Characterization of the Soluble Immune Complex (EIC) of the Amplified Enzyme-Linked Immunosorbent Assay (a-ELISA) and an Evaluation of this Assay for Quantitation by Reaction Stoichiometry

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Abstract
The molecular composition of the soluble enzyme immune complex (EIC) of alkaline phosphatase (AP) and anti-AP which comprises the detection system of the amplified ELISA (a-ELISA) was investigated. The EIC appeared relatively homogenous in sucrose density gradients and sedimented as a protein of 600–650 K daltons. Based on size and the results of double-lable experiments, the EIC was shown to be composed of two moles of anti-AP and three moles of AP. During reaction with substrate at pH 9.6, greater than 50% of the AP is released as free enzyme and the released enzyme has the same activity as enzyme found in the EIC. The maximum yield of EIC is produced by solubilization of the antibody-AP equivalence precipitate with a 9-fold excess of the amount of AP required for precipitation at equivalence. EICs show no significant loss of activity when stored for one year at 4°C, -20°C or -70°C. The EIC is most stable during long term storage (five years) in 50% glycerol at -20°. Over the linear region of titration curve for dimeric and monomeric M315, the ratio of AP or EIC to M315 fails to show a constant stoichiometry. Using 131I-EIC and 125I-M315, it was determined that the lack of a constant stoichiometry in the linear region was due to differences in the amount of enzyme bound. Hence, stoichiometric quantitation of the primary antibody is not possible using the current a-ELISA.