GB1560570A - Method of obtaining and using antigen from neisseria gonorrheae and products prepared therefrom - Google Patents

Description

(54) METHOD OF OBTAINING AND USING ANTIGEN FROM NEISSERIA GONORRHEAE AND PRODUCTS PREPARED THEREFROM (71) We, FISHER SCIENTIFIC COMPANY, a corporation organised and existing under the laws of the State of Pennsylvania, United States of America, 711 Forbes Avenue, Pittsburgh, Pennsylvania, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention resides in the fields of serology, microbiology and immunology and specifically relates to the preparation of antigen from whole cells and/or the culture medium in which the cells were grown, the use of the antigen so obtained and products containing the antigen.

According to one aspect this invention provides method of obtaining a membrane associated and/or surface antigen of Neisseria gonorrhoeae which comprises the steps of: (a) growing Neisseria gonorrhoeae whole cells in a medium: (b) separating said cells from the medium; (c) recovering from said cells and/or said medium Neisseria gonorrhoeae antigen which consists essentially of a membrane associated and/or surface antigen; steps (a), (b) and (c) being conducted while maintaining the structural integrity of the Neisseria gonorrhoeae cells with only minimal leakage of the cytoplasmic components.

A further aspect of the invention provides a method of preparing an antigen reagent for the detection of Neisseria gonorrhoeae infection which comprises the steps of: (a) adsorbing an antigen prepared by the method defined above, onto cholesterollecithin particles; and (b) dying the antigen-cholesterol-lecithin complex with a lipid solution dye.

Another aspect provides a method for carrying out an agglutination test for infection with Neisseria gonorrhoeae characterized by contacting a test spot on a card having a smooth surface with: a) a test serum; and b) an antigen reagent prepared by a method in accordance with the first aspect of the invention as aforesaid.

Yet another aspect of the invention provides a kit for carrying out an agglutination test for detection of infection with Neisseria gonorrhoeae which comprises at least one test card having a test spot thereon adapted to receive the test serum and antigen reagent; a container of an antigen reagent prepared by a method in accordance with the invention as aforesaid; a container of reactive control serum: a container of weakly reactive control serum; and a container of non-reactive control serum.

In the practice of a particular application of the present invention antigen obtained from Neisseria gonorrhoeae cells grown in a liquid culture medium and from the culture medium itself have been obtained and attached onto cholesterol-lecithin particles. The particles then serve as a vehicle for agglutination of the antigen in the presence of serum containing antibodies against Neisseria gonorrhoeae. Agglutination visibility of the antigencholesterol-lechithin complex is enhanced by dying the complex with a lipid soluble dye. In the practice of a preferred aspect of the invention a macroscopic test may be used with human serum to detect antibodies against Neisseria gonorrhoeae, the causative agent in gonorrhea. Such antibodies indicate the presence of past presence of gonorrhea. The present test generally utilizes heated serum from venous blood and indicates those afflicted with gonorrhea and those who are carriers of the disease. Test results conducted in warm-blooded animals have demonstrated that the antigen obtained from Neisseria gonorrhoeae cells and the culture medium in which they were grown also produces a vigorous antibody response in warm-blooded animals and that the antigen may be useful as in immunizing agent against gonorrhea.

Embodiments of the present invention described below specifically illustrate the growth of the organism or cells in a liquid culture medium and extraction of the antigen from the whole cells and liquid medium in which the cells were grown. However, the invention may be applicable to the growth of organisms and cells on a semi-solid or solid matrix such as gelatin, agar, agarose and mixtures thereof followed by extraction of the antigen from both the cells and semi-solid or solid matrix on which the cells were grown. In this case, the cells are scraped off the growth medium such as agar and the agar liquified by, for example, a blender, and the anigen then extracted as disclosed herein from both the cells and liquified agar.

In the practice of the present invention the solubilization of membrane bound and/or surface antigen is effected while maintaining the structural integrity of the cell with only minimal leaking of cytoplasmic components. The antigens used in the crude form (without acid precipitation and enzyme digestion) or processed further with acid precipitation and enzyme digestion may be absorbed onto cholesterol-lecithin particles to give a preferred form of diagnostic antigen reagent preparation in accordance with this invention. The usable antigen which can be used to prepare the antigen reagent includes the crude antigen from the cells which has not been acid precipitated and enzymatically digested, the acid precipitated antigen and enzymatically digested antigen from the cells, the crude antigen from the culture medium which has not been acid precipitated and enzymatically digested precipitated antigen and enzymatically digested antigen from the culture medium, or pooled mixtures thereof.

Antigens prepared according to the method of the present invention have demonstrated both in vivo and in vitro activity. The Neisseria gonorrhoeae test specifically illustrated herein is one example of the in vitro and in vivo activity of an antigen prepared according to the invention. Test results conducted in warm-blooded animals have demonstrated that antigen prepared according to the invention produces antibody response in warm-blooded animals and that such an antigen may be used as an immunizing agent, such as a vaccine, in humans. Enzyme digested Neisseria gonorrhoeae antigen have been inoculated into mice and rabbits in combination with Freunds incomplete adjuvant. Both the mice and rabbits responded serologically, producing antibodies which strongly agglutinated the antigen when attached to the cholesterol-lecithin particles. The above would indicate that enzyme digested Neisseria gonorrhoeae antigen obtained herein, may be used as a skin test antigen to detect gonorrhea in humans. For instance, in one form of test the antigen would be adjusted to the desired level of reactivity and incorporated into a dipping mixture similar to that used for the time test for tuberculosis. The tines would be dipped into this mixture and the dosage needed to elicit a skin reaction would adhere to the tips of the tine. When the points of the tine are applied to or pierce the surface of the skin. a portion of the antigen would be retained in the tissue. If there is an immune cellular response to the antigen, then a wheal and area of erythema would be evident as is seen with other skin test antigens.

Alternatively, the antigen could be inoculated intradermally and elicit the same type of response.

In preferred embodiments the invention is concerned with a method of obtaining antigen from Neisseria gonorrhoeae which comprises killing and fixing cells prior to separating said cells from the medium and in which the separated cells are extracted with an aqueous alkaline buffered alkali metal salt solution having a pH of from 6.0 to 11.0, wherein the concentration of the buffer and salt is at least 0.01 molar. In a more preferred form the aforesaid extraction step is followed by concentrating the buffered alkali metal salt extract; acid precipitating the antigen from the concentrate with an aqueous acid or acid buffer solution having a pH of from 1.0 to 6.0, wherein the concentration of the acid or buffer is at least 0.01 molar; and solubilizing the acid-precipitated antigen with an aqueous alkaline buffer solution having a pH from 6.0 to 11.0. wherein the concentration of the buffer is at least 0.01 molar before digestion of the solubilized antigen with the proteolytic enzyme.

In another preferred aspect, the recovery of the antigen is carried out by concentrating the medium containing the antigen: this recovery may include the additional steps of: (i) acid precipitating antigen from said concentrated medium with an aqueous acid or acid buffer solution having a pH of 1.0 to 6.0, wherein the concentration of said acid or buffer is at least 0.01 molar; and (ii) solubilizing said acid precipitated antigen with an aqueous buffer solution having a pH of from 6.0 to 11.0, wherein the concentration of said buffer is at least 0.01 molar.

It is generally preferred that the recovered antigen be completely digested by treatment with a proteolytic enzyme for 4 hour to four hours at 0 C to 45"C, after which digestion is stopped.

The practice of the method of the invention, in obtaining antigen from Neisseria gonorrhoeae cells grown in a liquid culture medium may, for example, comprises the steps of (a) killing and fixing Neisseria gonorrhoeae whole cells; (b) separating said cells from the liquid culture medium; (c) extracting antigen from said cells with an aqueous alkaline buffered alkali metal salt solution having a pH of from 8.2 to 8.8 wherein the concentration of the buffer is 0.1 molar; (d) acid-precipitating the concentrated antigen with an aqueous acid or acid buffer solution having a pH of from 2.0 to 4.0 wherein the concentration of said acid or buffer is 0.1 molar; (e) solubilizing the acid-preciptated antigen with an aqueous alkaline buffer solution having a pH of from 8.2 to 8.8, wherein the concentration of the buffer is 0.05 to 0.1 molar; (f) completely digesting the solubilized antigen with a proteolytic enzyme for 1/4 to 4 hours at from OOC to 45"C.; and (g) stopping the enzyme digestion of the antigen. By fixing the cells with formaldehyde it was found that the antigen became very stable; could be kept at 40C.; and remained fully active for extended periods of time.

In one aspect of the practice of the present invention, strains of Neisseria gonorrhoeae are grown in an aerated liquid culture medium for about 4 to 72 hours. The cells are then killed with a fixing agent. The preferred fixing agent is formalin. Other suitable fixing agents include any of those agents known and commonly used to fix cells or tissues for histological and/or microscopic examination. The N. gonorrhoeae cells are then removed or separated from the liquid culture medium by centrifugation, filtration, decantation, or other known means, and the culture medium retained. A preferred form of separation involves pelleting by centrifugation.

Extraction of the antigen from the killed and fixed cells is accomplished with an alkaline buffered salt solution wherein the concentration may range from at least 0.01 molar. or greater, for both the alkaline buffer and salt, optionally 0.1 molar for the alkaline buffer and 1.0 molar for the salt. A preffered alkaline buffer is sodium phosphate, however. other suitable alkaline buffers which may be used include, for example, tris (hydroxymethyl) aminomethane, Hepes(N-2-hydroxymethyl piperazine N-2-ethane sulfonaic acid), borates etc. The preferred salt is potassium chloride, however, other alkali metal salts containing potassium, sodium or lithium are deemed suitable. The pH of the alkaline buffered salt solution should be between 6.0 to 11.0. optimally, 8.2-8.8, especially 8.2-8.5.

After extraction of the antigen from the cells with the aqueous alkali buffered salt solution, the cells are separated from the extractant and the extractant concentrated, for example, by ultrafiltration, drying and/or dialysis. The concentrated extractant is then treated with an aqueous acid or acid buffer to precipitate the antigen. The antigen may be precipitated with an aqueous acid such as In hydrochloric acid or with an aqueous acid buffer at a pH of from 1.0 to 6.0 optimally pH 2.0 to 4.0, especially pH 3.6. The molar concentration of the acid buffer can range from 0.01 molar to 1.0 molar for example, optimally 0.1 molar. Suitable acid buffers include for example, acetate, McIlvanine, barbitol-acetate, etc. The preferred acid buffer is sodium acetate-acetic acid buffer. The acid precipitated antigen may then be separated from the acid or acid buffer by, for example, centifugation, washed one or more times with acid or acid buffer and the acid precipitated antigen solubilized in an aqueous alkaline buffer solution having a pH of from 6.0 to 11.0, optionally pH 8.2-8.8, especially pH 8.2-8.5. The molar concentration of this buffer can range from 0.01 molar to 1.0 molar; optionally 0.05 molar to 0.1 molar. As in the extraction step, suitable alkaline buffers include, for example. sodium or potassium phosphate, tris (hydroxymethyl)aminomethane, Hepes (N-2-hydroxymethyl piperazine, N-2-ethane sulfonic acid), borates, etc. The preferred buffer is a mixture of hydrochloric acid and tris (hydroxymethyl) aminomethane such as, for example. 0.05 molar tris (hydroxymethyl) aminomethane hydrochloride buffer at pH 8.2-8.5.

Digestion of the antigen is accomplished with the proteolytic enzymes such as the proteinases, for example. pepsin, trypsin, chymotrypsin. papain, chymopapain, etc. The preferred enzye is pepsin. There is no upper limit to the concentration of the enzyme other than what practicality dictates. The lower limit for the enzyme should be 0.01 molar per OD units of antigen at 280nm. The time and temperature ranges for digestion can range from 1/4 hor at 4 hours at from 0 C. to 450C. The preferred time and temperature for pepsin is 1 1/4 hours at about 37"C. The pH of the alkaline solubilized antigen is adjusted, if necessary and/or appropriate, for the particular enzyme used for digestion. For example, for digestion with pepsin, the solubilized antigen is adjusted on the acid side. that is, to from pH 1.0 to pH 6, preferaby pH 3.2. To stop digestion. the pH of the pepsin is returned to the alkaline side, that is, to pH 6.0 to 11.0, particularly pH 8.2 to 8.8. With other enzymes, the digestion to stopped with enzyme poisons such as mercury and cyanide, if pH adjustment is not appropriate.

The practice of the invention in obtaining antigen from the liquid culture medium in which Neisseria gonorrhoeae cells were grown may, for example, comprise the steps of (a) separating the cells from the medium; (b) concentrating the medium; (c) acid-precipitating the antigen from the medium with an aqueous acid or acid buffer solution having a pH of from 2.0 to 4.0 wherein the concentration of the acid or buffer is about 0.1 molar; (d) extracting antigen from the acid-precipitate with an aqueous alkaline buffered alkali metal salt solution or aqueous alkaline buffer solution having a pH of from 8.2 to 8.8 wherein the concentration of the buffer is 0.1 molar and that of the salt is 1.0 molar; (e) repeating step (c); (f) solubilizing the acid-precipitated antigen with an aqueous alkali buffer solution having a pH of from 8.2 to 8.8 wherein the concentration of the buffer is 0.05 to 0.1 molar; (g) completely digesting the solubilized anigen with a proteolytic enzyme for 4 hour to about 4 hours at from about O"C. to about 45"C.; and (h) stopping the enzyme digestion of the antigen.

In the preferred practice of the invention, in obtaining the Neisseria gonorrhoeae antigen from the liquid culture medium in which the cells were grown, the medium is generally concentrated such as by ultrafiltration, drying or dialyzing. If dried, the concentrate is rehydrated in aqueous acid buffer having a pH from about 1.0 to 6.0, optimally about pH 2.0-4.0, preferably about pH 3.6. The antigen obtained here is usable or it may be further purified. To do so, the insoluble material is separated from the liquid and washed several times with the acid buffer, to remove soluble material. The sediment is suspended in the acid buffer and the sediment removed by centrifugation, filtration or other acceptable methods. The sediment is dissolved in alkaline buffer, pH to 11.0, preferably pH 8.2-8.8.

Enzyme digestion is effected as set forth herein. If not dried, the material is dialyzed exhaustively against saline (0.001 to 3M, preferably 0.15M). This material may be used as the antigen or it may be further purified. To do so, the pH is adjusted 4.8-5.5 with an acid, preferably hydrochloric. The precipitate is discarded and the supernatant fluid adjusted to pH 3.6. The precipitate is removed and washed with acid buffer, pH about 3.6. The precipitate is dissolved in an alkaline buffer pH 6.0 to 11.0, preferably pH 8.2-8.8. Enzyme digestion is effected as set forth herein. Suitable acid and alkaline buffers are those referred to hereinabove.

Any of the antigen(s) obtained hereinabove from either the cells or the culture medium in which they were grown may be used alone or pooled to make the antigen reagent preparation of the invention.

In a further aspect, the invention is specifically concerned with a method of preparing an antigen reagent that is specific for the detection of infection with Neisseria gonorrhoeae which comprises: (a) absorbing the antigen(s) obtained hereinabove onto cholesterollecithin particles; and (b) drying the antigen-cholesterol-lecithin particles so obtained with a lipid soluble dye. Generally the amount of antigen employed to make the antigen reagent can range from 0.01 to 4.0 O.D. (280 nm), the amount of cholesterol from 0.7% to 1.4it, and the amount of lecithin from 0.1% to 2.0%. The amount of lipid soluble dye used to make the antigen reagent can vary from 1 mg. to 100 mg. percent. Representative dyes include, for example, Sudan Black B; Basic Brown; Amethyst Violet; Nile Blue A; Azocarmine G; and mixtures thereof. The dyes and mixtures thereof are present in the novel antigen reagent in conjunction with so]vents, buffers, etc., in a manner as is known in the art. These solvents, buffers, etc. are used as described in known antigen systems.

Usually the two components of the novel antigen reagent, i.e., antigen-cholesterollecithin complex and dye, are employed in concentrations ranging from 0.1 mg. percent to 1 gm. percent to about 100 mg. percent, by weight, of the dye, said weights based on the total weight of the resultant mixture including solvents, e.g. water, alcohol, etc., and buffer.

In the preparation of the cholesterol-lecithin particles or mixture, commercially available ash free cholesterol is generally dissolved in absolute alcohol (200 proof). Usually enough ash free cholesterol is dissolved in absolute alcohol to prepare 1.2 percent solution.

Commercially available egg, beef or vegetable lecithin, such as soy bean lecithin, is solubilized in absolute alcohol. The two constituents are then mixed with absolute alcohol in a ratio to produce a final concentration of cholesterol of from about 0.7% to 1.1% preferably about 0.9 percent, and a concentration of lecithin of from about 0.10 to about 2.0 percent, preferably about 0.22%.

The optimum amount of lecithin for the antigen suspension is determined by having successive increments of lecithin in 0.9 percent cholesterol. Antigen suspensions are prepared with each of these cholesterol-lecithin mixtures by the procedure described above.

The ratio of lecithin to 0.9 percent cholesterol which gives optional reactivity to sera from culturally positive individuals and from assumed non-infected individuals is used to prepare the antigen.

The antigen extracted as set forth hereinabove is placed in a container and the cholesterol-lecithin particles prepared as noted hereinabove are added. In current practice the bottle is agitated and the mixture allowed to stand from at least 5 minutes. The particulate matter is removed by centrifugation and resuspended in USR suspended medium. The suspension is then dyed with the desired lipid soluble dye or mixtures thereof.

In another aspect, the invention is concerned with a method for carrying out an agglutination test for infections with N. gonorrhoeae which in broad forms comprises mixing a test serum with the antigen reagent prepared hereinabove. And, in a still further specific aspect, the invention is concerned with a method for carrying out an agglutination test for Neisseria gonorrhoeae infections using a test card having thereon a test spot, such as an area delineated by a circle, adapted to receive a test sample and antigen reagent prepared as set forth hereinabove, said method comprising contacting said test spot with (a) a test serum and (b) an antigen reagent prepared as hereinabove.

The test utilizing the antigen reagent of the instant invention preferably entails the use of a test card. The test card should be smooth in order to render the test results easily discernible. Although it has been previously indicated in the prior art that the surface must be wettable, it is found that such is not the case and a wettable surface is merely preferred.

The card may be composed of well calendered paper or cardboard and may be absorbable, however, only small degrees of absorbability are preferred. One feature of the novel antigen reagent is that the test may be read whether the spot is wet or dry and therefore results can be ascertained more rapidly than when using other techniques. The card may also be a laminate of paper or cardboard base having a water-permeable or waterimpermeable material coated thereon such as polyethylene. The paper itself or the coating, howevern, should be of color which is contrasted with regard to the color of the dye employed in the test. Basically, any card similar in construction and composition to that disclosed in United States Patent No. 3,074,853 may be used.

In carrying out the test using the test card, about 0.05 ml. of serum which has been heated at about 62-63"C. for about 2 hour and about 1/60 ml. of antigen reagent is spread within the confines of a circle (e.g. about 18 mm) printed on a commercially available plastic coated card. The card is rotated on a mechanical rotator for between 4 to 20 minutes at from 80 to 180 rpm. However, hand shaking may also be effective. The card is examined for the presence of absence of characteristic clumping.

As mentioned above one aspect of the invention is concerned with a kit or package for carrying out an agglutination test for Neisseria gonorrhoeae infection which chiefly comprises at least one test card having thereon a plurality of test spots. such as areas delineated by a circle, adapted to receive the antigen reagent and the control sera; a container of antigen reagent prepared as set forth hereinabove; a container of reactive serum; a container of weakly reactive control serum; and a container of nonreactive contro serum. Other components may aso be included in the kit such as sampling tubes, rubber bulbs, etc.

As pointed out herein, tests in warm-blooded animals have demonstrated that enzyme digested Neisseria gonorrhoeae antigen obtained as set forth herein produces antibody to the antigen in warm-blooded animals and thus points toward the potential use of the anitgen as an immunizing agent in humans, it is envisaged that the antigen prepared in accordance with the invention can be used to immunize a human against Neisseria gonorrhoeae infection by administration of an immunologically effective amount fo an anitgen prepared as set forth herein. Administration may be parenterally. Antigen pepared in accordance with our invention may also be used in illiciting a response in warm-blooded animals by inoculation with said antigen.

The following examples of preferred embodiments will serve to illustrate the invention in more detail.

EXAMPLE 1 Preparation of Neisseria Gonorrhoeae Antigen from cells and medium A seed culture of Neisseria gonorrhoeae strain B370, deposited at AMERICAN Type Culture Collection, accession number 21824. is initiated from a lyophilized aliquot. After rehydrating with sterile distilled water, the fluid is transferred to chocolate eugon agar and incubated in a candle extraction jar at 36"C. for 24 hours. A two-liter volume of peptone medium is inoculated and placed on a shaker at 100 rpm in a 36"C. warm room. 1300 Ml. of liquid peptone medium is used to inoculate the fermentor (30 liter capacity). All ingredients of the media are sterilized in the fermentor except glucose which is added separately. After approximately 24 hours the culture is killed and fixed by the addition of formaldehyde to a final concentration of 2% (v/v).

The cells are separated from the culture fluid by centrifugation and the supernatant fluid is saved. The cells are extracted with 20 volumes (w/v) of 1.0 M KC1 in 0.1 M Na2HPO4, pH 8.8, overnight at 40C. and the cells removed by centrifugation. The extractant material is reduced, approximately 100 x by ultrafiltration, and then to apparent dryness by dialyzing against polyethylene glycol 6000. The dialysis bags are rinsed thoroughly and placed in 0.1 molar sodium acetate-acetic acid buffer, pH 3.6, to rehydrate and precipitate the antigen.

After centrifugation the acid precipitated antigen is solubilized in 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 8.5.

After concentrating about 70X by ultrafiltration, the culture supernate is dialyzed to dryness, using polyethylene glycol 6000. The dialysis tubes are placed in several changes of 0.1 M sodium acetate acetic acid buffer to partially rehydrate the contents and to precipitate the antigen. The sediment is removed by centrifugation and the pellet extracted with 10 volumes of 1 M KCL containing 0.1 M Na2HPO4 (pH 8.8). The supernatant is acidified with HC1 to pH 3.6 and the acid precipitated antigen is removed by centrifugation.

Solubilization of the antigen is accomplished using 0.1 M tris(hydroxymethyl)aminomethane buffer, pH 8.5.

The Tris solubilized antigen(s) extracted from the cells and the culture supernate are combined and subjected to pepsin digestion. The pH of the antigen solution is dropped to pH 3.2 by the addition of 12 N HC1. Then 10 mg. of pepsin (in 0.2 m acetic acid at 1 mg/ml) is added to 56 ml. of antigen (36 OD units of 280 nm) and incubated at 36 for 1-) hour. The reaction is terminated by the addition of 10 N NaOH until the pH is 8.5. Following inactivation of the pepsin, the digested antigen(s) are diluted 1:1 with 0.05 M tri(hydroxymethyl)aminomethane buffer, pH 8.5. The antigen prepared as above is ready for use in the preparation of the antigen reagent.

EXAMPLE 2 Preparation of Neisseria gonorrhoeae Antigen from Medium The formalin fixed cells are removed from the culture fluid by centrifugation. Fifty liters of supernatant is concentrated 100-fold using ultrafiltration. The concentrate is dialyzed against 0.85% saline and the volume restored to 500 ml. by ultrafiltration. The pH is adjusted to 5.2 and the precipitate removed by centrifugation. The supernatant is adjusted to pH 3.8 and the precipitate removed by centrifugation. The precipitate is resuspended in 100 ml. of tris(hydroxymethyl)-aminomethane buffer, pH 8.5.

EXAMPLE 3 Preparation of In Vitro Neisseria Gonorrhoeae Antigen Reagent The cell extract out and culture supernate extracted antigen prepared as in Example 1 and 2 are pooled and the pooled antigen absorbed onto the cholesterol-leci-thin particles in the following manner.

A 130 ml. solution of absolute ethanol containing 0.9% cholesterol and 0.225% egg lecithin is added dropwise to 130 ml. of solubilized antigen rotating at 160 rpm in a flat bottom container. The reaction mixture is then incubated at room temperature for 15 minutes. Following this the floc is pelleted by centrifugation at 3,000 x g for 3 hour at 4".

The flox is suspended in 1040 ml. of USR buffer and 4.55 ml. of Sudan Black B dye (10 mg/ml in absolute ethanol) is added to it while it is stirred on a magnetic stirrer. The dyed floc antigen is stored in a stoppered glass bottle at 4". The antigen reagent prepared as above is ready for use in the in vitro test.

EXAMPLE 4 In Vitro Test for Detection of Antibody to Neisseria Gonorrhoeae The test is conducted on serum which has been heated at 62-630C. for 2 hour. Five hundredths (0.05) ml. of serum and 1/60 ml. of antigen reagent prepared as in Example 3 is spread within the confines of a 18 mm diameter circle on a commercially available white test card. The card is placed on a clinical rotator for 8 minutes at 130 rpm. In a positive reaction, bright blue aggregates are visible under an incident beam of light when antibodies to the antigen are present in the serum. A negative reaction is one in which the reagent does not aggregate into discrete particles but remains as a homogeneous suspension.

EXAMPLE 5 The following illustrates the invention in kit form for the in vitro detection of Neisseria gonorrhoeae infection. The kit contains 500 determinations.

Each kit contains: 1 Antigen Reagent, 10 ml.

1 Reactive Control Serum, 1 ml.

1 Weakly Reactive Control Serum, 1 ml.

1 Nonreactive Control Serum, 1 ml.

35 Test Cards 500 Sampling Tubes Materials necessary to set up the test are included as disposable items in the kit which is designed to utilize a minimum of space. The items which need refrigeration may be separated from those which need refrigeration may be separated from those which can be stored at room temperature. A mechanical rotator (adjustable to 130 rpm) and a good light source are pieces of equipment which must be supplied by the laboratory. Refrigeration of the antigen reagent is suggested. A shelf life of more than 6 months was found when the antigen was stored at 2-10"C. Exposure of the antigen to bright sunlight should be avoided.

The antigen should be allowed to warm to room temperature (72-78"F.) before use.

Antigen used immediately after its removal from the refrigerator may not be as sensitive as properly warmed antigen. Upon completion of the daily tests, place the special cover over the tip of the dispensing needle. Place the capped bottle in the refrigerator Antigen Reagent Bottle To open the antigen reagent bottle, twist the cap in the direction of the arrow. This releases a spring loaded needle which pierces the diaphragm. The antigen dispensing system is then ready to use as described hereinafter.

Cards The cards used in the test have a special coating which permits the antigen-serum mixture to be easily spread over the test area. The cards should be handled near the edges so that the fingers do not touch the test areas since oil from the fingers may cause the improper spreading of the test within this area.

Sampling Tubes The sampling tubes are used to transfer serum when performing the Screening Test. The tube is designed to deliver approximately 0.05 ml. of serum. An unused sampling tube must be used for each test specimen.

Mechanical Rotator A rotator used for the VDRL slide test is satisfactory for use with this test if the speed can be adjusted to 130 rpm [Operating requirements: Platform to circumscribe a circle of 4 inch in diameter on a horizontal plane.] Light Source for Reading Tests A light, such as fluorescent desk lamp with two 15 watt bulbs or a high intensity light, is satisfactory. Most all reactions are visible with the light available in an adequately lit laboratory. However, marginal reactions may be missed if adequate light is not used.

Control Sera It is recommended that control sera be used to test the reactivity of the antigen. The sera are: Designation Reactivitv NR Nonreactive WR Weakly reactive SR Strongly reactive Serum Collect blood by venipuncture into clean, dry tubes and allow to clot. If necessary, centrifuge the specimens with sufficient force to sediment cellular elements. Remove serum from the clot and place in a clean, dry tube. Just before testing, heat to 62-630C. for 2 hour.

Test Procedure 1. Hold the plastic sampling tube between the thumb and forefinger near the sealed end. Compress the tube and insert the open end into the sample and release the finger pressure to draw up the sample, being careful not to disturb the clot or sedimented materials.

2. Hold the plastic sampling tube in a perpendicular postion over the test circle. Do not touch the card surface. Squeeze the sampling tube and allow 1 drop to fall onto the test area. Discharge the remaining sample back into the specimen tube.

3. Invert the plastic sampling tube, and with the stirring end (the sealed end) spread the specimen within the confines of the circle.

4. Gently shake antigen dispensing bottle before use. Hold in a vertical position and dispense 3 drops in a corner of the card. Then place 1 free-falling drop on each test area.

Pick up the antigen from the corner of the card.

5. Place the sample on a mechanical rotator under a humidity cover and rotate at 130 rpm for 8 minutes.

6. Examine the sample under bright light. Strongly and moderately reactive sera are obvious by the presence of bright blue aggregates. Minimally reactive sera are detected by moving the drop of sample back and forth to observe the small aggregate.

7. Report as: Reactive: Any characteristic clumping from minimal to strongly reactive. (Compare with control sera).

Nonreactive: No clumping or roughness.

EXAMPLE 6 In Vivo Antibody Response to Neisseria Gonorrhoeae Antigen Two rabbits were treated by subcutaneous inoculation with enzyme digested N.

gonorrhoeae antigen. The inoculum had been dialyzed against 0.85% NaC1 prior to its use.

One-half ml. of antigen was given subcutaneously in divided dosage in several sites. The animal received a booster inoculation. Fourteen days after the final inoculation the sera from the animals was tested using an enzyme digested N. gonorrhoeae antigen on cholesterol lecithin particles. Titers ranged up to 1:128. This example illustrates in vivo antibody response to the antigen of this invention and its potential use as an immunizing agent in humans.

EXAMPLE 7 In Vivo Antibody Response to Neisseria Gonorrhoeae Antigen Two goats were treated by subcutaneous inoculation with acid precipitated Neisserie gonorrhoeae antigen. The inoculum had been dialyzed against 0.85% NaC1 prior to it use.

Two ml of antigen was given subcutaneously in divided dosage in several sites. The animals received a booster inoculation one week later. Fourteen days after the initial inoculation the animals were tested using the Neisseria gonorrhoeae antigen on cholesterol-lecithin particles. Titers were 1:4 and 1:8 dilutions of serum. Over three weeks bleeding titers ranged from 1:8 to 1:16. One goat was treated by i.v. inoculation of the Neisseria gonorrhoeae antigen and developed a titer of 1:32. This example illustrates in vivo anit-body response to the antigen of this invention and its potential use as an immunizing agent, e.g. vaccine in man.

WHAT WE CLAIM IS: 1. A method of obtaining a membrane associated and/or surface antigen of Neisseria gonorrhoeae which comprises the steps of: (a) growing Neisseria gonorrhoeae whole cells in a medium; (b) separating said cells from the medium; (c) recovering from said cells and for said medium Neisseria gonorrhoeae antigen which consists essentially of a membrane associated and/or surface antigen; steps (a), (b) and (c) being conducted while maintaining the structural integrity of the Neisseria gonorrhoeae cells with only minimal leakage of the cytoplasmic components.

2. A method according to Claim 1 including the step of killing and fixing said cells prior to separating said cells from the medium.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (15)

**WARNING** start of CLMS field may overlap end of DESC **. Serum Collect blood by venipuncture into clean, dry tubes and allow to clot. If necessary, centrifuge the specimens with sufficient force to sediment cellular elements. Remove serum from the clot and place in a clean, dry tube. Just before testing, heat to 62-630C. for 2 hour. Test Procedure 1. Hold the plastic sampling tube between the thumb and forefinger near the sealed end. Compress the tube and insert the open end into the sample and release the finger pressure to draw up the sample, being careful not to disturb the clot or sedimented materials. 2. Hold the plastic sampling tube in a perpendicular postion over the test circle. Do not touch the card surface. Squeeze the sampling tube and allow 1 drop to fall onto the test area. Discharge the remaining sample back into the specimen tube. 3. Invert the plastic sampling tube, and with the stirring end (the sealed end) spread the specimen within the confines of the circle. 4. Gently shake antigen dispensing bottle before use. Hold in a vertical position and dispense 3 drops in a corner of the card. Then place 1 free-falling drop on each test area. Pick up the antigen from the corner of the card. 5. Place the sample on a mechanical rotator under a humidity cover and rotate at 130 rpm for 8 minutes. 6. Examine the sample under bright light. Strongly and moderately reactive sera are obvious by the presence of bright blue aggregates. Minimally reactive sera are detected by moving the drop of sample back and forth to observe the small aggregate. 7. Report as: Reactive: Any characteristic clumping from minimal to strongly reactive. (Compare with control sera). Nonreactive: No clumping or roughness. EXAMPLE 6 In Vivo Antibody Response to Neisseria Gonorrhoeae Antigen Two rabbits were treated by subcutaneous inoculation with enzyme digested N. gonorrhoeae antigen. The inoculum had been dialyzed against 0.85% NaC1 prior to its use. One-half ml. of antigen was given subcutaneously in divided dosage in several sites. The animal received a booster inoculation. Fourteen days after the final inoculation the sera from the animals was tested using an enzyme digested N. gonorrhoeae antigen on cholesterol lecithin particles. Titers ranged up to 1:128. This example illustrates in vivo antibody response to the antigen of this invention and its potential use as an immunizing agent in humans. EXAMPLE 7 In Vivo Antibody Response to Neisseria Gonorrhoeae Antigen Two goats were treated by subcutaneous inoculation with acid precipitated Neisserie gonorrhoeae antigen. The inoculum had been dialyzed against 0.85% NaC1 prior to it use. Two ml of antigen was given subcutaneously in divided dosage in several sites. The animals received a booster inoculation one week later. Fourteen days after the initial inoculation the animals were tested using the Neisseria gonorrhoeae antigen on cholesterol-lecithin particles. Titers were 1:4 and 1:8 dilutions of serum. Over three weeks bleeding titers ranged from 1:8 to 1:16. One goat was treated by i.v. inoculation of the Neisseria gonorrhoeae antigen and developed a titer of 1:32. This example illustrates in vivo anit-body response to the antigen of this invention and its potential use as an immunizing agent, e.g. vaccine in man. WHAT WE CLAIM IS:

1. A method of obtaining a membrane associated and/or surface antigen of Neisseria gonorrhoeae which comprises the steps of: (a) growing Neisseria gonorrhoeae whole cells in a medium; (b) separating said cells from the medium; (c) recovering from said cells and for said medium Neisseria gonorrhoeae antigen which consists essentially of a membrane associated and/or surface antigen; steps (a), (b) and (c) being conducted while maintaining the structural integrity of the Neisseria gonorrhoeae cells with only minimal leakage of the cytoplasmic components.

2. A method according to Claim 1 including the step of killing and fixing said cells prior to separating said cells from the medium.

3. A method according to Claim 2, including extracting the membrane associated

and/or surface antigen from the killed and fixed cells with an aqueous buffered alkali metal salt solution having a pH of from 6.0 to 11.0, wherein the concentration of said buffered salt is at least 0.01 molar.

4. A method according to Claim 3, wherein said recovery is conducted by: (a) concentrating said buffered alkali metal salt extract; (b) acid precipitating said antigen from said concentrate with an aqueous acid or acid buffer solution having a pH of from 1.0 to 6.0, wherein the concentration of said acid or buffer is at least 0.01 molar; and (c) solubilizing said acid precipitated antigen with an aqueous buffer solution having a pH of from 6.0 to 11.0, wherein the concentration of said buffer is at least 0.01 molar.

5. A method according to Claim 1, wherein said recovery is conducted by concentrating the medium containing the antigen.

6. A method according to Claim 5, wherein said recovery includes the additional steps of: (a) acid precipitating antigen from said concentrated medium with an aqueous acid or acid buffer solution having a pH of 1.0 to 6.0, wherein the concentration of said acid or buffer is at least 0.01 molar; and (b) solubilizing said acid precipitated antigen with an aqueous buffer solution having a pH of from 6.0 to 11.0, wherein the concentration of said buffer is at least 0.01 molar.

7. A method according to any one of the preceding Claims, including the step of completely digesting the recovered antigen with a proteolytic enzyme for 4 hour to four hours at from OOC to 45"C.

8. An antigen prepared in accordance with any one of Claims 1 to 7.

9. A method of preparing an antigen reagent for the detection of Neisseria gonorrhoeae infection which comprises the steps of: (a) adsorbing an antigen according to Claim 8, onto cholesterol-lecithin particles; and (b) dying the antigen-cholesterol-lecithin complex with a lipid solution dye.

10. An antigen reagent prepared according to Claim 9.

11. A method for carrying out an agglutination test for infection with Neisseria gonorrhoeae characterized by contacting a test spot on a card having a smooth surface with: a) a test serum; and b) an antigen reagent in accordance with Claim 10.

12. A kit for carrying out an agglutination test for detection of infection with Neisseria gonorrhoeae which comprises at least one test card having a test spot thereon adapted to receive the test serum and antigen reagent; a container of an antigen reagent of Claim 10; a container of reactive control serum; a container of weakly reactive control serum; and a container of nonreactive control serum.

13. A method of obtaining a membrane associated and/or surface antigen from Neisseria gonorrhoeae, substantially as described with reference to Examples 1, or 1 to 3.

14. A method according to Claim 11, substantially as described herein, with reference to any one of Examples 4 or 5.

15. A kit according to Claim 12, substantially as described herein, with reference to Example 5.