JPS6256980B2 - - Google Patents

Description

[Detailed description of the invention]

An object of the present invention is to provide a novel reagent for quantifying antibody d against esterase a derived from pathogenic streptococci, that is, anti-esterase antibody d. As an example of conventionally quantifying anti-esterase antibody d, Hayano, one of the present inventors, reported that
and Immunity 15(1) 295-299 (1977) is known. In this method, which can be called repetitive counterelectrophoresis, anti-esterase antibody d and esterase a, which is an antigen, are spotted close to each other in parallel on a gel plate, subjected to electrophoresis, and left overnight. After that, electrophoresis is performed again, and then a coloring reagent is sprayed onto the gel plate, and the main point is to determine the amount of anti-esterase antibody from the size of the moving spots that appear. This method requires a small amount of sample and has the advantage of being able to save the results on the board, but it is cumbersome and takes about 48 hours to obtain results, and the results are determined based on the size of the spot. This is, so to speak, a qualitative method. On the other hand, the method using the quantitative reagent of the present invention is an excellent method that not only requires a small amount of analyte but also provides highly quantitative results in a short time of about 1 to 2 hours. The present invention relates to a reagent for quantifying anti-esterase antibody d consisting of the following reagents 1, 2 and 3. Reagent 1 Esterase a derived from pathogenic Streptococcus Reagent 2 Protein b capable of binding to antibody d against esterase a derived from Pathogenic Streptococcus and binding an insoluble carrier. Reagent 3 Reagent c for quantitative determination of esterase a Here, esterase a (hereinafter also referred to as antigen) used as reagent 1 is prepared by the method described in JP-A-49-132288, for example. Obtained from culture. Examples of pathogenic streptococci include hemolytic streptococcus [Streptococcus pyogenes] [known] described in Examples below.
However, the present invention is not limited to these, and any bacteria can be used as long as it has not lost its esterase production ability, regardless of whether it is hemolytic or not, and can be obtained from various bacterial depository institutions. Esterase a is A- depending on the type of pathogenic streptococcus.
They are classified into serologically different types such as type A, type B, type C, etc., but all of these can be used in the present invention. By using the reagent of the present invention, it is possible to differentially quantify anti-esterase antibodies d according to the serological type of esterase. For example, anti-A-type esterase antibody d can be quantified using A-type esterase a, but anti-A-type esterase or anti-B-type esterase antibody d cannot be quantified, and anti-B-type esterase antibody d cannot be quantified. Type B esterase a must be used for this purpose.
The ability to perform such fractional quantification can be counted as one of the features of the present invention. In addition, as a protein that can bind to the anti-esterase antibody d used as reagent 2 and is bound to an insoluble carrier (hereinafter also referred to as a capture agent), for example, a known insoluble carrier such as a bacterial cell wall or glass beads can be bound to the insoluble carrier. Antibodies against the anti-esterase antibody d, which have been purified, or bacterial cell walls containing "Protein-A" that can bind to immunoglobulins such as Staphylococcus aureus can be used as they are, but isolated and purified "Protein-A" and Those bound with an insoluble carrier such as Cephadex (manufactured by Pharmacia) can also be used. Antibodies against anti-esterase antibody d can be obtained by parenterally administering the antibody d to an animal different from the animal that produced the antibody d,
Preferably, it is obtained by parenterally administering the immunoglobulin of the animal that produced the antibody d to another animal, and then separating the produced antibody. The resulting antibody against anti-esterase antibody d can be bound to an insoluble carrier, for example, according to the disclosure of JP-A-52-117419. The isolation and purification method of "Protein-A" and the binding of "Protein-A" to an insoluble carrier are described, for example, in JP-A-49-133597 and JP-A-49-133597.
It can be carried out according to the description in JP-A-133518. In addition, "Protein-A" is Staphylococcus
It is a protein that exists in the cell wall of bacteria such as P. aureus Cowan and can nonspecifically bind to immunoglobulins. Therefore, the "protein-A" portion in the bacterial cell wall as described above corresponds to a protein that can bind to anti-esterase antibody d, and the remaining portion corresponds to an insoluble carrier. The esterase quantitative reagent used as reagent 3 reacts with a substrate for esterase a such as S-acetylthiophenol, and a decomposition product of the substrate such as 5,5'-dithiobis-2-nitrobenzoic acid [hereinafter referred to as DTNB]. color forming agent,
Known reagents include enzyme reaction terminators such as acetone, buffering agents, and the like. It is convenient to handle these reagents 1, 2, and 3 if they are made into a kit in the form of liquid or powder, depending on their properties. The anti-esterase antibody d in a specimen can be quantified using the reagent of the present invention by sequentially performing the following steps. First step [Antigen-antibody reaction step] A step of adding reagent 1 containing esterase a, which is an antigen, to the subject and causing a reaction [antigen-antibody reaction] to generate an antigen-antibody complex e. Second step [Antigen-antibody complex capturing step] A step of reacting the antigen-antibody complex e with reagent 2 [capture agent] and capturing the complex e as a precipitate. Third step [Esterase activity measurement step] A step of measuring the activity of esterase in the precipitate obtained in the previous step. The first step can be carried out by reacting a specimen suspected of containing anti-esterase antibody d with esterase a, preferably with an excess amount of esterase a. This reaction is usually carried out in a buffer [PH6-8]
for 5 to 40 minutes at 20 to 40°C, preferably at 37°C.
This is done by incubating at a temperature around ℃. In order to remove unreacted esterase a, the subsequent antigen-antibody complex capturing step [second step] of the present invention involves adding reagent 2 [capture agent] to the anti-antigen antibody complex e produced in the first step. This can be carried out by reacting and then separating the resulting precipitate by means such as centrifugation. This reaction is usually carried out using a buffer solution [PH6~
8) for 5 to 40 minutes at a temperature of 20 to 40°C, preferably around 37°C. The final esterase activity measurement step [third step] is carried out using a known esterase quantitative reagent c.
This can be done by using For example, in this step, the precipitate obtained in the previous step is reacted with S-acetylthiophenol, which is a substrate, and the produced thiophenol is reacted with DTNB, which is a thiol-based coloring reagent, and the resulting yellow pigment is color-compared. Can be implemented. Furthermore, in quantifying anti-esterase antibody d using the antibody quantification reagent of the present invention, reagent 2 [capture agent] is added to the sample to capture the anti-esterase antibody d in the sample as a precipitate, and reagent 1 [capture agent] is added to the sample to capture the anti-esterase antibody d in the sample as a precipitate. It can also be carried out by adding esterase a] to perform an antigen-antibody reaction, and then measuring the esterase activity in the precipitate. This method is a method in which the order of addition of reagents 1 and 2 to the sample is reversed in the previously described method. Furthermore, anti-esterase antibody d can also be quantified by simultaneously adding reagents 1 and 2, incubating the mixture, and then measuring the esterase activity in the resulting precipitate. This method is easier to operate than the above two methods, and the results of both methods are in good agreement. Thus, the present invention is useful as a reagent for quantifying anti-esterase antibody d, and as a diagnostic agent for pathogenic streptococcal infections, especially hemolytic streptococcal infections (hereinafter referred to as hemolytic streptococcal infections). Next, the present invention will be explained in more detail with reference to Examples. Example 1 Production of esterase [antigen] 0.025% glucose, 0.3% beef heart infusion, 2% polypeptone, 0.2% salt, 0.2%
Streptococcus pyogenes in a medium [PH7.4] consisting of dry yeast and 0.4% dibasic sodium phosphate.
SS 379 [A-40 type] was inoculated and cultured for 16 hours at 37°C, followed by centrifugation to remove the bacterial cells. Add solid ammonium sulfate to the liquid to make it 60% saturated.
Centrifuge for a minute to obtain the precipitate. A small amount of this
Dissolve in 0.02M phosphate buffer (PH7.0), dialyze against the same solution for 8 hours, and freeze-dry to obtain 1.2 g of crude A-type esterase. Similarly, Streptococcus pyogenes 69882
[A-49 type] and Streptococcus sp H36B [B
-1-b type] to obtain A-type and B-type esterases, respectively. Example 2 Production of bacterial cell wall [capture agent] 2% bactocazamino acid, 2% yeast extract, 0.7
Consisting of % sodium lactate, 2% sodium glycerophosphate, 0.02% magnesium sulfate, 0.001% manganese sulfate, 0.32% ferrous sulfate, 0.32% sodium citrate, 0.4% potassium phosphate and 6.25% dibasic sodium phosphate. Medium [PH7.0] 50
Staphylococcus in a 100 volume fermentation tank containing
aureus Cowan and at 37℃ every minute.
Incubate for 20 hours at 50 °C aeration and 150 revolutions/min stirring speed. The culture was centrifuged to separate the bacterial cells, dispersed in water, crushed with a Dyno Mill, washed twice with 1M saline and then with water, and freeze-dried to obtain 58 g of protein-A-containing cell walls. obtain. Example 3 Production of insoluble antibody (capture agent) 0.1 M of anti-human immunoglobulin rabbit antiserum obtained by immunizing rabbits with human immunoglobulin was added
Dilute by adding 150 ml of potassium phosphate buffer [PH7.0], gradually add 200 ml of saturated ammonium sulfate solution, stir on ice for 20 minutes, and centrifuge at 1100 x g for 20 minutes. Then, dissolve the precipitate in 0.1M potassium phosphate buffer [PH7.0] to make a total volume of 100ml. 43 ml of saturated ammonium sulfate solution was gradually added to this, and the mixture was further stirred for 20 minutes under ice cooling, and then centrifuged again. Remove the supernatant, gradually add 39 ml of saturated ammonium sulfate solution, stir for 20 minutes under ice cooling, and centrifuge to separate the precipitate. This precipitate was dissolved in 0.02M potassium phosphate buffer [PH
7.0〕Dissolve in 100ml and put in dialysis tube.
Dialyze overnight against 0.02M phosphate buffer 10.
After performing this dialysis twice, the internal solution was adjusted with the same buffer so that the protein concentration was 1 g/50 ml.
Next, 20 ml of an aqueous suspension containing 1 g of Lactobacillus plantarum ATCC-8014 cell wall [insoluble carrier] was added.
Add 10 ml of 1M sodium acetate buffer [PH5.0] and mix. 120 ml of 0.8% glutaraldehyde aqueous solution was added little by little at room temperature while stirring, and stirring was continued for an additional 2 hours. The reaction mixture was then centrifuged at 6000 x g for 10 minutes to remove the precipitate at 0.1
Suspend in 500 ml of %BSA-0.9% NaCl-0.04M potassium phosphate buffer [PH7.0], sonicate at 19KC for 30 seconds, and centrifuge to collect the precipitate. Add 100 ml of the same buffer solution to this precipitate and repeat the sonication and centrifugation again. After performing this operation twice, the precipitate was added to 100 ml of the above BSA-NaCl-potassium phosphate buffer and treated with ultrasound to prepare bacterial cell wall-bound immunoglobulin/rabbit antibody [insoluble antibody].
Obtain a suspension. This insoluble antibody suspension was used as a capture agent in the Examples below. Example 4 Anti-A-type esterase antibody quantitative reagent 1: A-type esterase 50 obtained in Example 1
Dissolve mg in water to make a total volume of 10 ml. Reagent 2: Staphylococcus aureus obtained in Example 2
Disperse 750 mg of Cowan's cell wall in water and dissolve the entire amount.
10ml and eggplant. Reagent 3-1: 0.3mM-DTNB-0.05M Tris-HCl buffer [PH8.0] Reagent 3-2: 10mM-S-acetylthiophenol-ethanol solution Reagent 3-3: Acetone buffer: 0.02M phosphate buffer Solution [PH7.0] Buffer: 0.02M Tris-HCl buffer [PH8.0] Sera from patients with streptococcal infection [A and B] thought to contain anti-esterase antibody d were incubated at 57°C for 30 minutes.
Heat for a minute. Add 0.1ml of serum diluted 20 times with buffer to the test tube, and add 50μ of reagent 1 [antigen].
1 and incubated at 37°C for 10 minutes to allow antigen-antibody reaction to occur. Next, add 0.1ml of reagent 2 [capture agent].
Add and incubate again at 37°C for 30 minutes. Then, centrifuge while washing twice with buffer. The precipitate was dispersed in 1 ml of reagent 3-1 [coloring agent], and then added with reagent 3.
-2 [Substrate] Add 40 μl and incubate at 37°C for 30 minutes. Next, 2 ml of reagent 3-3 [enzyme reaction stopper] was added and centrifuged at 1700 x g for 10 minutes, and the absorbance of the yellow supernatant was measured at 412 nm in a cell with a 1 cm optical path. The titer of anti-A-type esterase antibody was 380 for serum A and 320 for serum B. The antibody titer is 5 μl.
Obtained by enzymatic reaction for 30 minutes using serum of
It is expressed as the absorbance at 412 nm multiplied by 1000. Example 5 Quantification of anti-A-type esterase antibody Hemolytic streptococcus was isolated in the same manner as in Example 4, except that the A-type esterase obtained in Example 1 was used as reagent 1, and the insoluble antibody obtained in Example 3 was used as reagent 2. The titers of anti-A-type esterase antibodies in the infectious disease patient sera [C and D] were determined and values of 852 and 662 were obtained, respectively. Example 6 Quantification of anti-B esterase antibody The titer of anti-B esterase antibody in serum E of a patient with streptococcal infection was determined in the same manner as in Example 4, except that the B esterase obtained in Example 1 was used as reagent 1. It was quantified and a value of 924 was obtained. Example 7 Differential determination Three types of esterases were prepared in the same manner as in Example 4, except that A-type, A-type, or B-type esterase was individually added as reagent 1 to streptococcal infection patient serum F and normal human serum G. The titer of anti-esterase antibody was quantified and the results shown in the following table were obtained.

[Table] Example 8 Quantification of anti-A-type esterase antibody Sera from patients with streptococcal infection [H and J] were incubated at 57°C.
Heat for 30 minutes. This was mixed with the buffer of Example 4 for 20 minutes.
Dilute it twice, take 0.1 ml of it into a test tube, add 0.1 ml of reagent 2 [capture agent] from Example 4, and hold it at 37°C.
The resulting precipitate is centrifuged and centrifugally washed with the buffer of Example 4. Add 0.1 ml of the buffer solution of Example 4 to this and stir, and then add 50 μl of Reagent 1 of Example 4 and incubate at 37°C. After 10 minutes, the precipitate was washed with the buffer of Example 4 while being centrifuged, and the esterase activity of the resulting precipitate was measured in the same manner as in Example 4. As a result, the titer of anti-A-type esterase antibody in the subject was found to be lower than serum H. For serum J, it was 720, and for serum J it was 430. Example 9 Quantification of anti-A-type esterase antibody Serum K of a patient suffering from streptococcal infection is heated at 57°C for 30 minutes. This was diluted 20 times with the buffer solution of Example 4, 0.1 ml of it was put into a test tube, 50 μl of Reagent 1 of Example 4 and 0.1 ml of Reagent 2 were added, and the mixture was heated at 37°C for 10 minutes.
After incubation for a minute, the resulting precipitate was treated in the same manner as in Example 4 to obtain an antibody titer of 475. Example 10 Comparison of various quantitative methods The titer of anti-A-type esterase antibodies in serum L, M, N, O, and P of patients with streptococcal infection was determined using the method described in Example 4, Example 8, and Example 9. The results are shown in the following table.

[Table] Represents.
Example 11 Quantification of anti-A-type esterase antibody The anti-A-type esterase antibody was quantified in the same manner as in Example 4 using the serum F used in Example 7 diluted multiple times with the buffer of Example 4. The results were obtained.

【table】

Claims (1)

[Scope of Claims] 1. A reagent for quantifying antibodies against esterase derived from pathogenic streptococci, comprising the following reagents 1, 2 and 3: Reagent 1 Esterase a derived from pathogenic streptococci Reagent 2 Esterase a derived from pathogenic streptococci Protein b capable of binding to antibody d and bound to an insoluble carrier. Reagent 3 Reagent c for quantitative determination of esterase a