Conjugate polysaccharide antigen with polyethylenimine as immunosorbent assay for the detection of streptococcal and pneumococcal infections

 

(57) Abstract:

Usage area: immunology, biotechnology. The inventive synthetic immunosorbent obtained by processing the polysaccharide antigen by periodate sodium and polyethylenimine, allows to determine pneumococcal and streptococcal infections. Immunosorbent has a mol.m. about 300 million daltons, which allows its use in agglutination test in the detection of pneumococcal and streptococcal infections. table 2.

The invention relates to immunobiotechnology, namely the conjugates and immunosorbents.

Immunosorbent can be used for diagnosis of infectious and inflammatory diseases.

Known activated matrix for immobilization of proteins (Senyk A. A. and co-authors. Activated matrix for immobilization of proteins, ed.St. USSR N 1280834, CL G 01 N 33/50) formula

P-O-CH2--CH2-NH-(CH2)nwhere P is the remainder of agarose, Sephadex or cellulose; n=2 or 3 for immobilization of proteins.

Use polysaccharide matrix, which will receive in three stages.

1. Activation of the polysaccharide carrier epichlorohydrin.

2. The disclosure epoxy cycle of aminokislot proteins, containing a primary amino group, for example, transcortin, sexstoriesanime globulin immune globulins.

However, the unsuitable matrix for immobilization of polysaccharide antigens, as the latter is unable to communicate with the connecting element.

Known conjugate, in which the active group of the protein antigen associated sulfonium or alkylating or allermuir agents (agglutination reagent and the method of its production, the application of EPO N 280561, CL G 01 N 33/546).

However, binding agents suitable only for protein antigens, which are often not sufficiently specific and, therefore, does not allow to identify the disease, which reduces the accuracy of diagnosis.

Known also immunosorbent [1], which is selected as a prototype. Is a conjugate antigen is a polysaccharide-protein. Polysaccharide and protein covalently linked through

-CH2- protein. Low agglutinating properties do not allow it to be used in the solution. Small adhesive properties allow its use only with the activated solid phase carriers. In particular, the conjugate can not be used with inactivated solid phase carriers, for example, latausha good agglutinating properties, that will allow you to use it in the solution, and sufficient adhesive properties that allow its use with non-activated solid phase. This significantly extends the use of the conjugate.

The technical result is that the aldehyde group of the polysaccharide is associated polyethylenimine.

The essence of the invention lies in the fact that the conjugate has the formula

< / BR>
where n = 103-106< / BR>
From the work described in the introductory part of the description, it is known that polysaccharide is used as the antigen and activated media. Using polyethylenimine as the link is not known. Known only use it as a polycation to bind sulfo-, carboxy - and other acidic groups (Haars, A., Zonnez S. Hutterma A. Quantitative determination of lignosulfomates from sulfite spend liguors using presipitation with polyethyleneimine. Holzforsch 35(2); 59-65. 81).

Therefore, there is an inventive step.

The conjugate was prepared as follows.

Polysaccharide antigen, for example the C-polysaccharide of pneumococcus (SPSH) or A-polysaccharide of Streptococcus gr. And( APSH) is treated with periodate sodium, then add a solution of polyethylenimine. Full reaction scheme UB>2+4O2+2Na++2H+< / BR>
2.(C6H6O3(HO)2)n+ (C4H9N)n(NH2) where n = 103-106< / BR>
P R I m m e R. Dosage of the ingredients are listed per 1 mg of the polysaccharide. 1.1 mg SPCH - pneumococcus antigen dissolved in 0.1 ml of 0.1 M bicarbonate buffer pH 8. Add 0.1 ml of periodate sodium (2 mg/ml). Incubated for 1 h in the dark. Then add a drop of glycerin to remove free peerdata sodium) and by vigorously shaking pour of 0.45 ml of polyethylenimine, 2 mg/ml (mol.m. 30-40 kDa) in 0.1 M bicarbonate buffer pH 8. Incubated for 2 h

2. 1 mg of group-specific polysaccharide of Streptococcus group a antigen of Streptococcus dissolved in 0.1 ml of 0.1 M bicarbonate buffer pH 8. Add 0.1 ml of periodate sodium (2 mg/ml) in bicarbonate buffer and incubated for 1 h in the dark. To remove free periodate sodium pour in a drop of glycerine. Then add 0.45 ml of a solution of polyethylenimine (2 mg/ml) and incubated in the dark for 2 h

Thus prepared conjugates lead phosphate buffer to 40 ml and poured into wells for assay 100 ál into each well. Stand for 12 h at 4aboutC, washed on the elemental analysis.

Since the polysaccharide antigen is specific for acute infectious-inflammatory diseases, immunosorbent suitable for the diagnosis of this class of diseases. Thus sorbent with SPSH - pneumococcus antigen suitable for the diagnosis of diseases caused by pneumococcus (such as acute pneumonia, with APSH - antigen of Streptococcus group A - for the diagnosis of diseases caused by Streptococcus group A (rheumatism, tonsillitis, glomerulonephritis)), with Re - glycolipid a antigen of gram-negative microorganisms for the diagnosis of diseases caused by gram - negative organisms enterobacteria (pyelonephritis, enteritis, adnexitis, etc). For diagnostic purposes in wells with conjugate make 100 ml of 0.5% solution of bovine serum albumin in phosphate buffer, incubated 1 h at 37aboutWith, and then remove the contents from the wells and make them 100 μl of the test serum in dilutions from 1:5 to 1:640. Incubated 1 h at 37aboutC, washed 4 times add to the wells, 100 μl of antibodies to human immunoglobulins conjugated to horseradish peroxidase at a dilution of 1:200 and incubated 1 h at 37aboutC, washed 5 times, make 100 ál of substrate - orthophenanthroline (1 mg/CMA. As an example, the results of studies immunosorbent SPCH five patients.

1. Patient d, acute pneumonia. From sputum sown pneumococcus. The titer of antibodies to SPSH identified using the developed conjugate as immunosorbent assay, 1:320.

2. Sick Yu, acute pneumonia, bronchial asthma. From sputum sown pneumococcus. The titer of antibodies to conjugate with SPSH identified using the proposed immunosorbent assay, 1:640.

3. Patient C, tuberculosis of the lungs. The antibody titer of 1:40.

4. Patient N., sarcoidosis. The antibody titer of 1:10.

5. Patient G, chronic bronchitis, sown from sputum aureus. The titer of antibodies to conjugate with SPSH identified using the developed immunosorbent assay, 1:20.

These examples show that high titers of antibodies were observed in patients in the acute phase of the disease caused by pneumococcus.

In the case of use of a sorbent solution to 50 μl of the suspension of sorbent granules with media add 50 ál of test serum, diluted saline solution from 1:1 to 1:1024, incubated for at least 15 min, the reaction account for the formation of a colored precipitate.

As an example, robotopia (standard) and using the proposed immunosorbent assay in patients with streptococcal infection. The results are shown in table. 1.

From table. 1 shows that when using standard it is impossible to establish the degree of reliability at different activity of the process, since the data obtained with its help, do not correspond to the state of the process. Using this proposed immunosorbent assay marked a highly reliable difference between groups of patients.

Parallel research was conducted in the titer of antibodies on the proposed invention and prototype. Investigated the serum of patients shown in the examples described above. The results are shown in table. 2.

Analysis of the data table. 2 shows that the sorbent-prototype is not suitable due to their low molecular weight to work, as it does not have agglutinating properties. The proposed conjugate can be used as immunosorbent assay in agglutination reactions.

CONJUGATE POLYSACCHARIDE ANTIGEN WITH POLYETHYLENIMINE AS IMMUNOSORBENT ASSAY FOR THE DETECTION OF STREPTOCOCCAL AND PNEUMOCOCCAL INFECTIONS.

Conjugate polysaccharide antigen with polyethylenimine General formula

< / BR>
where n = 103- 106,

as immunosorbent assay for the detection of streptococcal and pneumococcal infections.

 

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