Use of quantitative immunofluorescence for determining the adsorption capacity of magnetic immunosorbents

The adsorption capacity of microgranulated polyacrylamide magnetic immunosorbents has been studied by the method of quantitative immunofluorescence as applied to the causative agents of plague, cholera, and melioidosis. Similar regularity in the dynamics of antigen adsorption on the granules of magnetic immunoadsorbents has been established. This regularity consists in the direct relationship between this process and the concentration of infective agents interacting with magnetic sorbents.     

Isolation of antilymphocytic antibodies with the aid of cellular immunosorbents]

The authors suggest a method of obtaining purified antilymphocytic antibodies by mean of a specific immunosorbent of human or mouse lymphocytes fixated with glutharic aldehyde Such immunosorbents subjected to special treatment could be used repeatedly; their sorptive capacity was retained in such case. Only from 5 to 12% of the activity could be obtained from immunosorbents sorbed from the serum with 77--93% activity. In comparison with the initial serum, purification was from 6- to 15-fold. Thus, the suggested method provided considerable purification of the antilymphocytic preparations and permitted to obtain highly active antilymphocytic antibodies.     

Separation of antigens by immunological specificity: Use of cellulose-linked antibodies as immunosorbents

1. Immunosorbents were prepared by coupling activated aminocellulose with the γ-globulin concentrates of antisera prepared against ovalbumin and human serum albumin. 2. The immunosorbents were low in solubility, but high in capacity for homologous antigens. 3. The high specificity of these immunosorbents was demonstrated by their use in fractionating various mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

Separation of antigens by immunological specificity: Use of disulphide-linked antibodies as immunosorbents

1. γ-Globulin concentrates of antisera prepared against ovalbumin and human serum albumin were thiolated and cross-linked to form insoluble polymers. 2. These immunosorbents were of low solubility and of high capacity for homologous antigen. 3. The high specificity of these immunosorbents was demonstrated by fractionation of various binary mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

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.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

Use of immunoenzyme analysis for determining antibacterial antibodies in diphtheria infection

A diagnostic EIA system for the detection of antibacterial antibodies in diphtheria infection has been developed. As antigen, homogeneous membrane protein (mol. wt. 64 KD) obtained from Corynebacterium diphtheriae cell walls has been used. This protein antigen has been prepared with the use of nonionic detergent NP-40.     

Use of a polyvalent erythrocyte diagnostic agent for determining antibodies to Pseudomonas aeruginosa in clinical practice

The use of polyvalent erythrocyte diagnosticum prepared on the basis of 5 polysaccharide antigens of P. aeruginosa slime, isolated from strains belonging to the most widespread serovars, makes it possible to check up the humoral response of donors after their immunization with P. aeruginosa polyvalent corpuscular vaccine with the aim of obtaining anti-P. aeruginosa donor plasma. Antibody titers, determined in the passive hemagglutination test with the use of the proposed diagnosticum and corresponding to a serum dilution of 1:320 and greater, age tentatively diagnostic, which may be indicative of P. aeruginosa in the development of purulent septic complications in patients. The use of the passive hemagglutination test with the newly developed polyvalent erythrocyte diagnosticum makes it possible to check up the specific response of patients having P. aeruginosa infection in the process of their treatment with anti-P. aeruginosa hyperimmune plasma used as a part of complex therapy.     

Absorption of anti-placenta serum by means of immunosorbents]

An anti-placenta serum was absorbed by means of immunosorbents to remove antibodies against human serum proteins. The absorption met with some difficulties, because the anti-placenta serum contained antibodies against several human serum proteins. 12 different methods were compared for their suitability to adsorb these antibodies against human serum proteins. Most suitable is human serum cross-linked by glutardialdehyde. Good results were obtained too with human serum linked to Enzacryl, Polyaminostyrene or CNBr-activated Sephadex.     

Use of cooperative monoclonal antibodies in immunoenzyme analysis for determining human tumor necrosis factor

A cooperative sandwich enzyme immunoassay (EIA) based on the newly produced pair of cooperative monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was developed and characterized. It was found that, when used simultaneously, cooperative mAbs was capable to bind TNF from its preformed complexes with soluble TNF receptors (sTNF-R), thus providing the effective TNF detection in ex vivo samples by the respective one-step cooperative EIA. While demonstrating typical analytical characteristics regarding variability, dynamic range and specificity, a cooperative EIA offers an advantageous combination of high sensitivity (< 2 pg/ml) and short-time TNF capture protocol (1 hour). Application of cooperative EIA for TNF detection in clinical samples has demonstrated an increased serum TNF levels in patients with the mixed connective disease and infectious endocarditis that positively correlated with severity of systemic inflammatory reactions. Production and EIA application of cooperative mAbs would be promising in development of standardized and clinically applicable immunoassays for cytokines.     

Erythrocytic diagnostic agents for determining antibodies to Proteus O antigens

Highly sensitive and specific erythrocyte diagnostic agents (ED) for the determination of antibodies to Proteus O-antigens have been obtained by the sensitization of formolated sheep red blood cells (SPBC) with activated lipopolysaccharides (LPS) without the use of mediators. The tannin treatment of formolated SRBC and/or the increase of temperature from 45 degrees C to 100 degrees C in the process of the preparation of ED have been found to produce no increase in effectiveness. Antibody ED permitting the detection of Proteus O- and H-antigens has been obtained by the sensitization of formolated chick red blood cells with immunoglobulin preparations to Proteus hydroxylamine antigens, carried out with the use of amidol. The experiments have shown the possibility of using this antibody ED for the determination of O-antibodies in the antigen neutralization test with nonactivated LPS used as an agglutinating agent. The passive hemagglutination test with antibody ED has proved to be a more sensitive method for the detection of O-antibodies than the antigen neutralization test with antigenic ED. The determination of Proteus etiology in the passive hemagglutination test with the use of antigenic ED has been shown to be highly effective in the examination of patients with chronic osteomyelitis at the stage of exacerbation.     

A simplified method of rapid hemagglutination test for determining the levels of tetanus antibodies]

A new, simplified methodology of preparation of lyophilized chrome-coated turkey blood cells (formalin-treated ) for rapid identification of tetanus antibodies in indirect hemagglutination test is described. Blood cells diagnostic preparations obtained in this way were easier and three times faster to prepare than tanned cell preparations . They maintained unchanged capability for specific agglutination during at least one year of storage when kept at 4 degrees C. proposed methodology enable to start a production of laboratory kits necessary for controlled prophylaxis of tetanus.     

Use of paper indicator discs for determining the minimal inhibitory concentration of antibiotics]

Sensitivity of clinical strains of Staphylococcus and some Enterobacteriaceae to a number of widely used antibiotics was compared simultaneously with the use of two methods, i. e. the agar diffusion method and the method of serial dilutions. Regularities in distribution of the staphylococcal strains according to their sensitivity to antibiotics, such as erythromycin, benzylpenicillin, levomycetin and others were also studied with respect to every year using indicator paper discs. Interrelation observed during the comparison of the microbial sensitivity with the use of the two assay methods provided elaboration of the criteria for classification of the strains as "resistant" or "sensitive". The differentiation boarder for these two groups was determined according to the principle of the assay error minimization. A necessity of using standard dry media for specification of individual characteristics of various drugs in estimation of the microbial sensitivity to them by the agar diffusion method is emphasized.     

Use of cellulose carbonate for the preparation of immunosorbents: the radioimmunoassay of follicle-stimulating hormone

The use of cellulose 2,3-carbonate as a matrix for the insolubilisation of biologically active molecules has been extended to the preparation of insoluble antigen and antibody (immunosorbents). Both human pituitary follicle-stimulating hormone (FSH) and its homologous antibody have been covalently attached to cellulose carbonate by the nucleophilic attack of their primary amino groups on the cyclic carbonate groups. Antibody to FSH retains its immunological reactivity on insolubilisation, and is therefore suitable for use in the solid phase radioimmunoassay of unknown amounts of FSH by competitive binding of radioactively labelled and unlabelled FSH. Acceptable inhibition curves can be obtained, and the low, non-specific absorption characteristics have advantages over other systems. FSH also retains immunological reactivity on insolubilisation, and the derivative holds potential for the radioimmunoassay of FSH as it can be layered immunologically with anti-FSH and then FSH itself.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.