Reagent for Streptococcal Anti-Esterase Assay

The present invention is illustrated by the following Examples but is not limited thereto. In the Examples, "%" is % by weight, unless specified otherwise.

Preparation of esterase (a) (antigen):

A culture medium (pH 7.4, 10 liters) composed of 0.025% of glucose, 0.3% of beef heart infusion, 2% of polypeptone, 0.2% of sodium chloride, 0.2% of dried yeast and 0.4% of disodium hydrogen phosphate was inoculated with Streptococcus pyogenes SS 379, type A-40, and the medium was subjected to stationary culture at 37.degree. C. for 16 hours. The resulting culture broth was centrifuged to remove the cells. To the filtrate was added solid ammonium sulfate to give 60% saturation, and the mixture was centrifuged at 6,000.times.g for 20 minutes. The resulting precipitate was collected and dissolved in a small amount of 0.02M phosphate buffer (pH 7.0), and the mixture was dialyzed against the same buffer solution for 8 hours and lyophilized to give a powder (1.2 g) containing type A-I esterase.

In the same manner as described above, Streptococcus pyogenes 69882, type A-49 and Streptococcus sp H36B, type B-I-b were cultivated, and there was obtained each powder containing type A-II or type B esterase, respectively.

Preparation of bacterial cell walls containing "protein-A" (immuno-adsorbent):

A 100 liter fermentor containing a culture medium (pH 7.0, 50 liters) composed of 2% of Bactocasamino acid (manufactured by Difco), 2% of yeast extract, 0.7% of sodium lactate, 2% of sodium glycerophosphate, 0.02% of magnesium sulfate, 0.001% of manganese sulfate, 0.32% of ferric sulfate, 0.32% of sodium citrate, 0.4% of potassium dihydrogen phosphate and 6.25% of disodium hydrogen phosphate was inoculated with Staphylococcus aureus Cowan I, and the medium was cultivated for 20 hours at 37.degree. C. (aeration rate, 50 liters/min.; agitation speed, 150 r.p.m.). The resulting culture broth was centrifuged to isolate the cells. The cells thus obtained were suspended in water and were disrupted, and the resulting mixture was washed with 1M aqueous sodium chloride solution (twice) and with water using a centrifuge, and lyophilized to give cell walls containing "protein-A" (58 g).

Preparation of insoluble antibody (immuno-adsorbent):

An anti-human immunoglobulin rabbit antiserum (50 ml), which was prepared by immunizing rabbit with human immunoglobulin, was diluted with 0.1M potassium phosphate buffer (pH 7.0, 150 ml), and thereto was gradually added a saturated aqueous ammonium sulfate solution (200 ml). The mixture was stirred under ice-cooling for 20 minutes and then centrifuged at 11,000.times.g for 20 minutes. The resulting precipitate was collected and dissolved in 0.1M potassium phosphate buffer (pH 7.0) to make a total volume of 100 ml. To the solution was gradually added a saturated aqueous ammonium sulfate solution (43 ml), and the mixture was stirred under ice-cooling for 20 minutes and again centrifuged. The supernatant fluid was separated and thereto was gradually added a saturated aqueous ammonium sulfate solution (39 ml), and the mixture was stirred under ice-cooling for 20 minutes and then centrifuged. The resulting precipitate was collected and dissolved in 0.02M potassium phosphate buffer (pH 7.0, 100 ml). The mixture was dialyzed against 0.02M phosphate buffer (pH 7.0, 10 liters) in a dialysis tube overnight. The dialysis was repeated twice.

The dialyzed inner solution was adjusted with the same buffer solution to give a protein concentration of 1 g/50 ml. The resulting mixture was mixed with an aqueous suspension (20 ml) containing cell walls of Lactobacillus plantarum ATCC-8014 (1 g, an insoluble carrier) and 1M sodium acetate buffer (pH 5.0, 10 ml). To the mixture was gradually added with stirring 0.8% aqueous glutaraldehyde solution (120 ml) and the mixture was stirred an for additional 2 hours. The reaction mixture was centrifuged at 6,000.times.g for 10 minutes. The precipitate was collected and suspended in 0.1% bovine serum albumin (BSA) - 0.9% NaCl - 0.04M potassium phosphate buffer (pH 7.0, 500 ml) and subjected to the sonication at 19 KC for 30 seconds, and the resulting mixture was centrifuged. The resulting precipitate was collected and added to the same buffer as used above (100 ml), and the mixture was again sonicated and centrifuged. The procedure was repeated twice, and the resulting precipitate was collected and added to the same BSA-NaCl-potassium phosphate buffer as above (100 ml), and the mixture was sonicated again to give a suspension containing bacterial cell wall-bound immunoglobulin rabbit antibody (an insoluble antibody).

The suspension of an insoluble antibody thus obtained was used for the determination of anti-esterase antibody in the following Examples.

Determination of anti-type A-I esterase antibody:

reagent 1: A solution (10 ml) which was prepared by dissolving the type A-I esterase (50 mg) obtained in Example 1 in water

reagent 2: A suspension (10 ml) which was prepared by suspending the cell walls of Staphylococcus aureus Cowan I (750 mg) obtained in Example 2 in water

reagent 3-1: 0.3 mM-DTNB-0.05M Tris-HCl buffer (pH 8.0)

reagent 3-2: Substrate solution, 10 mM S-acetylthiophenol in ethanol

reagent 3-3: Acetone

buffer solution I: 0.02M Phosphate buffer (pH 7.0)

buffer solution II: 0.02M Tris-HCl buffer (pH 8.0)

Sera containing the anti-esterase antibody (d) obtained from patients infected by hemolytic streptococci (two boys of 6 and 4 years old) were used as the test material. The sera were heated at 57.degree. C. for 30 minutes.

Each serum was diluted to 20 fold with buffer solution I, and the diluted serum (0.1 ml) was added to a test tube, and thereto was added Reagent 1 (antigen) (50 .mu.l), and the mixture was incubated at 37.degree. C. for 10 minutes to proceed to antigen-antibody reaction. To the reaction mixture was added reagent 2 (immuno-adsorbent) (0.1 ml), and the mixture was again incubated at 37.degree. C. for 30 minutes. The reaction mixture was washed with buffer solution II twice and centrifuged. The resulting precipitate was collected and suspehded in reagent 3-1 (coloring agent) (1 ml) and thereto was added Reagent 3-2 (substrate) (40 .mu.l), and the mixture was incubated at 37.degree. C. for 30 minutes. After adding reagent 3-3 (enzymatic stopping reagent) (2 ml), the reaction mixture was centrifuged at 1,700.times.g for 10 minutes. The absorbance of the resulting yellow supernatant fluid was measured at 412 nm in a cell (light path: 1 cm). As a result, the serum from the boy of 6 or 4 years old showed a value of anti-type A-I esterase antibody of 380 or 320, respectively.

The absorbance multiplied by a factor of 1000 was used as the value of antibody (1000.times.A.sub.412 /5 .mu.l serum/30 min. at 37.degree. C.).

In the same manner as described in Example 4 except that the type A-II esterase obtained in Example 1 was used as reagent 1 and the insoluble antibody obtained in Example 3 was used as reagent 3, two sera obtained from patients infected by hemolytic streptococci (girl of 7 years old and boy of 4 years old) were assayed. As a result, the sera showed a value of anti-type A-II esterase antibody of 852 and 662, respectively.

In the same manner as described in Example 4 except that the type B esterase obtained in Example 1 was used as reagent 1, a serum obtained from a patient infected by hemolytic streptococci (girl of 8 years old) was assayed. As a result, the serum showed a value of anti-type B esterase antibody of 924.

Differential determination of various types of antibody:

In the same manner as described in Example 4 except that either type A-I, type A-II or type B esterase was added as reagent 1, a serum obtained from a patient infected by hemolytic streptococci (girl of 7 years old) and a serum obtained from a non-infected healthy boy (6 years old) were assayed, by which each of three anti-esterase antibodies was differentially measured. The results are shown in Table 1.

Determination of anti-type A-I esterase antibody:

Sera obtained from patients infected by hemolytic streptococci (girl of 5 years old and boy of 6 years old) were used as the test material. The sera were heated at 57.degree. C. for 30 minutes.

Each serum was diluted to 20 fold with buffer solution I as used in Example 4, and the diluted serum (0.1 ml) was added to a test tube, and thereto was added reagent 2 (immuno-adsorbent) (0.1 ml) as used in Example 4, and the mixture was incubated at 37.degree. C. for 30 minutes. The resulting precipitate was collected by centrifugation and washed with buffer solution II as used in Example 4 with a centrifuge. To the resulting precipitate was added Buffer solution I (0.1 ml) as used in Example 4, and the mixture was stirred and thereto was further added reagent 1 (50 .mu.l) as used in Example 4, and the mixture was incubated at 37.degree. C. After 10 minutes, the resulting precipitate was washed with buffer solution II as used in Example 4 with a centrifuge. The activity of esterase in the precipitate was measured in the same manner as in Example 4. As a result, the serum of the girl of 5 years old or the boy of 6 years old showed a value of anti-type A-I esterase antibody of 720 or 430, respectively.

Determination of anti-type A-I esterase antibody:

A serum obtained from a boy (5 years old) infected by hemolytic streptococci was heated at 57.degree. C. for 30 minutes. The serum was diluted to 20 fold with buffer solution I as used in Example 4. The diluted serum (0.1 ml) was added to a test tube, and thereto was added reagent 1 (50 .mu.l) as used in Example 4 and reagent 2 (0.1 ml) as used in Example 4, and the mixture was incubated at 37.degree. C. for 10 minutes. The resulting precipitate was treated in the same manner as described in Example 4, and the antibody value was measured likewise. As a result, the serum showed an antibody value of 475.

Comparison of the methods in Examples 4, 8 and 9:

With respect to several sera obtained from several children patients infected by hemolytic streptococci as mentioned in Table 2, the anti-type A-I esterase antibody was measured in the same manner as described in Examples 4, 8 and 9, respectively. The results are shown in Table 2.

Determination of anti-type A-I esterase antibody:

The serum obtained from a girl (8 years old) as used in Example 7 was diluted 2 fold successively with buffer solution I as used in Example 4, and values of the anti-type A-I esterase antibody were determined in the same manner as described in Example 4. As shown in Table 3, there was a linear relationship between the antibody values and dilution folds.

Correlation between the value of anti-type A-I esterase antibody and ASO value:

With respect to sera obtained from 102 child patients infected by hemolytic streptococci, the value of anti-type A-I esterase antibody was determined in the same manner as described in Example 4 and the data were compared with the value of an antibody against Streptolysin O (ASO value) determined by the method of Rantz-Randall [cf. Proc. Soc. Exp. Biol. Med., 59, 22 (1945)]. The correlation of both data is shown in the accompanying FIG. 1.