Principles, problems, and strategies in the use of antigenic mixtures for the enzyme-linked immunosorbent assay

Abstract

Competition between proteins and other macromolecules for adsorption sites on plastic was studied with the enzyme-linked immunosorbent assay (ELISA) to determine effects of the use of antigenic mixtures or extracts of organisms on assays of antibodies and antigens by ELISA. A comparison of a number of different polystyrene microplates with bovine albumin and human immunoglobulin G (IgG) as antigens showed two major classes of plates: those which adsorbed albumin poorly and those which adsorbed albumin well. IgG adsorbed well on all plates, but plates which adsorbed albumin best also gave significant background levels of nonspecific binding of conjugate. When mixtures of IgG and bovine serum albumin were used as coating antigens, significant competition was observed; the component present at 1% or less in the mixture was essentially undetectable unless excessive amounts of conjugate were used. The important factor was the ratio of competitor to antigen, not the absolute amount. Other proteins (ovalbumin, rabbit albumin, human albumin, and gelatin) were equally effective competitors for adsorption sites on plastic. Nonionic detergents (Tween 20, and Triton X-100) were strong competitors even at 10:1 competitor-to-antigen ratios. In antigen capture assays, normal serum components blocked attachment of antigen-specific IgG, but this competition could be lessened to a degree by the use of strongly binding polystyrene plates. In indirect ELISA for measurement of serum antibody, the use of antigenic mixtures gave significantly lower antibody titers when the desired antigen was less than 1% of the total protein coated. Therefore, the use of mixed or crude antigens in ELISA presents significant problems concerning the sensitivity and specificity of tests. The enzyme-linked immunosorbent assay (ELISA) (9) is a major tool for detecting antigens and antibodies in a wide variety of diseases (20, 21). Although the method was initially devised for measurement of antibodies to individual proteins, a large number of assays for individual disease agents have been devised using partially purified or even unpurified antigens. Such assays are not uniformly successful, and when they are it is not possible to define the reacting antigen(s). If we were to wait until suitably purified antigens were available, a number of currently useful assays would not be available for serodiagnosis. It has been recognized that components of antigenic mixtures (i.e., an extract of a microorganism) likely compete with each other for the limited sites on plastic surfaces (4, 5, 13, 16). Thus, studies to determine the nature of competition are necessary to define conditions under which ELISAs can best be performed with partially purified or unpurified antigens. Proteins apparently attach to plastic surfaces by hydrophobic interactions (12, 19), and the

Cross‐Reactive Antibodies to Mycoplasmas Found in Human Sera by the Enzyme‐Linked Immunosorbent Assay (ELISA)

“…High CF sera showed significantly high ELISA values not only for M. pneumoniae (P<0.001), but also for M. hyorhinis (P<0.001) and M. pulmonis (P=0.001). It is suspected that the main factor causing background readings is the nonspecific adsorption of serum and the conjugate on ELISA antigens or on the surface of plate wells (7,13,16). However, our results showed that the background readings found in ELISA for measuring antibodies to M. pneumoniae were not due to nonspecific adsorption of human IgG on the plate wells and on medium components which might have contaminated mycoplasmal ELISA antigens.…”

Specific antibody responses to subgingival plaque bacteria as aids to the diagnosis and prognosis of destructive periodontitis*

“…An antigen in excess will block the binding of another in much smaller amounts leading to failure of detection of the minor antigen(s). This aspect of ELISA technology where antigen mixtures are assayed with single serum samples has been illustrated by Kenny & Dunsmoor (1983). Some antigens may not even bind at all but the use of single antigen assays would obviate this problem.…”

Problems in the Detection of Complement‐fixing Immune Complexes

“…Coating was performed in 0.1 M carbonate buffer pH 9.6 at a final concentration of 0.5 mg/ml of antibodies. The microtitre plates were thoroughly washed with PBS containing 3% Tween 20 (10). Samples diluted 1:20 in PBS containing 3% Tween 20 were tested in duplo, avoiding the outermost wells.…”