IVIG EFFECTS ON ELISA PRA COMPARING ANTI-Fc TO PROTEIN G AS A SECONDARY ANTIBODY FOR DETECTING HLA ALLOANTIBODIES.

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IVIG EFFECTS ON ELISA PRA COMPARING ANTI-Fc TO PROTEIN G AS A SECONDARY ANTIBODY FOR DETECTING HLA ALLOANTIBODIES.
R. F. McAlack, PhD , D. A. Sesok-Pizzini, MD , D. H. Lee, MS and D. Cervellini BS . Philadelphia PA, Albert Einstein Medical Center, 19141, Transplant Immunology and Philadelphia PA, University of Pennsylvania, 19104, Pathology and Laboratory Medicine .

IVIG has an inhibitory effect on IgG alloantibodies function in sensitized renal transplant recipients. Difficulties monitoring IVIG therapy arise as standard antibody (PRA) tests don’t show the inhibitory effects of IVIG. NIH-CDC shows inhibition while AHG-CDC and ELISA do not. A mechanism for IVIG is inhibition of complement binding and prevention of a lytic attack. For ELISA testing we used Protein G as it binds to the Fc piece of IgG close to the binding site of complement component C1q. By substituting Protein G for the standard anti Fc gamma secondary antibody in an ELISA procedure we expect to find results similar to the NIH-CDC.
Sera from 10 patients with a range of Class I PRA from 20 to 100% were chosen. To control for dilution normal human sera was added to one set at 40% by volume. To the second set Polygam™ S/D(Baxter) was added at 40% by volume. The NIH-CDC and AHG-CDC PRA testing was performed by standard methods. ELISA was performed with LAT trays(One Lambda) with the usual secondary antibody, an anti-Fc gamma and Protein G, both conjugated with alkaline phosphatase.
IVIG inhibition is seen with the complement assay, NIH-CDC and in the ELISA assay using Protein G. Inhibition is very dependent on the serum titer and the strength or avidity of the alloantibody. The PRA for Protein G follows the inhibition pattern as demonstrated by the NIH-CDC results and confirms that IVIG's ability to block C1q also will block Protein G.
In vitro IVIG can block complement binding to IgG as demonstrated by the NIH-CDC PRA procedure. In ELISA, blocking of the complement binding site can be demonstrated by using Protein G as the secondary antibody. Blocking by IVIG is dependent on the antibody titer for Class I and decreases as the PRA increases.