1

NEPHRITIS STRAIN ASSOCIATED PROTEIN

It has been observed that acute post-streptococcal glomerulonephritis (APSGN) results from infection with a limited number of Group A streptococci. More specifically, not all streptococcal infections have APSGN as a necessary sequela.

It has been observed further, especially in the third world nations, that the incidence of risk for APSGN is greatest in early childhood, i.e. from about the third to the tenth year. It appears that up to the age of about 3, children are protected by residual maternal immunity. After the age of about 10 most children will have developed their own immunity. It is desirable, therefore to have a screening method of testing children from the age of about 3 to 10 to determine if they are harboring an APSGN infection. When such children are found to be negative to a screening test, they can be protected by prophylactic treatment with, for example penicillin. A positive test indicates that the child has already been exposed and developed immunity.

A screening test which would not cause an unacceptable number of false positives could be based on the use of an antigen to the antibodies produced by the child's own immune system in response to the specific streptococcus which give rise to APSGN.

There are several reports of the isolation of an extracell protein (NASP) from Group A streptococci obtained from patients diagnosed as having developed APSGN. See for example Villareal et al., J. Exp. Med 1 9, 459 (1978) and Ohkuni et al., Clin. Exp. Immunol. S _, 185 (1983). The protein, however, has not been isolated except in experimental quantities associated with a number of impurities and, because of the isolation Drocedure, in denatured form.

A procedure has now been developed which permits the isolation of useful quantities of the nephritis associated protein from group A, C and G streptococci in purified non- denatured form suitablefor the production of test kits where false positives are not unacceptably high. This procedure makes possible the production of diagnostic kits based on NSAP and useful to screen large populations for APSGN. The purified NSAP is also useful to produce monoclonal antibodies to itself. These can be used as absorbents for the isolation of additional NSAP. NSAP can also be modified to produce a product which retains the ability to generate an immune response but does not result in overt infection. The modified NSAP would, therefore, be a useful vaccine.

There follows a typical procedure for the isolation of NSAP utilizing as a source a nephritis positive strain of streptococci. The procedure is illustrated with a strain identified as A374 in the collection at the Rockefeller University. It was originally isolated from an APSGN positive patient. The procedure is applicable to any APSGN positive strain.

Strain A374, pre-adapted to growth in the chemically defined medium of (CDM) Van de Rijn et al. , J. Exp. Med. 149, 459(1980), was inoculated into 2 liter volumes of CDM to an initial optical density of 0.05 (550 nm) . Cultures were grown at 37°C in roller bottles (Falcon 3027, VWR, South Plainfield, NJ) and rotated at 1 rpm. The pH of the culture was monitored by sterile removal of 10 ml aliσuots at hourly intervals; a pH of 6.8 was maintained by addition of sterile 5N NaOH. At early stationary phase of growth, iodoacetamide (1 mg/ml) , phenylmethylsulfonylfluoride (ImM) and ethylenediaminetetracetic acid (5 mM) were added. The culture ws centrifuged at 10,000 x g for 25 min at 4°C the supernatant was decanted and filtered through a 0.22 mu

cellulose acetate membrane (Millipore Corp, Bedford, MA).

Solid ammonium sulfate (Type III, Signa, St. Louis, MO) was added to the filtered supernatant to a final concentration of 70% saturation. After stirring at 4°C for 18 h the suspension was centrifuged at 10,000 x g at 4°C for 25 min. The pellet was washed twice with 70% saturated ammonium sulfate. The washed precipitate was suspended in 50 mM TrisOCHl buffer pH

7.5 and dialyzed against several changes of same buffer until all traces of ammonium sulfate were removed. Residual sulfate in the dialysate was tested by adding 100 ul 1%

(w/v) barium acetate, and 200 ul IN HCl to 1 ml dialysate.

Dialyzed material was applied to 1.5 cm x 20 cm column of DEAE-Sepharose 6B-C1 (Pharmacia, Piscataway, NJ) equilibrated with 50 mM Tris-HCl buffer, pH 7.5. After application of sample and elution with four column volumes of 50 mM Tris- CH1 buffer, pH 7.5 a sodium chloride gradient (0- 0.5 M) in 50 mM Tris-HCl buffer pH 7.5 was applied. Fractions were collected and assayed for absorbance at 280 nm and NSAP by Dot-blot analysis using monoclonal antibody to NSAP (Mab NSAP) prepared as described hereinafter. Fractions which were Mab-NSAP reactive were pooled and dialyzed against 25 M piperazine-CHl buffer, pH 5.5. After dialysis the sample was applied to a 0.7 cm x 50 cm column of polybuffer exchanger (PBE 94, Pharmacia, Piscataway, NJ), previously equilibrated with 25 mM piperazine-CHl buffer, pH 5.5. After application of sample and elution with five volumes of start buffer, 12 volumes of a 1:10 dilution of Polybuffer 7 4 (Pharmacia, Piscataway, NJ), titrated to pH 4.0 with 1 N HCl were added. The column was eluted at a constant flow rate of 15 cm h~l. Fractions were monitored at 280 nm and by Dot-blot analysis using Mab-NSAP as a probe. Fractions containing NSAP were pooled and dialyzed against 0.1M NaHC03, pH 8.0 prior to isolation of the desired product by lyophilization.

The lyophilized product thus obtained is non-denatured and highly purified. The yield of pure product is approximately

10,000 % higher than the yield of impure product obtained by previous methods.

Mab-NSAP are prepared from the product of the invention in the usual way. The product is injected into BALB/c mice and spleen cell/myeloma hybrids are prepared. The hybrid supernatants are screened against the extracellular products from a nephiritic streptococcal strain, for example by enzyme linked im unoassay (ELISA) to locate the positive clones.

The NSAP of this invention can be utilized in a screening test for APSGN. In the test sera from the patient under test is reacted with NSAP, normally in a diagnostic composition such as a buffered aqueous composition containing a diagnosticall effective amount of NSAP and thereafter, in the case of a positive test, detecting the presence of a reaction product.

Any of a large number of clinical tests may be employed utilizing the NSAP of this invention. Typical tests include radioimmunoassay, enzyme linked immunoassay, precipitation, agglutination, direct and indirect immunofluorescence, and complement fixation. These tests may employ competitive and sandwich type assays. The tests may employ detectable labels in accordance with standard practice. Useful labels include fluorescent labels such as fluorescein, rhodamine or auramine. Radiosotopes such as l^C, 131j _/ 125j _an( j 25s may be employed. Enzyme labels which may be utilized include, for example, - glucamidase, ^-glucosidase, ^-D-galactosidase, urease, glucose oxidase plus peroxidase, and acid or alkaline phosphatase. Methods for labeling biological products such as cells, antibodies, antigens and antisera are well known and need not be described. A "diagnostically effective amount" of NSAP will vary appreciably with a number of factors well known to those skilled in the art. These include, for example, the sensitivity of the test employed, the instrumentation available and the amount of sample under test.

All of the tests which may be usefully employed in accordance with this invention involve the formation of a detectable reaction product which includes the NSAP of the invention and an antibody present in a positive serum. Of course there may be other components such as an antiantibody in the detectable reaction product. Either the NSAP or the antiantibody may be labeled.

In a typical test, blood or serum aliquots from the individual under test are added to a physiological saline solution, which is then added to a mixture containing NSAP in labeled form. If there is a positive reaction, the reaction product can be detected visually or by using an instrument adapted to the selected label.

A wide variety of diagnostic kits can be visualized utilizing the purified NSAP of the invention. Generally those kits will include NSAP adsorbed . to a substrate.

Any of a variety of adsorbents can be used. These include for example glass, plastic or other inert surfaces which may be the inner surfaces of test tubes or a surface of a test plate. Typical examples of flat surfaces especially useful in the enzyme linked immunoassay procedure (ELISA) or the radioimmunoassay procedure (RIA) include glass, nitrocellulos paper or plastics such as polystyrene, polycarbonate or various polyolefins. Particles which can be used for macroscopic procedures wherein the reaction product can be detected visually, e.g., the hemagglutination procedure, include biological particles such as sheep red blood cells or human group 0 red blood cells, and biologically inert particles such as charcoal, bentonite or latex beads. The latter can be formed of polystyrene, polyvinylpyrrolidone or various polyolefins.

A test kit could comprise lyophilized NSAP in a glass vial. The NSAP would be removed from the vial in a coating buffer, e.g., bicarbonate buffer at a pH of from about 9.2 to 10. Aliquots of the composition containing about lOug/ml of NSAP can be placed in the wells of a test plate and, after appropriate inclubation and washing, reacted with the suspected sera.

An alternate test kit would be a test plate with absorbed NSAP which had already been treated with a coating buffer. The sera under test can be added to the well.

One extremely useful test kit comprises a polystrene or other inert absorbent in the form of a "dip stick". The stick can be utilized as a prob by inserting into the test sera. A positive aggulation reaction, if there is one, can be visually observed. The same results can be accomplished with small particles, such as polystyrene beads coated with NSAP. These can be suspended in the plasma and the results observed.

The common characteristic of the test kits is that they comprise NSAP absorbed on an inert surface.

The NSAP of this invention has a molecular weight of about 46k daltons. It will activate plasminogen to produce plasmin. Above pH 9 it loses its ability to activate plasminogen but retains its antigenic activity. The first amino acids on the amino terminus are

10 Ile-Ala-Gly-Pro-Glu-Trp-Leu-Leu-Asp-Arg

20 -Pro-Ser-Val-Asn-Asn-Ser-Gln-Leu-Val-Val-Ser

It is believed to be a streptokinase, but it is unique in that no other streptokinase has all of the same chemical, physical and immunogenic properties.