0, and again with 0.1 m phosphate buffer at pH 7 with 1%
albumin. The washed conjugate is resuspended in 0.1 m phosphate buffer at pH
7 with 1% albumin in a volume equal to the volume of the original antigen solution.
The antigen-carrier conjugate may be used in en~yme immunoassay or radioimmunoassay in the manner described above. It has been observed ~hat when these conjugates are employed in the testing procedures there is less background in~erference in the reading of the testing instruments with result-ing better differentiation between positive and negative tests.
The following non-limiting examples are given by way of illustration only:

INDIRECT IMMUNOFLUORESCENT ANTIBODY TEST
Preparation of the Antigen 1. Fresh isolates of N. gonorrhoeae with appropriate antigen profile or a lyophil~zed subculture of the standard strains (B-370, B-585 or B-104) are subcultured to maintenance medium (Table III), The cultures are maintained at 37C. in a 4 - 10% CO2 atmosphere and subcultured to fresh maintenance medium once a month.
2. The strains are subcultured as needed to freshly prepared chocolate agar slants ~rabbit blood) ~Table II).
3. The slants are incubated at 36C. for 18 - 24 hours and the growth is suspended in physiological saline and adjusted to 3 - 4 x 106 organisms/ml.
4. A small drop of the suspension is placed on each end of an alcohol cleaned fluorescent antibody slide and smeared with a loop.
5. Slides are air dried and fixed in 1% formalin for 10 minutes.
6. Slides are rinsed 10 X in distilled water, air dried and stored at lOS;~Z~8 -20C. until needed.
Preparation of the Pa~ient's Serum 1. Dilute patient's serum 1:10 in physiological saline.
2. 0.5 ml. of the 1:10 dilution is heated for 30 minutes in a 59C.
water bath.
Staining 1. Slides are ta~en out of the freezer 10 - 15 minutes before use and labelled with patient's name or number.
2. Add one dsop of heated diluted serum to one end of the prepared antigen slides and one drop of the unheated serum to the other end.
3. Allow to incubate at room temperature for 20 minutes in a humid chamber.
4. Wash 10 times rapidly in 0.1 M phosphate buffered saline pH 7.6.
5. Wash for 10 minutes in a fresh bath of the same buffer.
6. Rinse 10 times in distilled water and gently blot dry.
7. Stain with working dilution of fluorescein isothiocyanate conjugated anti-human immunoglobulin G for 20 minutes followed by a washing cycle as before (steps 4 to 61.
8. Mount the slides in a glycerol-carbonate bicarbonate buffer pH 9Ø
Readings The slides are examined with a fluorescent microscope fitted with an HBO-200 light source, 3 mm BG-12 exciter filter, a BG-38 red excluding filter, and OG-l ocular filter, a dark field condenser, and a 100 X oil immersion objecti~e.
Interpretation of Results The degree of the peripheral fluorescence of the bacterial cells in the smear stained with the heated serum specimen is compared with the smear stained with unheated serum. Specimens that show 1 - 2~ fluorescence or more with no or insignificant reduction after heating, are considered positlYe and interpreted to indicate current or recent past infection.

1()5~,Z68 EXA~LE 2 COLORIMETRIC ENZYME-LINKED IMMUNOASSAY
-Preparation of the Antigen 1. Fresh isolates of N. gonorrhoeae with appropriate antigenic profile or a lyophilized subculture of the standard strains (B-370, B-585 or B-1094) are subcultured to maintenance medium (Table III)~ The cultures are main-tained at 37C. in a 4 - 10% C02 atmosphere and subcultured to fresh main-tenance medium once a month.
2. The strains are subcultured as needed to freshly prepared chocolate agar slants (rabbit blood) (Table II)~
3. The slants are incubated at 36C. for 18 - 24 hours and the growth is suspended in physiological saline and adjusted to 3 - 4 x 108 organisms/ml.
4. Rabbit IgG in 0.1 M phosphate buffered pH 7.0 is added to the bacterial suspension to a final concentration of 50 mg/ml.
5~ Aqueous solution of 2~5% gluteraldhyde is added dropwise with gentle stirring to a final concentration of 10 mg. gluteraldhyde per 100 mg. protein in solution.
6. Leave at room temperature for 1 hour.
7~ Disperse the insoluble protein-bacterial gel in 0.2 M phosphate buffered saline pH 7.2~
8. Centrifuge for 15 minutes at 3000 rpm at 4C.
9. Repeat steps seven and eight two times.
10. Resuspend the protein-bacterial gel in a volume of 0.2 M phosphate buffered saline pH 7.2 four times the volume of the rabbit protein added in step 4.
11. Dispense the suspension in 0.25 ml, aliquots and store in refrigerator until used.
i Preparation of the Patient's Serum 1, Dilute patient's serum in 0.2 M phosphate buffered saline pH 7.2.
1:10, 1:100, and 1:1000~
2. Heat the different dilutions for 30 minutes in a 59C. water bath.

iOS'~Z68 Procedure l. Add 0.25 ml, of each dilution to the antigen preparation in separate tubes and mix by shaking manually.
2. Incubate at 37C. for 15 minutes in a water bath.
3. Centrifuge at 3000 rpm for 5 minutes and tiscard the supernatant.
4. Wash the pellet 2 times with 5 volume of 0.2 M PBS pH 7.2.
5. Add 0.1 ml. of working dilution of horse-radish peroxidase (HRP) conjugated anti-human immunoglobulin G.
6. Incubate at 37C. for 15 minutes in a water bath~
7. Add 5.0 ml. of 0.2 M PBS pH 7.2, centrifuge and wash the pellet as before ~steps 5 and 6~.
8. Add 3.0 ml~ of the substrate tTable IV~ and mix by shaking.
9. After 5 minutes the reaction is stopped by adding one drop of 6N
sulfuric acid~
10. Tho 0,D. is measured at 400 m~
Interpretation Reactive sera are indicated by comparison with a standard curve prepared with known positi~e and negati~e sera.
Contrals run with each batch should include:
a~ Positive controls b) Negative cont~ols c) Weekly positive (borderline controls) d1 Antigen control e~ Substrate control Enzyme control ENZYME-LINKED IMMWNOASSAY
~ Preparation of Antigen ; :
1. Fresh isolates of N. gonorrhoeae with appropriate antigenic profile or a ly~philized subculture the standard strains tB~370, B-585 or B-1094) are subcultured to maintenance medium (Table III). The cultures are maintained at 37C. in a 4 10% C02 atmosphere and subcultured to fresh maintenance lOS'~Z68 medium once a month.
2. The strains are subcultured as needed to freshly prepared chocolate agar slants ~rabbit blood) Crable II).
3. The slants are incubated at 36C. for 18 - 24 hours and the growth is suspended in physiological saline and adjusted to 3 - 5 x 108 organisms/ml.
4. Store the suspension in the refrigerator till used.
Preparation of Patient's Se_a 1. Dilute patient's serum in 0.2 M phosphate buffered saline pH 7.2.
1:10, 1:100 and 1:1000.
2. Heat the different dilutions for 30 minutes in a 59C. water bath.
Procedure 1. 0.2 ml. of the bacterial suspension is placed on a disposable filter.
2, 0.2 ml. of the pt. serum is added and mixed with cells by manual shaking.
3. Incubate at 37C. for 15 minutes.
4. Wash thrice by suction using 5.0 ml. of 0~2 M PBS pH 7.2.
5. Atd 0.1 ml. of working dilution of horse-radish peroxidase conjugated anti-human immunoglobulin G.
6. Incubate at 37C. for 15 minutes.
7. Wash as in step 4 and check the final washing for enzyme activity and repeat the washings if necessary.
8. Add 1.0 ml~ of the substrate. The substrate is prepared by adding 1.0 ml. of 30% hydrogen perQxids to 99 ml. of distilled water to form a 0.3%
solution. This solution (1 ml.) is added to 99 ml. of 0~01 M phosphate buffer pH 6.0 (0.003S) and 0.2 ml. of 1~ o-dianisidine in methyl alcohol is added to 24.0 ml. of thus prepared 0.003% hydrogen peroxide.
9. Reactive sera give reddish-brown pigmentation of the filter.

PREPARATION OF ANTIGENIC COMPOSITIONS
The cell growth Prom step 2 of Example 1, above is harvested and suspended in ph~siological saline solution. It is then filtered through cheesecloth at a te~perature of 5 - 10C~, which temperature is maintained -16_ -lOS'~Z~8 throughout subsequent manipulations. The residue on the filter is washed twice with physiological saline solution and the filtrate centrifuged at 10,000 rpm for 10 minutes~ The sediment is resuspended in physiological saline and washed once by centrifugation. The sediment containing the antigen is weighted to calculate the wet weight.
A total of 6.0 ml. of 0.3% SDS in physiological saline is added to the precipitate from the centrifugation per gram of wet weight of bact~rial sediment, and the mix is incubated at room temperature for 10 minutes. The mix is centrifuged at lO,000 rpm for lO minutes and the supernatant collected.
The setiment is treated with 6~0 ml/gm. of 0.1% SDS in physiological saline.
The mix is incubated at rosm temperature for lO minutes, centrifuged as before, and a second supernatant collected. The two supernatants are recentrifuged to remove any cells or large fragments and pooled.
At this pointJ the antigen unit value is 100-200 Ag. u/mg of protein.
The pooled supernatant is column chromatographed over Sepharose 4B
and eluted with 0.002 molar phosphate buffer at pH 7.6. Appropriate fractions ~usually 5 - 10 ml) are collected and the protein concentration monitored by the Lowry technique. The antigen composition with the best Ag. u. value is fount in the first peak which is made up of 5 - 10% of the protein. With fresh preparations, the value i5 from lOO0 - 2000 Ag. u./mg.

CONJUGATION TO NON-ANTIGENIC CARRIER
Equal volumes of antigen solutions containing a minimum of lO0 Ag.
u./mg. of protein and DEAF-cellulose in 0.1 molar phosphate buffer at pH 8 are mixet and left standing overnight in the refrigerator. The mix is $iltered and washed 3 times with an equal volume of 0.3 molar phosphate buffer at pH 7 followed b~ an additional 3 washings with equal volumes of o.l molar phosphate buffer at pH 7 with 1% albumin. The washed conjugate of antigen to cellulose is resuspended in o.l molar phosphate buffer at pH 7 with 1%, albumin in a Yolume equal to that of the original solution.
This material is sui~able for use in any of the various testing procedures described above.

105~Z68 COLORIMETRIC ENZYME-LINKED IMMUNOASSAY WITH ANTI~ENIC COMPOSITION
For each sample of patient's serum to be tested, an 0.6 micron filter is first wet with 0.1 molar phosphate buffer at pH 7 with 1% albumin.
A total of 0.5 ml. of patient's serum (1:10 dilution) is added together with 0.2 ml. of the final suspension from Example 5. The mix is incubated at room temperature for 60 minutes and washed thoroughly 6 times with the same phos-phate bufer.
A total of 0.2 ml. of the appropriate dilution of horse-radish peroxidase (HRP~ conjugated with anti-human IgG is added followed by 4.0 ml.
of the same buffer, The mix is incubated for 30 minutes at room temperature and washed until the filtrate is free of conjugate.
The filter with the precipitate is placed in a test tube containing the substrate (Table IV~ and incubated for 5 minutes. The reaction is then stopped by the addition of 2 drops of 6 N sulfuric acid. The optical density of the solution is read at 400 manometers. The test results are interpreted by comparison with controls which are known to be positive or negative.

COLORIMETRIC ENZYME-LINKED IMMWNOASSAY
1. Mix 1 gram of cyanogen bromide activated Sepherose IV particles (CNB-Sepherose activated-Pharmacia Fine Chemicals) with 5 ml. portions of 10 3 M. HCl.
2, Pour the frac~ion containing at least 100 Ag. u./mg. of protein onto the filter bed and recycle filtrate several times until the protein is adsorbed maintaining at all times a temperature of 5 - 10C.
3, Block any remaining active sites on the Sepherose by adding 5 - 10 ml. of 0.5% Bovine Serum Albumen in lM. glycine buffer (pH 8.2), recirculate filtrate overnight by continuous pumping.
4. Let filtrate drain out and transfer adsorbed particles to centrifuge, suspend in 10 ml~ saline solution~ centrifuge at about 10,000 rpm for 10 minutes. Decant and discard supernatant liquid~
5. Repeat washing as in step 4 and recover precipitate.

iO5Z2f~8 6. Suspend particles in about 10 ml~ of saline solution.
7. Dilute patient's serum one to ten.
8. Mix 0.1 ml. of particle suspension (from 6) with 0.5 ml. of the diluted serum.
9. Incuba~e at room temperature for 45 minutes.
10. Centrifuge at about 5000 rpm for 10 minutes at room temperature. Dis-card supernatant liquid.
11. Resuspend the precipitate in 0.1 ml. of rabbit anti-human IgG con-jugated through gluturaldehyde to horse-radish peroxidase. (50 micrograms proteln per ml.)
12 Incubate 30 minutes at room temperature.
13. Centrifuge at 5000 rpm for 10 minutes and discard supernatant.
14. Wash precipitated particles twice by centrifugation as în 13 with 0.01 M phosphate buffer, pH 7Ø
15. To the washed precipitate, add 2 ml, of the substrate mixture of Table IV.
16. Incubate for 15 minutes at room temperature~
17. Add 2-3 drops of 6 N sulfuric acid to stop reaction.
18. Measure adsorption at 4000 Angstroms (400 mm) using a Gilford 2400 instrument.
A positiYe serum will giYe a reading of about 100, a negative serum of about 40.
The following Tables illustrate the ~arious media utilized in the description of this invention.
TABLE I
FERMENTATION MEDIUM
Starch gelatin agar, infusion-free, with indicator and carbohydrate (for Neisseria gonorrhoeae and Neisseria meningitidis~
Agar ............. ~ ............................................... ....3 grams Gelatin ,,..~ . 10 gra~s Sodiu~ chloride .. ~...... ~.~.~......... ,.~.. ~.................... ~ 5 grams Peptone ~Difco proteose No. 3) .,~ .. 10 grams
-19--lOS~i8 Starch, soluble, powdered ... ..~.... ~.~...... ~.......... ~......................... 5 grams Water to Make ... ~...... ~.... ~.~.... ~.~...... ~.......... ~...................... 1000 grams Phenol red, 0.02 percent .... ..~.... ~........ ~............................. 30 ml. per kg.
Carbonhydrate (Sucrose, Glucose or Maltose) ............................. 10 grams per kg.
Dissolve the agar in half the water by autoclaving; the gelatin, salt, peptone, and starch in the remainder with heat. Combine and make up to total weight. Adjust pH to 7.4-7.6. Filter through cotton. Weigh the filtrate recovered and add the indicator and carbohydrate. Dispense 2.5 ml.
amounts in 11 by 75 mm. tubes and autoclave at 115C. for twelve minutes.
Trim the plugs and seal with paraffin.
TABL~ II
_ROWTH MEDIUM
Glucose agar, infusion free, with coagulated blood tfor Neisseria gonorrhoeae and Neisseria meningitidis?

.
Agar .............. ~....................................... ......... ~............ 15 grams Sotium chloride .............. ~.......... ~....... ~................................ 5 grams Disotium phosphate, Na2HP04 .. ,............................ .....~................. 5 grams Peptone, (Proteose peptone ~3) .~ .......................................... 20 grams Glucose ................. ~........ ~............ ~........... ........~............ 0.5 grams Distilled water to make .,................................................ 1000 grams _ Rabbit blood, defibrinated, sterile ...................................... 10 ml. per 100 m Dissolve the agar in half the water by autoclaving; the salts, peptone, and glucose in the remainder with heat. Combine and make up to 1 kg.
and ad~ust p~ to 7.4-7.6. Dispense 1500 ml. amounts in 3L gauged neck flasks.
Autoclave thirty minutes. Store.
As required, melt the agar base. Admix the blood aseptically 15-20 ml. amounts in glass-covered Petri plates. Deliver without incubation.
TABLE III

INTENANCE MEDIUM
Beef-infusion agar with ascitic fluid Beef infusion, concentrated , ........ ...... , ~... ,........................ 500 grams
-20-105;~Z~8 Agar ............... ,.~. .~.... ~.,... .,~,.. ~,..... ,,.... .. ,~ .~, 5 grams Sodium chloride .~.. .. ... ,.~.... ~ ....... ~.................. ........5 grams Peptone ............ ~............................... ~..... ,............ ........10 grams Water ..... .... ......... ... .............. ~..... ~........... .......500 gra~s Ascitic fluid, sterile .................................................. .......an equal volume Dissolve the agar in the water by autocla~ing, and the peptone and salt in the infusion. Combine. Make up to total weight. Adjust pH to 7.5 wîth lN NaOH. Fil$er by asperation. Usually dispense 400 ml. amounts in 2 liter flasks and autoclave thirty minutes. Store. Melt thc agar base and cool to 50C.
Warm the ascitic fluid to 50C. and combine aseptically. Mix well and dispense with ~septic precautions 4-6 ml. amounts in 15 by 125 mm. tubes.
Cover the medium aseptically with about 4 ml. of sterile mineral oil. Cool in an upright position. Incubate forty-eight hours at 35qC. - 37C. and ninety-six hours at 20C. - 27C. Inspect and store.
TABLE IV
SUBSTRATE FOR PEROXIDASE TESl a) Dilute 3% hydrogen peroxide solution with water to make a one to one hundred dilution.
b) Dilute the solution with phosphate buffer ~0.01 molar at pH 7) to another one to one one huntred dilution.
c) To 24 ml. of the ~hus diluted hydrogen peroxide, add 0.2 ml. of a 1% solution of o-anisidine in methanol.
-21-

Claims (35)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A serological method for determining the presence of Neisseria gonorrhoeae antibodies in human serum which comprises diluting the serum to be tested in physiological saline at a dilution of about 1 : 2 to 1 : 1000, heating the diluted serum at about 56°C to 65°C for from about 15 to 30 minutes thereafter incubating with a composition containing a heat labile antigen produced from a growth culture of Neisseria gonorrhoeae to form an antigen-antibody conjugate when said antibodies are present, and detecting whether any such antigen-antibody conjugate forms the antigen being charac-terized as follows:
1. inactivated by heating at about 56°C for about 30 minutes;
2. inactivated by suspension in an aqueous medium at pH values up to 5;
3. stable in an aqueous medium at pH values of 7 and above, 4. stable when incubated with the following enzymes:
deoxynucleic acidase (Enzyme No. 3.1.4.5) ribonucleic acidase (Enzyme No. 3.1.4.22) lipase (Enzyme No. 3.1.1.3) dextranase (Enzyme No. 3.2.1.11) neuraminidase (Enzyme No. 3.2.1.18) lysozyme (Enzyme No. 3.2.1.17), and 5. inactivated when incubated with pronase (Enzyme No. 3.4.24.4), trypsin (Enzyme No. 3.4.21.4) or chymotrypsin (Enzyme No. 3.4.21.1).
2. A serological method as in claim 1 wherein the presence of said conjugate is detected by reaction with an anti-human IgG labelled with a chemi-cal which fluoresces when exposed to ultraviolet light.
3. A method as in claim 2 wherein the chemical is selected from the group consisting of fluorescein, rhodamine and auramine.
4. A method as in claim 2 wherein the antigen composition has an anti-gen unit value of at least 100 Ag. u./mg of protein.
5. A method as in claim 2 wherein the antigen unit value is at least 1000.
6. A method as in claim 2 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21823.
7. A method as in claim 2 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21824.
8. A method as in claim 2 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21825.
9. A serological method as in claim 1 wherein the presence of said conjugate is detected by reaction with an anti-human IgG labeled with an enzyme.
10. A method as in claim 9 wherein the enzyme is selected from the group consisting of peroxidase (Enzyme No. 1.11.1.7), .beta.-glucuronidase (Enzyme No. 3.2.1.31), .beta.-D-glucosidase (Enzyme No. 3.2.1.21), .beta.-D-galacto-sidase (Enzyme No. 3.2.1.23), urease (Enzyme No. 3.5.1.5), glucose oxidase (Enzyme No. 1.1.3.4) plus peroxidase, galactose oxidase (Enzyme No. 1.1.3.9) plus peroxidase, and acid phosphatase (Enzyme No. 3.1.3.2).
11. A method as in claim 9 wherein the antigen composition has an antigen unit value of at least 100 ag. u./mg. of protein.
12. A method as in claim 9 wherein the value is at least 1000.
13. A method as in claim 9 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21823.
14. A method as in claim 9 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21824.
15. A method as in claim 9 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21825.
16. A serological method as in claim 1 wherein the presence of said conjugate is detected by reaction with an anti-human IgG labeled with a radioactive element.
17. A method as in claim 16 wherein the radioactive element is selected from the group consisting of 14C, 125I, 131I, and 35S.
18. A method as in claim 16 wherein the chemical is selected from the group consisting of fluorescein, rhodamine and auramine.
19. A method as in claim 16 wherein the antigen composition has an antigen unit value of at least 100 Ag. u./mg. of protein.
20. A method as in claim 16 wherein the antigen unit value is at least 1000.
21. A method as in claim 16 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21823.
22. A method as in claim 16 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21424.
23. A method as in claim 16 wherein the antigen is produced by a culture of Neisseria gonorrhoeae ATCC 21825.
24. An antigen composition comprising a fixed cell suspension from a Neisseria gonorrhoeae growth culture characterized by the presence of a heat labile antigen which will form an antigen-antibody conjugate when in-cubated with sera containing heat stable Neisseria gonorrhoeae antibodies;
the antigen being characterized as follows:
1. inactivated by heating at about 56°C for about 30 minutes;
2. inactivated by suspension in an aqueous medium at pH values up to 5;
3. stable in an aqueous medium at pH values of 7 and above;
4. stable when incubated with the following enzymes:

deoxynucleic acidase (Enzyme No. 3.1.4.5) ribonucleic acidase (Enzyme No. 3.1.4.22) lipase (Enzyme No. 3.1.1.3) dextranase (Enzyme No. 3.2.1.11) neuraminidase (Enzyme No. 3.2.1.18) lysozyme (Enzyme No. 3.2.1.17), and 5. inactivated when incubated with pronase (Enzyme No. 3.4.24.4), trypsin (Enzyme No. 3.4.21.4) or chymotrypsin (Enzyme No. 3.4.21.1); the antigen unit value of the composition being at least 100 Ag. u./mg. of protein.
25. A composition of claim 24 wherein the value is at least 1000.
26. An antigen slide with two separate dried drops of a suspension of claim 24.
27. A slide as in claim 26 wherein the drops are fixed with 1 to 3 percent formalin.
28. A slide as in claim 26 wherein the drops are fixed with a 10 : 90 : 5 mixture of 10 percent formalin in phosphate buffered saline pH 7.6, 95 percent ethanol and glacial acetic acid.
29. A composition as in claim 24 wherein the growth culture is Neisseria gonorrhoeae ATCC 21823.
30. A composition as in claim 24 wherein the growth culture is Neisseria gonorrhoeae ATCC 21824.
31. A composition as in claim 24 wherein the growth culture is Neisseria gonorrhoeae ATCC 21825.
32. lyophilized products from a suspension of claim 24.
33. A product as in claim 32 wherein the growth culture is Neisseria gonorrhoeae ATCC 21823.
34. A product as in claim 32 wherein the growth culture is Neisseria gonorrhoeae ATCC 21824.
35. A product as in claim 32 wherein the growth culture is Neisseria gonorrhoeae ATCC 21825.
CA180,020A 1972-08-31 1973-08-30 Serological test for neisseria gonorrhoeae antibodies Expired CA1052268A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28512872A 1972-08-31 1972-08-31
US38586373A 1973-08-06 1973-08-06

Publications (1)

Publication Number Publication Date
CA1052268A true CA1052268A (en) 1979-04-10

Family

ID=26963002

Family Applications (1)

Application Number Title Priority Date Filing Date
CA180,020A Expired CA1052268A (en) 1972-08-31 1973-08-30 Serological test for neisseria gonorrhoeae antibodies

Country Status (6)

Country Link
JP (1) JPS5825978B2 (en)
CA (1) CA1052268A (en)
CH (1) CH604720A5 (en)
DK (3) DK550777A (en)
IT (1) IT1012069B (en)
SE (1) SE7611560L (en)

Also Published As

Publication number Publication date
DK550677A (en) 1977-12-09
IT1012069B (en) 1977-03-10
JPS4992223A (en) 1974-09-03
CH604720A5 (en) 1978-09-15
DK550777A (en) 1977-12-09
DK550877A (en) 1977-12-09
JPS5825978B2 (en) 1983-05-31
SE7611560L (en) 1976-10-18

Similar Documents

Publication Publication Date Title
US4029756A (en) Serological procedure for determining presence of neisseria gonorrhoeae antibodies
US4618576A (en) Diagnostic test for Streptococcus A
EP1107773B1 (en) Method for detection of legionella bacteria employing purified antigen-specific antibodies
JPH0782021B2 (en) Non-immunochemical binding of lipopolysaccharide and its sandwich analysis method
US4241045A (en) Purified antigen to test for Neisseria gonorrheae antibodies
Van Vuurde et al. Comparison of immunofluorescence colony-staining in media, selective isolation on pectate medium, ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in cattle manure slurry
US4351761A (en) Purified antigen to test for Neisseria gonorrhoeae antibodies
Somerville et al. Urea-mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other species
US5399484A (en) Use of blocking protein with high pH extraction in method to determine a microorganism associated with periodontal disease and kit useful therefor
US4298689A (en) Gonorrhea diagnostic test
US4186182A (en) Serological test for Neisseria gonorrhoeae antibodies
CA1052268A (en) Serological test for neisseria gonorrhoeae antibodies
US4666851A (en) Specific mycoplasma membrane antigen and antibody and their clinical applications
EP0439210A1 (en) Differentiation of microorganisms associated with periodontal diseases , article and kit useful therein
Wong et al. Typing of heat-stable and heat-labile antigens of Campylobacter jejuni and Campylobacter coli by coagglutination
US5672517A (en) Methods and compositions for diagnosis and treatment of interstitial cystitis
EP0439212A1 (en) Device, test kit and sandwich assay for the detection of Bacteroides intermedius, Bacteroides gingivalis or Actinobacillus actinomycetemcomitans
SU1673973A1 (en) Method for diagnosis of plant diseases, caused by pseudomonas syringae of iv serogroup
US5707878A (en) Method for detecting blood component using conidiobolus hemagglutinin
KR101341994B1 (en) A method of manufacturing antigen slide for indirect immunofluorescence antibody assay against Legionella species and a micro-multivalent antigen slide for simultaneous detection of antibodies manufactured by using the same
US8642275B2 (en) Immunological tests for the presence of bacteria which make use of antibodies obtained using a specific method
CA2033005A1 (en) Direct binding assay for the determination of a bacteroides organism
Scheel et al. Detection of Chlamydia trachomatis in the urine of young Norwegian males by enzyme immunoassay
CA1310903C (en) Immunological diagnosis of gonococcal infection using a conserved surfaceprotein antigen of neisseria gonorrhoeae
CA2032112A1 (en) Screening assay for microorganisms associated with periodontal diseases, article and kit useful therein