Method of analysis of non-competitive enzyme immunoassays for antibody quantification.
A computerized analysis of a quantitative enzyme-linked immunoadsorbent assay (EIA) using a non-specific immunoglobulin (IgG) of known concentration as the standard has been developed for measuring specific antibody levels in serum without the need for affinity purification of the positive control antibody. The computer program utilized logit-log linear regression analysis of sigmoid serial dilution curves plus a weighted least-squares best curve fit analysis and an iterative manipulation to eliminate errant data points. The EIA was performed using serial dilutions of standard and unknown antibodies, and a double sandwich technique. A comparison of antibody levels determined by EIA using non-specific IgG as a standard relative to antibody levels determined using affinity-purified specific antibody as a standard were 1.04, 0.53, 0.48, and 0.97 for four different polyclonal antibody systems. Five monoclonal antibodies to carcinoembryonic antigen gave ratios as described above of 1.07, 1.59, 1.73, 2.32, and 2.42. The corresponding antibody affinity constants (1/mol) were 1.0 X 10(8), 3.8 X 10(8), 5.5 X 10(9), 1.8 X 10(10), and 2.6 X 10(10) respectively. This method permits accurate quantification of serum antibody levels when affinity-purified antibodies are not readily available and avoids errors due to loss of antibody activity during affinity purification.
Beatty
J D
JD
Beatty
B G
BG
Vlahos
W G
WG
Hill
L R
LR
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies / Q000032:analysis*; D002138:Calibration; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis; D012710:Serum Albumin, Bovine / Q000276:immunology; D012984:Software
D000596:Amino Acids; D000906:Antibodies; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine