Comparison of Enzyme-Linked Immunosorbent Assay and Passive Hemagglutination Method for Quantification of Antibodies to Lipopolysaccharide and Tetanus Toxoid in Rats

Abstract

In a comparative study, the enzyme-linked immunosorbent assay, using peroxidase labeled anti-rat immunoglobulin M and immunoglobulin G, and the passive hemagglutination test were applied to determine the primary and secondary antibody response to lipopolysaccharide and tetanus toxoid in rats. In the enzyme-linked immunosorbent assay, the antigens were bound to the wells of polystyrene microplates, tetanus toxoid directly, and lipopolysaccharide after completing it with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled anti-immunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with their respective antigens. The enzyme-linked immunosorbent assay proved to be more sensitive than the hemagglutination reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, the enzyme-linked immunosorbent assay is a convenient method for measuring both immunoglobulin M and immunoglobulin G antibodies. At low serum dilutions of lipopolysaccharide antisera, inhibition of the reaction in the enzyme-linked immunosorbent assay occurred. This phenomenon could be prevented by heating the sera at 560C for 30 min. Lipopolysaccharide was immunogenic in rats over an extremely wide dose range (from 10 pg to 1 mg); the optimal immunogenic dose of lipopolysaccharide for young adult rats was 0.1 to 1,000 ,ug when administered intravenously, and that of tetanus toxoid was 5 to 10 lines of flocculation, as determined by the Ramon flocculation test.

An ELISA for measuring serum antibodies to Actinobacillus actinomycetemcomitans

“…However, there was generally good agreement among the techniques concerning the relative antibody activity in the 9 serums tested. Two recent reports comparing similar enzyme-linked assays and passive hemagglutination, to analyze antibodies reactive with Escherichia coli LPS and tetanus toxoid (Vos et al 1979) and antibodies to Leishmania braziliensis (Luzzio, McRoberts & Euliss 1979), support the present findings of increased sensitivity of the ELISA. However, the most important advantage of the ELISA over the PHA is that ELISA permits antibody quantitation and isotype determination.…”

Immunohistochemical investigation of the presence and distribution of cementum‐associated lipopolysaccharides in periodontal disease

“…The LPS solution was used as the antigen in an ELISA system according to the method of Vos et al (1979). Briefly, microtitre plate wells were coated with the LPS at a concentration of 25 //g/ml (optimum concentration determined by trial experiments), incubated with the test serum, foliowed by a peroxidase-conjugated rabbit anti-human immunoglobulins serum and an o-phenylenediamine/H2O2 substrate.…”

Quantitation of anti‐tetanus and anti‐diphtheria antibodies by enzymoimmunoassay: Methodology and applications

“…The EIA methodology applied to our assay for antibodies to TT and DT is not significantly different from the methodology used by some other groups (18,20), although every published procedure differs from the others in one or another detail. The EIA that we have developed is relatively simple to perform and shows remarkable reproducibility and very good sensitivity.…”