Standard sera in solid-phase immunoassays

Area of Science:

• Immunology
• Biochemistry
• Assay Development

Background:

• Antibody titers from solid-phase immunoassays (SPIA) are commonly converted to weight unit concentrations.
• This conversion relies on standard sera with known antibody concentrations.
• Systematic studies validating this conversion method are lacking.

Purpose of the Study:

• To investigate the magnitude of errors in converting antibody titers to weight concentrations using standard sera.
• To evaluate the impact of using homologous versus heterologous standard sera.
• To assess the effectiveness of using single versus multiple standard sera for conversion.

Main Methods:

• Antibody concentrations were determined using quantitative precipitation or equilibrium dialysis.
• Thirteen sera or ascites fluids were analyzed.
• Solid-phase radioimmunoassay (SP-RIA) and enzyme-linked immunosorbent assays (ELISA) were employed using standard sera for conversion.

Main Results:

• Conventional solid-phase assay conversions using a single homologous standard serum resulted in up to fourfold errors.
• Using a combination of two standard sera reduced these errors.
• Using heterologous standard sera (directed to different antigens) did not introduce significantly greater errors than homologous standards.
• Combining two heterologous reference sera further reduced errors, but requires antigen coat binding capacity checks.

Conclusions:

• The conversion of antibody titers from solid-phase immunoassays to weight concentrations is prone to significant errors.
• Employing multiple standard sera, including heterologous ones, can mitigate these errors.
• Careful validation, including checking antigen binding capacity, is crucial when using heterologous standards for accurate antibody quantification.