Determination of antithyroid antibodies by an immunoenzymatic method; an adaptation for expressing results in international units]

Solid phase immunoenzymatic methods (ELISA) have been developed for the determination of antithyroglbulin (ATG), antimicrosomal (AMc) and antimembrane (ATMA) antibodies in blood serum. The results have been expressed in international units (IU). The level of nonspecific reaction was determined on the basis of 30 samples of blood serum obtained from healthy donors. The double standard deviation values amounted to 8 IU for antithyroglobulin antibodies, 17 IU for antimicrosomal antibodies and 53 IU for antimembrane antibodies at the serum dilution of 1:100. The values of double standard deviation obtained for the healthy donors correspond to the borderline between the positive serum samples and those containing no autoantibodies. The level of autoantibodies in patients with autoimmune diseases of the thyroid varied considerably ranging from complete absence to several thousand units per milliliter in single cases. Antithyroglobulin antibodies were determined simultaneously by using the described method and the commercial kit (Walker, Cambridge) and the results obtained by the two methods were compared. A linear correlation with the correlation coefficient r = 0.93, p < 0.001 was obtained. A good but nonlinear correlation was demonstrated with the methods expressing the results in titre values.     

Determination of Mycoplasma pneumoniae antigens and antibodies by the immunoenzyme method in patients with respiratory tract diseases

The optimum conditions for carrying out the enzyme immunoassay (EIA) with a view to determine M. pneumoniae antigen and antibodies to them in the sera of patients with different respiratory diseases were established. The use of the specially modified EIA technique made it possible to reveal that patients with tuberculosis and chronic pneumonia showed similar occurrence of M. pneumoniae (35.7% and 35.0% of cases, respectively), while in patients with pulmonary sarcoidosis M. pneumoniae occurred in 27.2% of cases. At the same time the occurrence of antibodies in patients with chronic pneumonia and sarcoidosis was more than three times greater than in tuberculosis patients.     

Determination of meningococcal antibodies by a radial hemolysis method

The possibility of using, on principle, the reaction of radial hemolysis for the determination of antibodies to meningococci has been shown. The sensitivity and resolution of this method has been found to depend on the dose of the antigen used for the sensitization of erythrocytes, on the concentration of the erythrocyte suspension introduced into the gel and on the amount of complement. The optimum conditions for the reaction of optimum hemolysis, used for the determination of antibodies to serogroup A Neisseria meningitidis polysaccharide, have been established: the sensitizing dose of the antigen must be 50-100 micrograms/ml, the concentration of sensitized erythrocytes 25%, and the amount of complement 20-40 HU.     

Place of thyroglobulin antibodies assay in laboratory diagnostic of autoimmune thyroid diseases

Tyroglobulin and thyroid peroxidase antibodies have been estimated in patients with thyroid autoimmune diseases. In a group of 109 patients with Hashimoto's thyroidities 85.53% and 78.89% were positive for Tyroglobulin antibodies and anti-TPO antibodies respectively. The anti-Tg antibodies has not been detected in 14.67% and anti-TPO in 21.1% patients. Both antibodies have not been detected in 1.83% of patients. In a group of 79 patients with Graves' disease 62.02 and 91.13% were positive for anti-Tg and anti-TPO antibodies respectively. The anti-Tg antibodies has not been detected in 37.97% and anti-TPO in 8.66% patients. Both antibodies have not been detected in one patients with exophtalmos (1.26%). Our results indicate that anti-tyroglobulin antibodies should be estimated only in patients suspected for thyroid autoimmune disease and negative for thyroid peroxidase antibodies.     

Determination of antibodies to cross-reacting antigens of microorganisms in oncological diseases

It was studied antibodies to saprophytic microorganism Bacillus megaterium H glycoprotein in healthy, oncological, non-oncological gastrointestinal patients and animals. High level of antibodies to microbial glycoprotein in blood sera was revealed in cancer and precancer patients. Analogical results were obtained in mice A/Sn and Balb/c with inductive or transplanted tumors. It has been suggested the use of Bacillus megaterium H glycoprotein (M. m. 65-70 kD) for immunological monitoring.     

Determination of membrane antigens by a covalent crosslinking method with monoclonal antibodies

Monoclonal antibodies that recognize cell surface proteins may serve as very useful tools for the study of the biological functions of membrane proteins. However, solubilization of the antigens with detergents may lead to major conformational changes of the protein, making their determination with monoclonal antibodies by immune blot or ordinary immunoprecipitation methods difficult. This is especially evident when the monoclonal antibodies recognize tertiary structures of the proteins in the membrane. We have generated two monoclonal antibodies which are specific for the cell surface antigens of multidrug-resistant human cell lines. However, the antigens of both monoclonal antibodies were difficult to detect by either immune blot or ordinary immunoprecipitation methods. We used a cleavable crosslinking reagent dithiobis(succinimidyl propionate) to covalently link the monoclonal antibody with its antigenic determinant in the membrane of intact cells. By this method, we were able to detect the antigens for these two monoclonal antibodies following solubilization, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method should have wide applicability in determination of membrane antigens recognized by monoclonal antibodies when immune blot or ordinary immunoprecipitation methods are not successful.