The determination of IgG subclass concentrations in serum by enzyme-linked immunosorbent assay: establishment of age-related reference ranges for cord blood samples, children aged 5-13 years and adults.

We describe a simple enzyme-linked immunosorbent assay for the measurement of immunoglobulin G (IgG) subclasses in serum. The microtitre plate is coated directly with diluted samples, standards or controls, blocked with phosphate-buffered saline containing 1% Tween to prevent non-specific protein binding to the plate, and each subclass assigned with specific mouse monoclonal antibody. The specific antibody is detected with alkaline-phosphatase linked anti-globulin and the colour development of the paranitrophenyl phosphate indicator reagent is measured at 405 nm until the highest concentration of standard gives an absorbance in excess of 1.4. Coefficients of variation measured at three concentration levels were less than 7% for within-assay variation and less than 10% for between-assay variation. Regression analysis of total IgG with the sum of the measured IgG subclasses gave a correlation coefficient of 0.932 (P < 0.001). The assay has been used to establish age-related reference ranges for serum IgG subclasses; these are found to be slightly different from those established for radioimmunodiffusion methods. Our study does not confirm the previously reported absence of IgG4 in normal individuals.