Effects of reaction variables on nephelometric and turbidimetric responses for the immunochemical reaction of immunoglobulin G.
Here we report a kinetic study of the immunoprecipitin reaction involving immunoglobulins G, A, and M. We used stopped-flow mixing adapted for simultaneous monitoring of nephelometric and turbidimetric signals for some studies and centrifugal mixing for others. The variables having the most significant effects on the kinetic responses in the regions of excess antibody and excess antigen are polyethylene glycol concentration, ionic strength, and the ratio of antigen to antibody. We document effects of these variables on maximum velocities and signal changes over a fixed-time interval for both monitoring modes and on the maximum signal change for nephelometry. We use response-surface methodology to help identify interactive effects among these variables (polyethylene glycol, NaCl, and antibody concentrations) and to select the best combination to use to quantify antigen in all regions of the immunoprecipitin curve. We also observe that turbidimetric responses are more reproducible and much simpler than are nephelometric responses. Implications of these results for quantification of the immunoglobulins are discussed.
Skoug
J W
JW
Department of Chemistry, Purdue University, West Lafayette, IN 47907.
Pardue
H L
HL
D000941:Antigens / Q000032:analysis; D006801:Humans; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis*; D007075:Immunoglobulin M / Q000032:analysis; D007163:Immunosorbent Techniques; D007700:Kinetics; D009391:Nephelometry and Turbidimetry; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology
D000941:Antigens; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011092:Polyethylene Glycols; D012965:Sodium Chloride