Attempt to isolate antierythrocyte antibodies by the method of affinity chromatography. I. IgM--complete antibodies

It follows from the results of the present study that erythrocyte stromata can serve as a suitable insoluble immunosorbent of complete antibodies. When using optimum conditions, such as laboratory incubation timperature, suitable buffers for both washing the column prior to absorption and for elution, it is possible to obtain, by means of the chromatographic column with 0.5 ml of erythrocyte stromata suspended in 40 g of silica glass beads (ballotini), fractions devoid of complete antibodies and subsequently to obtain the latter in purified form by elution. In complete anti antibodies, the elution yield amounted to an average of 35% of the initial amount of antibodies. Although the elution yield is not high and non-specific absorption has been demonstrated, the method can be recommended for the isolation of complete antibodies and thus for the production of monospecific sera because it is easy, rapid and inexpensive due to the regeneration ability of the mentioned columns and the relatively long duration of the immunoabsorption capacity of the incorporated erythrocyte stromata.     

Suppression of the formation of cells secreting antibodies and antigen-dependent nonspecific immunoglobulins in mice receiving isologous antierythrocyte immunoglobulins

Mice injected with syngeneic cellulose-conjugated immunoglobulins (Ig) containing antibodies to sheep red blood cells (SRBC) develop a specific non-responsiveness to SRBC. Such animals demonstrate a sharp decrease not only in the formation of anti-SRBC antibody producers but also of the cells secreting antigen-dependent nonspecific Ig. The inhibition of both these processes is antigen-specific. It is suggested that inhibition of the cells forming antigen-dependent nonspecific Ig is due to suppression of either hypothetic inductors or precursors of these cells expressing an idiotype spectrum similar to that of anti-SRBC antibody producers.     

Use of immunosorbents for determining antibodies to histaglobulin]

A determination was made of antibodies to histaglobulin and gamma-globulin with the aid of immunosrobents in 178 sera of children suffering from allergic diseases, during the histaglobulin therapy. Results of investigations showed antibodies to histaglobulins to be absent in children untreated by this preparation. But they appeared in 57% of cases after the first course of histaglobulin treatment, and their incidence and their average level increased with an increase in the number of the courses of treatment carried out. gamma-Globulin antibodies were found at the initial condition in 42% of cases; this percentage rose to 68 after the first course of histoglobulin treatment. The authors believe that determination of histaglobulin antibodies during the treatment with this preparation could serve as an auxiliary immunological criterion of the efficacy of histaglobulin therapy.     

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.     

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.     

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.     

Peptide-specific antibodies employed in determining the interspecies immunological cross-reactivity of haemopexin

1. Antibodies were raised in rabbits against nine peptides analogous to sequences of the human serum beta-glycoprotein haemopexin, and seven peptides were very antigenic. 2. One of these affinity-purified peptide-specific antibodies interacted with a highly conserved sequence of the haemopexin of five of the seven species tested. 3. Another antibody bound pig haemopexin even better than human haemopexin. 4. The overall, arbitrarily assessed, immunological cross-reactivity between the haemopexin of human and other species follows the order: rabbit greater than mouse greater than chicken greater than pig greater than rat greater than cow.     

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.     

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.     

A radioimmunological method for determining IgA-class antimeningococcal secretory antibodies in human saliva

The data on the content of secretory IgA antibodies to group A Neisseria meningitidis protein antigen in the saliva of persons, both having had contact with N. meningitidis culture and having had no such contact, are presented. The results were obtained by the method of radioimmunoassay, developed specially for the determination of N. meningitidis protein antigen.     

Micromethod of determining antibodies to nerve tissue galactocerebrosides in the complement fixation reaction

A micromethod has been developed for the determination of serum antibodies to galactocerebroside, a specific antigen of myelin membranes. The method is based on complement fixation test and is used for the analysis of sera from healthy rabbits and those immunized with galactocerebroside.     

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O⁺ erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.