Method of determining iga-proteinase activity

FIELD: medicine.

SUBSTANCE: immunoglobulin-A is sorbed in microplate wells, then after incubation and treatment, a solution containing a proteolytic enzyme is introduced in the microtablet wells. It is followed with incubation; the contents of the wells are poured out, and then an enzyme conjugate, e.g. peroxidase, with immunoglobulin-A Fc-fragment antibodies and a substratum of this enzyme are introduced. After incubation of a reaction mixture, reaction results are accounted by a spectrophotometre. IgA-protease activity is calculated by the amount of hydrolysed substratum of the enzymatic reaction.

EFFECT: use of the invention allows facilitating determination of the IgA-protease activity level, the method exhibits good result reproducibility, high sensitivity, low substratum consumption and the absence of necessity to use of immunoglobulins of certain specificity.

1 dwg, 1 tbl, 4 ex

 

The invention relates to the field of biochemistry, and in particular to methods IgA-proteinase activity, and can be used in Enzymology, Microbiology, pharmacology, clinical biochemistry for screening and quantitative estimation of activity of proteolytic enzymes.

Proteases of pathogenic and conditionally pathogenic microorganisms play a significant role in the pathogenesis of the lesions. Much attention is paid to microbial proteolytic enzymes, which break down molecules of immunoglobulins. To detect the activity of these enzymes have been developed appropriate methods [1, 2, 3]. However, a semiquantitative estimate of the proteolytic activity of these methods are not amenable to standardization and therefore difficult to apply in practice. The most promising of modern methods of IgA-proteinase activity is enzyme immunoassay due to its sensitivity, selectivity and the possibility of automation of the analytical process. However, the method enzyme immunoassay IgA-by activity, is closest to our invention [4], requires the manufacture and sorption on the microplate bacterial antigens, specific to the used bacterial antigen immunoglobulins or, otherwise, high flow pool especificaciones, carrying out the enzymatic reaction in a separate container.

The purpose of the claimed invention is to develop a method that allows to quantify the level of IgA by the activity of enzyme-linked immunosorbent assay without the use of bacterial antigens with carrying out the enzymatic reaction directly in the wells of the microplate.

This object is achieved through the development of a detection method, which involves sorption in the wells of the microplate immunoglobulin a, then the introduction into the wells of the microplate solution containing proteolytic enzymes, conducting incubation, resulting in the cleavage of immunoglobulin molecules and their desorption, pouring the contents of the hole, and then adding the enzyme conjugate with antibodies against the Fc fragment of immunoglobulin a, the substrate of this enzyme and the calculation of IgA-proteinase activity on the amount of hydrolyzed substrate enzymatic reaction.

The technical result of the claimed invention to provide a simplified method of determining the level of IgA-proteinase activity characterized by good reproducibility, high sensitivity, low consumption of the substrate and the possibility of using antibodies regardless of their specificity.

PR is measures 1. Determination of IgA-proteinases the activity of trypsin. Dissolve human IgA in 0.05 M sodium phosphate buffer, pH 7.4, at concentrations of 0.1-1 µg/ml and contribute 50 ál of solution in each well of flat-bottomed polystyrene 96-well plate. Close the lid and leave overnight at 4°C. Three times washed tablet 0.15 M sodium phosphate buffer solution, pH 7.4, containing 0.05% tween-20, filling 100 ál in each well, then the tablet dry by shaking out the remaining liquid. In wells of a number of bring in 100 μl of 0.05 M Tris-Hcl buffer solution with pH 8.0. In one of the wells contribute 100 ál of trypsin solution in the same buffer at a concentration of 1 µg/ml. Then produce a double dilution of trypsin, using more of the following hole. After incubation in an incubator for 1 h at 37°C, three cleaning 0.15 M sodium phosphate buffer solution, pH 7.4, containing 0.05% tween-20, and drying the tablet into each hole making 100 μl of peroxidase conjugate with antibodies against the Fc fragment of immunoglobulin a in the same buffer at selected breeding. After incubation in an incubator for 1 h at 37°C, three times washing with detergent and drying the tablet into each hole making 100 ál of substrate solution (5 mg OPD in 0.1 M sodium citrate buffer, pH of 9.0, and 8 μl of hydrogen peroxide). After 30 min incubation in the dark, the reaction is stopped by the bearing to each well 50 µl of 25% sulfuric acid. The results of the reaction consider using a spectrophotometer with a vertical beam of measuring light absorption at 492 nm. The results are shown in table 1. IgA-proteinase activity calculated from the standard curve (see drawing), using the formula

A=(Ck-Co)/tCf,

where a is the activity of the enzyme (in the usl. units) activity

Ckthe concentration of immunoglobulin in control,

Withaboutthe concentration of immunoglobulin in the hole, where he made a solution of enzyme

T - time proteolysis,

Cfthe concentration of enzyme in the sample solution.

Example 2. Determination of IgA-proteinase activity carried out analogously to example 1, but instead of an enzyme solution used culture liquid culture of bacteria. IgA-proteinase activity count on the amount of protein in the culture fluid.

Example 3. Determination of IgA-proteinase activity carried out analogously to example 1, but instead of an enzyme solution used disintegrate bacterial cells. IgA-proteinase activity count on the amount of protein in desintegrate.

Example 4. Determination of IgA-proteinase activity carried out analogously to example 1, but instead of an enzyme solution is used, the suspension of bacterial cells. IgA-proteinase activity count on the number of cells in a liquid.

Table 1
According to the optical density (OD) of the concentration of trypsin.
Trypsin, ng/mlOD 492 nm
9760,075
4880,240
2440,318
1220,423
610,512
300,713
150,815
7to 0.900
01,100

LITERATURE

1. Bleeg H.S., Reinholdt j, Kilian M. Bacterial immunoglobulin A proteases monitored by continuous spectrophotometry //FEBS Lett. - 1985. - Vol.188. - No 2. - P.357-362.

2. Qiu J., Brackee G.P., Plaut A.G. Analysis of the specificity of bacterial immunoglobulin A (IgA) proteases by a comparative study of ape serum IgAs as substrates //Infect Immun. - 1996. - Vol.64. - No 3. - P.933-937.

3. Wiesner R., Troll, W. A new assay for proteases using fluorescent labeling of proteins //Ibid. - 1982. - Vol.121. - No 2. - P. 290-294.

4. Tyurin Y.A. Proteindna activity of the intestinal microflora in acute intestinal infections in children. //Abstract. dis. Kida. the honey. Sciences. - Kazan, 2003, p.21.

The method of determination of IgA protein is heat activity, characterized in that adsorb in the wells of the microplate immunoglobulin-a And then into the wells of the microplate make a solution containing a proteolytic enzyme, spend incubation, pour the contents of the wells, and then make the enzyme conjugate with antibodies against the Fc fragment of immunoglobulin a and the substrate of this enzyme, followed by calculation of IgA-proteinase activity on the amount of hydrolyzed substrate of the enzymatic reaction.



 

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