Elucidation of non-parallel EIA curves.

Quantitative determinations by EIA can be only obtained by reverse regression when linear portions of sample and standard curves are parallel. However, analysis of complex biological fluids often yields sigmoid curves displaying lower slopes, thus invalidating any quantitative interpretation. We hypothesized that this phenomenon was due to a competition effect between the target (for example an antigen) and related molecules for the binding sites (for example a capture antibody) immobilized onto the solid phase. This has been confirmed experimentally using various target-to-competitor ratios and formulated as a mathematical model. The slope decrease in target detection was related to the proportion of competitor, not in a linear, but in an exponential manner. This mathematical model has been computerized and can be used to correct aberrant sample curves provided the relevant parameters have been previously determined in the same systems.

Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses.

We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion. The assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes.

Rapid biotin-avidin method for quantitation of antiviral antibody isotypes.

A rapid and efficient method is described for isotype quantitation of antiviral antibodies in mice infected with Theiler's murine encephalomyelitis virus (TMEV). Serum antibodies were reacted with fluorochrome-labeled TMEV in a modified fluid-phase particle concentration fluorescence immunoassay (PCFIA). Biotin and avidin were used to attach anti-immunoglobulin isotype antibodies to polystyrene particles by the separate incubation of biotinylated goat anti-mouse isotypes (IgG1-, IgG2a-, IgG2b-, IgG3-, or IgM-specific) with avidin coupled polystyrene particles. These anti-isotype particles captured the virus-antibody complexes. Mouse myeloma proteins were used to quantitate and standardize isotype profiles of normal mouse serum using fluorescein isothiocyanate (FITC)-labeled, goat anti-mouse isotypes and polystyrene particles coated with goat anti-mouse. These assays quantitated the affinity-purified mouse serum antiviral antibodies for the standardization of antiviral isotype assays. Immunoglobulin of all serum isotypes as well as the amount of virus-specific isotypes can be quantitated rapidly and accurately.