Биологические науки / 9.Биохимия и биофизика

Galkin O.Yu.

National Technical University of Ukraine “Kyiv Polytechnic Institute”, Ukraine

Enzyme immunoassay for the quantitative determination of Human IgM

Determination of antibodies allows to evaluate the potential of the humoral immune response without antigenic specificity. Increasing concentrations of IgM can be observed at a number of infectious diseases (viral, intrauterine, early bacterial and parasitic, acute and chronic suppurative infection), autoimmune conditions (rheumatoid arthritis), chronic diseases of the hepatobiliary system, enteropathy, multiple myeloma etc. Acquired deficiency IgM-antibodies may be due to cytotoxic and radiation therapy, splenectomy, hastroenteropatiyeyu and burns, lymphomas. Congenital deficiency of immunoglobulin M occurs in Bruton's disease.

Often the concentration of immunoglobulins determine by nephelometry or turbidometry. However, these methods are sensitive enough to control production and determine the level of immunoglobulin secretion of B-cells such as lymphocytes in culture to the differential diagnosis of primary immunodeficiencies, or the degree of secondary immunodeficiencies caused by low levels of antibodies. Enzyme immunoassay method (ELISA) has high sensitivity, which is also necessary for such diagnostic tasks.

Thus, we faced the task of developing a highly sensitive enzyme immunoassay for the quantitative determination of human IgM. This problem can be solved using supersensitive biological reagents, such as monoclonal antibodies (McAbs).

To solve the task it was necessary to obtain a set of monoclonal antibodies to human IgM and conduct a comprehensive study of its biological properties. Monoclonal antibodies obtained by immunization of two ways - short and long term. Detailed survey data described in our previous works [1, 2].

In the study received McAbs as part of diagnostic kits for detection of specific IgM was obtained the following results. It was proved the use of conjugates consisting of two McAbs test kits for CMV diagnostics (conjugate 112H12-HRP) and Epstein-Barr virus (mixture of 112H12-HRP conjugates and 125C2-HRP), also consisting of three McAbs immunosorbents test kits for diagnosis of toxoplasmosis, rubella, and urogenital chlamydia (112C5.2 antibody) and herpes simplex virus (antibodies 125B5 and 126G6).

In the next stage of the selection performed couples McAbs for so-called IgM-«capture» ELISA. According epitop mapping A and B McAbs recognize most distant epitopes. As a result of studying the properties McAbs in ELISA for determination of specific IgM antibody epitopes A1 (111C2, 112C5.2) and B1 epitopes (125B5, 126G6) showed better results when used as part immunosorbent and A2 epitopes (112H12, 116C4) and B2 epitopes (125C5) - consisting of enzyme conjugate. So McAbs epitopes A1-B2 and B1-A2 are most likely to "sandwich"-combination for constructing non-competitive ELISA. For an optimal orientation McAbs in test system antibody epitopes A1, B1 tested as immunosorbent and epitopes A2, B2 in the form of HRP-conjugates. Sorption-detection ability of different pairs of McAbs correlated with their affinity, and was most pronounced for high affinity monoclonal antibodies 125B5 and 112H12. This pair of McAbs were used for further research.

After establishing the optimal configuration of McAbs in "sandwich"-ELISA, carried out optimization of production analysis: establishment of a working concentration of monoclonal antibodies and enzyme conjugate, time and setting conditions, sample volume and composition of the reaction buffer. The final protocol, below is the result of the work and is the optimized version of the "sandwich"-ELISA, for which analytical characteristics were determined.

Protocol of "sandwich" ELISA. McAbs specific to human IgM sorbs in 0.02 M carbonate-bicarbonate buffer at a concentration of 2 mg/ml in 96-hole plates for ELISA. Plates was incubated for 12 h at 4 °C, then washed three times by phosphate buffer saline with added 0.05% Tween-20 (PBT) and kept in a solution of BSA (10 mg/ml FSB) 1 h at 37 °C. After fourfold washing tablet by PBT holes filled 100 ml reaction buffer (0.05 M Tris-HCl buffer, pH 8.0, 0.15 M NaCl, 5 mM EDTA, 0.5 mg/ml BSA, 0,2% Tween-20, 25 mg/ml mouse McAbs, nonspecific to human IgM [3]), containing 500 ng/ml labeled with horseradish peroxidase McAbs to human IgM. Further in the hole brought to 20 ml of control samples IgM relatively standardized preparation Human IgM and serum samples of human blood previously diluted 1:1000 reaction buffer. Plates has been incubated for 2 h at 37 °C with and laundered 4 times. Further procedures were carried out as for indirect ELISA. Human IgM concentration was calculated using the gauge graphics standard (control) samples. If the concentration of IgM in the sample exceeded the value of 3.2 mg/ml should be re-studied sample analysis with additional dilution in 2-4 times.

As a result of the complex characteristics of monoclonal antibodies to human IgM was shown that antibody epitopes А1-В2 and В1-А2 are most likely to "sandwich"-combination for constructing non-competitive ELISA. Sorption-detection ability of different pairs of McAbs is correlated with their affinity. The most pronounced sorption-detection properties have high affinity McAbs 125B5 and 112H12.

Literature

1. Galkin O.Yu., Nikolayenko I.V., Dugan O.M. Obtaining and characterization of new monoclonal antibodies to human IgM // Problems of ecological and medical genetics and clinical immunology. - 2009. - Issue 5 (92). - P.105-118. (In Ukrainian).

2. Galkin O.Yu., Dugan O.M. Comparison of schemes immunization of Balb/c mice for obtaining of monoclonal antibodies to human IgM // Immunology and Allergology. - 2009. - № 1. - P. 68-73. (In Ukrainian).

3. Patent of Ukraine for utility model 26769 UA, IPC (2006) C12N5/00. Monoclonal antibody 4G11 to the vaccine strain of polio virus type II P712CH2ab / Nikolaenko I.V., Galkin O.Yu.; Nikolaenko I.V., Galkin O.Yu. - № u 2007 04447; declare. 23/04/2007, Publ. 10/10/2007. (In Ukrainian).