Detection of soluble antigens of blood group ABH using immunoenzyme analysis

The use of the indirect ELISA techniques did not ensure the sharp differentiation of the antigens of the blood groups A and B on the polystyrene sorbent by means of heteroimmune sera, though such differentiation could be achieved by means of monoclonal antibodies. The test system known as "the lectin-antibody sandwich" was found to have the optimum sensitivity and specificity permitting the detection of soluble ABH antigens. This variant of ELISA permitted the detection of blood group A antigen both in native biological materials and in traces of blood and saliva, thus making it possible to carry out its quantitative determination.     

Use of an artificial antigen simulating the Salmonella O-determinant 4 for determining antibodies to Salmonella group B O-antigen in immunoenzyme analysis

The use of the artificial antigen abequosylmannoside copolymer with acrylamide in the enzyme immunoassay for the determination of antibodies in the sera of salmonellosis patients has enhanced the specificity of the serological diagnosis of group B salmonellosis in comparison with the use of the natural antiren, S. typhimurium lipopolysaccharide.     

The use of immunoenzyme analysis in the laboratory diagnosis of plague]

The possibility of using the enzyme immunoassay (EIA) for the early diagnosis of pneumonic plague was studied in experiments on monkeys. EIA was shown to be more effective than the passive hemagglutination test. The diagnostic value of blood serum samples was found to be higher than that of nasopharyngeal mucus samples taken from the sick animals. The conclusion on the suitability of EIA for the early laboratory diagnosis of this disease was made.     

The use of immunoenzyme analysis and immunoblotting for the serological characterization of mycobacterial antigens]

The enzyme immunoassay and immunoblotting were used for the study of the serological activity of different mycobacterial antigens and the spectrum of antibodies to them in patients with different forms of tuberculosis and healthy persons. Antibodies in patients' sera were shown to bind antigens with different molecular weight. The level and spectrum of antibodies to purified protein fraction I made it possible to differentiate between patients with various forms of tuberculosis and healthy persons.     

The type subtyping of meningococci by means of whole-cell immunoenzyme analysis]

In this work the method of the whole-cell enzyme immunoassay, used for the serotype-subtyping of meningococci by means of specific monoclonal antibodies, is described. High specificity of the method, the simplicity of the assay procedure and evaluation of its results, as well as the availability of this method for practical use, have been demonstrated. The results of this investigation confirm the importance of the evaluation of type-subtype appurtenance of reference and laboratory strains used in experiments. Study of 72 meningococcal strains obtained from patients has revealed their polyclonal character in respect of their type-subtype signs.     

The immunoenzyme analysis of ceruloplasmin

A highly specific and sensitive enzyme immunoassay (EIA) system, suitable for the qualitative analysis of ceruloplasmin, has been developed. The possibility of its use for the examination of children with mononucleosis and pseudotuberculosis has been studied. An increase in the concentration of ceruloplasmin has been more pronounced in infectious mononucleosis (0.506 +/- 0.026 g/l) and pseudotuberculosis (0.421 +/- 0.157 g/l). The results of EIA coincided with the data obtained by radial immunodiffusion.     

Optimization of the parameters that permit improvement in plate sorption activity for solid-phase immunoenzyme analysis]

The possibility of increasing the sorption activity of polystyrene plates with an initially low capacity for sorption has been shown. The qualitative and quantitative parameters of the process of physical modification have been ascertained. The empirical formula for achieving the highest degree of the sorption activity of plates by their definite exposure to ultraviolet radiation has been obtained.     

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096 ng/ml, and the linear range was 0.1-3500 ng/ml with a regression coefficient (R²) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.     

Use of immunoenzyme analysis for the detection and differentiation of influenza viruses A, B and C

Test systems for ELISA, containing antibodies to internal or supercapsid proteins and intended for the detection and differentiation of influenza A, B and C viruses in small amounts, have been developed. The possibility of using these systems for the determination of viruses both in material obtained directly from humans and in chick embryo isolates has been demonstrated.     

Increased specificity of immunoenzyme analysis for diagnosing meningococcal infection

The ELISA test system for the detection of polysaccharide antigens of meningococci, groups A and C, on the basis of the neutralization of specific antibodies has been developed. The specificity of this reaction is determined by the chemically pure preparations of group A and C meningococcal polysaccharides. The sensitivity of this test system based on the neutralization of antibodies is not inferior to that of ELISA with the use of double antiserum.     

Determination of the O antigen of Shigella sonnei by a competitive immunoenzyme method

A technique for immunoenzymatic diagnosis of dysentery by Shigella sonnei O-antigen was developed. For induction of antibodies to O-antigen rabbits were immunized by intravenous administration of a commercial antidysentery vaccine. Specific antibodies to O-antigen belonging to class G immunoglobulins and not binding to O-antigens of Sh. flexneri and Salmonella typhimurium were obtained. beta-Lactamase of Bacillus licheniformis 749/c was used as a marker enzyme in the immunoenzymatic assay. To increase the sensitivity, beta-lactamase molecules were preliminarily linked with glutaric aldehyde into oligomers. Conjugates of Sh. sonnei O-antigen with the oligomers of B. licheniformis 749/c beta-lactamase were prepared with the periodate method by oxidizing O-antigen. The conjugate was used in competing solid phase immunoenzymatic assay for determination of Sh. sonnei O-antigen in blood serum of patients with dysentery. The sensitivity of the assay is 0.5-1 ng per 1 ml of O-antigen.     

Solid phase enzyme-linked competitive binding assay for riboflavin

A new solid-phase enzyme-linked assay for riboflavin (vitamin B2) is described. The assay is based on the competition between analyte vitamin molecules and a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate for a limited number of riboflavin-binding protein sites immobilized on Sepharose particles. Significant improvements in conjugate catalytic activity and thus detectability are achieved by optimizing the reaction conditions used to covalently link 3-carboxymethylriboflavin to the enzyme. Optimization experiments include studying the effects of reaction pH and organic solvent composition. Final assay detection limits and the sensitivity of the dose-response curves are dependent on the ratio of conjugate to binding protein sites utilized in an equilibrium assay protocol. Selectivity of the method correlates well with that predicted based on the known association constants of riboflavin-binding protein with flavin analogs. The assay is shown to offer adequate detection limits and selectivity for direct measurement of riboflavin in urine, infant formula, and vitamin capsules.     

A competitive binding assay for fructose 2,6-bisphosphate

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P2 to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol protomer) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6-[2-32P]P2 and samples containing varying concentrations of fructose 2,6-P2. The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P2 in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P2, ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. This new assay is simple, direct, rapid, and does not require pretreatment of tissue extracts.     

Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.     

Use of immunoenzyme analysis for evaluating the specificity of the antibody recognition of the antigenic determinants of Streptococcus group A

Investigations carried out with the use of a CELIA system have revealed that antibodies in the sera of patients with primary erysipelas and antibodies in rabbit antiserum to the ribosomes of group A streptococcus specifically bind with adsorbed streptococcal ribosomes, recognizing the antigenic determinants of streptococcal ribosomes, which differ from those of individual Gram-negative prokaryotes (Escherichia coli, Shigella sonnei, Shigella flexneri, Salmonella minnesota). The modified CELIA system used in this investigation has made it possible to find out that antibodies in the sera of patients with primary erysipelas and antibodies in rabbit ribosomal antiserum bind with different antigenic determinants of the ribosomes of group A streptococcus.     

Use of solid-phase immunoenzyme analysis for the serodiagnosis of pseudotuberculosis

The diagnostic test system based on the solid-phase enzyme immunoassay (EIA) for the detection of antibodies to Yersinia pseudotuberculosis in the sera of patients with the use of Soviet-made preparations and reagents has been developed. The test has been performed in microchambers for immunological reactions, thus making it possible to decrease the consumption of reagents 10-20 times in comparison with the traditional technique with the use of plates. The results of the titration of 42 sera in EIA and in the passive hemagglutination test (PHAT) are indicative of the presence of positive correlation (r = 0.78; p less than 0.05) between antibody titers in EIA and PHAT. A fourfold or greater increase in antibody titers has been determined by means of EIA in 80% of cases and with the use of PHAT in 55% of cases. The minimum diagnostic titer yielded by EIA has been determined: 1:256.     

The development of methods for obtaining monospecific ingredients for immunoenzyme analysis

The methods of the modification of Salmonella O- and H-antigens and the preparation of biologically active sorbents on their basis have been developed. The use of these sorbents has permitted the isolation of affinity antibodies with strictly defined specific activity. The work shows the possibility of the successful use of carriers obtained on the basis of porous glass, chemically modified by acrylic copolymers containing activated carboxylic groups, and intended for the immobilization of antigens of both protein and carbohydrate nature.     

Use of solid-phase immunoenzyme analysis for determining allergen-specific IgE antibodies

An ELISA system for the detection of allergen-specific IgE antibodies to ragweed allergen has been developed. The system is highly sensitive and specific. Ragweed pollen allergen has been obtained by the dialysis of water-soluble extract through a kidney membrane. The high molecular fraction of ragweed allergen, showing the whole of the allergenic activity detected by skin tests in untreated patients, has been used for coating polystyrene assay plates. To detect IgE antibodies to ragweed allergen, the conjugate of sheep anti-IgE antibodies with horse-radish peroxidase has been used. The level of allergen-specific IgE antibodies has been determined on the basis of the data on the optical density of the samples in comparison with that of the normal sera. The correlation factor of the results obtained in the assay of specific IgE antibodies with the newly developed assay system and with the commercial kit Phadezyme RAST manufactured by Pharmacia AB (Sweden) has proved to be 0.82 at n = 39, p less than 0.01, while the variation factor in the reproduction of the assay results has proved to be 12% at n = 40.