A comparison of ELISA and flow microsphere-based assays for quantification of immunoglobulins.
An automated microsphere-based flow cytometric assay (FlowMetrix system) was compared with a conventional enzyme-linked immunosorbent assay (ELISA) for quantifying Ig classes in serum and stool samples. The reproducibility of the process of coupling capture antibodies to microspheres was tested. The use of independently coupled microspheres did not increase the variation of assay results relative to using the same bead set in repeated assays. However, it is necessary to ensure quality control of the coupling process since slight variations in the coupling procedures can profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect assay precision. Although the ELISA was more sensitive and did not have the problems with instrument performance encountered with the FlowMetrix assay, the latter was more reproducible, had a greater dynamic range of measurement, and took considerably less preparation time than the ELISA. Greater reproducibility is especially important for measurement of fecal Ig, which is typically highly variable. Thus, in addition to its multi-analyte capability, the FlowMetrix assay system has definite advantages over a conventional ELISA. Mechanical problems such as microspheres settling to the bottom of wells during analysis by an automated plate reader will likely be overcome, and sensitivity improved as this technology develops.
Dasso
Joseph
J
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1892, USA. jdasso@caribsurf.com
Lee
Juliet
J
Bach
Hanh
H
Mage
Rose G
RG
D000418:Albumins / Q000032:analysis; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005243:Feces; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D007136:Immunoglobulins / Q000032:analysis*; D051379:Mice; D008863:Microspheres; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D013997:Time Factors
D000418:Albumins; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007136:Immunoglobulins; D011933:Reagent Kits, Diagnostic