Pl. Masson

Particle counting immunoassay (PACIA)—VI. The determination of rabbit IgG antibodies against myelin basic protein using IgM rheumatoid factor

PACIA, which is based on latex agglutination and instrumental counting of residual non-agglutinated particles, is a practical method for the determination of IgG antibodies against myelin basic protein (MBP). These antibodies agglutinated latex to which MBP had been covalently bound by carbodiimide. The agglutination was greatly enhanced by rheumatoid factor, the final reaction being directly proportional to the amount of IgG antibody attached to the latex bound MBP. To avoid interference by endogenous IgM rheumatoid factor each sample was reduced with dithiothreitol. The antibody content of a rabbit antiserum used as a calibrator for both PACIA and a solid phase radioimmunoassay (RIA) was estimated by the Farr precipitation technique using 125I-labelled MBP. The sensitivities of PACIA and RIA were similar i.e. 10 ng/ml IgG anti-MBP antibodies. However, the presence of serum proteins tended to decrease the agglutination reaction. The correlation coefficient between methods was 0.93. The worst coefficient of variation achieved by PACIA over daily analyses during 5 days was 23%. Despite this imprecision, which for antibody titration is still acceptable, PACIA seems an attractive method for large epidemiological studies because of its sensitivity, its short incubation time (30 min instead of 24 hr for RIA), facility of application and stability of reagents.

Typing of Subclasses and Light-chains of Human Monoclonal Immunoglobulins By Particle Counting Immunoassay (pacia)

The subclasses of monoclonal IgGs and IgAs were identified by particle-counting immunoassay. The principle of the test is the inhibition of the agglutinating activity of either specific antisera or monoclonal antibodies (for IgA only) on latex particles coated with a monoclonal IgG or IgA of known subclass. The feasibility of assay of polyclonal Ig subclasses was demonstrated. However, the anti-IgG2 antiserum cross-reacted with an allotype (nG4m(b)) of IgG4. The possibility of typing monoclonal Igs for light chains by the same technique was also demonstrated. Results are obtained in 30 min, and the method requires only small amounts of purified immunoglobulins (Igs) and antisera or monoclonal antibodies.