ENRICHED HIGH—TITER anti-HEPATITIS A VIRUS (HAV) IgM FOR POSITIVE CONTROLS IN DIAGNOSTIC DEVICES

Abstract

Hepatitis A refers to liver inflammation caused by infection with the hepatitis A virus (HAV). The HAV is one of several viruses that can cause hepatitis and is one of the 3 most common hepatitis viruses in the United States. The other 2 are hepatitis B and hepatitis C viruses. The presence of anti-HAV IgM in serum indicates the acute or early convalescent stage of HAV infection. Although several commercial tests are available to aid in diagnosis, the lack of available high titer plasma for positive control, due to the short window of the IgM response following infection (5-10 weeks after exposure) and the low U.S. incidence of the HAV (one in 20,000 or 5 cases per 100,000) is a persistent problem. Samples collected prior to 4 and later than 12 weeks from exposure to HAV often demonstrate anti-HAV IgM titers too low to be useful as a positive control. A low titer stock of anti-hepatitis A (HAV-IgM) with a signal-to-cut-off ratio of -3 is abundantly available. It requires at least a 5-fold enrichment before it can be used in medical diagnostic devices. In order to achieve this goal, we have developed a simple two-step process for enrichment. The first step consists of a fractionation of plasma proteins by ammonium sulfate (A/S) precipitation. The second step is the use of a Pellicon concentrator of molecular weight cut-off of 500 kD. Identity of the enriched anti-HAV IgM product was evaluated by SDSPAGE and Western blotting. The functionality of IgM was evaluated by ELISA, Abbott AxSYM HAVAB-M 2.0, Abbott AxSYM HAVAB 2.0, DiaSorin ETI-HAIGMK PLUS, and DiaSorin ETI-AB-HAVK PLUS assays. The enriched anti-HAV IgM was formulated in normal human plasma and was found to be stable for 11 days at 37°C. This was translated to 15 months of stability at 2-8°C by the Arrhenius plotting model (Kannen 1964). In addition to the enriched anti-HAV IgM product, the process should be amenable to production of IgM-positive controls from low-titer anti-HBcore IgM anti-Toxoplasma IgM, and anti-CMV IgM plasma, as well as from other disease-state plasma where high titer IgM plasma is in short supply.