Preliminary evidence for a diagnostic immunoglobulin G1 antibody response among culture-positive cows vaccinated with Brucella abortus strain 19 and challenge exposed with strain 2308.

The sera of cows inoculated with Brucella abortus have a characteristically high titer of immunoglobulin (Ig) G1 antibodies to a soluble brucella antigen compared with sera of noninoculated vaccinated cattle. Concentrations of antigen-specific IgG1 were greater than 10-fold higher than those for IgG2, even though total IgG2 concentrations were higher than total IgG1 concentrations. Increases in IgG1 antibodies to Brucella abortus soluble antigen were detected shortly after vaccination in those cows from which strain 19 was isolated and by 28 weeks in cows from which strain 2308 was isolated. Increases in specific antibodies were not paralleled by increases in either total IgG1 or total IgG2 concentrations. Rather, there was a 15-fold to greater than 200-fold increase in specific activity, with up to 16% of the IgG1 specific for the brucella antigen used in the assay. Thus, measurement of changes in total IgG1 concentrations is not a reliable method to identify brucellosis-associated anti-Brucella abortus soluble antigen activity. Only one cow in a panel of 10 selected for detailed study showed a false-positive IgG1 titer, whereas some serologic assays showed as many as 4 or 5 false-positives. Results of the complement-fixation test, among the battery of serologic tests used for detection of brucellosis, best agreed with the occurrence of increased IgG1 antibody levels.

Application of the amplified enzyme-linked immunosorbent assay: comparative quantitation of bovine serum IgG1, IgG2, IgA, and IgM antibodies.

The comparative quantitation of serum antibodies to a defined antigen, using the amplified enzyme-linked immunosorbent assay (a-ELISA), has been demonstrated in a model system in which bovine immunoglobulins (Ig) IgG1, IgG2, IgA, and IgM antibodies to human serum albumin (HSA) were measured. Comparative measurements are facilitated because the same enzyme-antibody complex is used for measuring all 4 isotypes. Serum dilutions from 1:100 to 1:50,000,000 were titrated and, when graphed logarithmically, yielded dose-response plots that contained a linear segment for all but IgA anti-HSA. Data obtained with whole serum and fractions enriched in IgA- and IgM-anti-HSA indicated that dimeric IgA antibodies may compete poorly with those of the IgM and IgG classes and that IgM antibodies are more avid than those of the IgG1 and IgG2 subclasses. Plateauing of complete a-ELISA titrations at optical density (OD)400 nm values lower than those observed for their standard-curve counterparts was interpreted to result from saturation of the antigen. Quantitation was accomplished through the use of standard curves prepared by adsorbing purified, radiolabeled Ig of the 4 isotypes directly to polystyrene. These plots were also valuable in evaluating antiglobulin specificity and potency and for ascertaining linearity in the absence of the primary antibody to be measured, as well as standard curves for determining antibody content in absolute terms. The absolute amounts of IgG1 and IgG2 antibodies to HSA were simliar to those determined by quantitative precipitation and constituted about 25% of the total IgG1 and IgG2 in a hyperimmune bovine serum. Only 5% of the serum IgM was specific antibody to HSA. The specificity of various anti-bovine globulin reagents was further shown by demonstrating the charactristic distribution of IgG1, IgG2, IgM, and IgA anti-HSA in serum fractionated on Sephadex QAE-50 and in sucrose density-gradients. Finally, data on the influence of enzyme-complex concentrations, complex-step incubation times, and the reaction kinetics of soluble antibody-enzyme complexes on a ELISA results are presented.

Amplification of the enzyme-linked immunosorbent assay (ELISA) in the detection of class-specific antibodies.

A modification of the standard enzyme-linked immunosorbent assay (ELISA) is described which circumvents the requirement for specifically purified antibodies from which antibody-enzyme complexes are made. The assay utilizes the principle of a soluble anti-alkaline phosphatase immune complex (AP-A-AP) and has been called the amplified ELISA. Methods for preparing and evidence for the specificity of rabbit anti-rat gamma-FC, IgM (mu) and IgA (alpha) are presented. These reagents are used to measure anti-DNP antibodies belonging to classes IgG, IgM and IgA in rat serum. Using antiglobulin and anti-enzyme reagents prepared in guinea pigs, anti-ovalbumin antibodies are measured in rabbit serum. Titration curves are similar when the amplified ELISA is compared to the standard ELISA. A change in slope suggesting an effect of saturation of antigen sites, occurs at the same input antibody concentration for both assays. Determination of the anti-DNP concentration of unknown sera by extrapopulation from titration graphs of a known serum suggests that the value is overestimated, i.e., amplified when the amplified ELISA is used. In addition, the amplified ELISA has an improved ability to detect low levels of antibody. Evidence is presented which illustrates how the use of optimally conjugated DNP-proteins, age of conjugates, and optimal dilutions of secondary antiglobulins and the AP-A-AP reduce non-specific binding in the amplified ELISA. The amplified ELISA is capable of detecting 2.4 ng of antibody to ovalbumin in a one: one million dilution of rabbit serum with high reproducibility and low background.