Optimal conditions of immune complex transfer enzyme immunoassays for antibody IgGs to HIV‐1 using recombinant p17, p24, and reverse transcriptase as antigens

Abstract

The immune complex transfer enzyme immunoassays for antibody IgGs to p17, p24, and reverse transcriptase (RT) of HIV‐1 were tested under various conditions. Antibody IgGs to HIV‐1 were reacted for up to 20 hr with 2,4‐dinitrophenyl‐bovine serum albumin‐recombinant HIV‐1 protein conjugates and recombinant HIV‐1 protein‐β‐D‐galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti‐2,4‐dinitrophenyl group) IgG by incubation at 4–30°C for up to 2 hr with shaking and were transferred onto polystyrene beads coated with (antihuman IgG γ‐chain) IgG in the presence of excess of ϵN‐2,4‐dinitrophenyl‐L‐lysine by incubation at 4–30°C for up to 2 hr with shaking. When serum randomly collected from an HIV‐1 seropositive subject and serum included in an Western blot kit were tested, the formation of the immune complex was almost completed within 1 hr for antibody IgG to 17, within 1–2 hr for antibody IgG to p24 and within 4 hr for antibody IgG to RT. Even for antibody IgG to p17, however, the immune complex continued to be formed for at least 2 hr, when serum samples at early stages of HIV‐1 infection were tested. Trapping and transferring of the immune complexes were faster at higher temperatures and were almost completed within 0.5–1.5 hr, although the amount of the immune complexes trapped and transferred at 25 and/or 30°C increased for 0.5–1 hr, but subsequently tended to decline. When the formation, trapping, and transferring of the immune complexes were performed for 0.5, 1, and 1 hr, respectively, with shaking followed by 1 hr assay of bound β‐D‐galactosidase activity, the sensitivities for antibody IgGs to p17, p24, and RT using 10 μl of serum samples were similar to or significantly higher than those of the corresponding previous immune complex transfer enzyme immunoassays using 10 μl of serum samples, in which the formation, trapping, and transferring of the immune complexes were performed for 3, 16, and 3 hr, respectively, without shaking, followed by 2.5 hr assay of bound β‐D‐galactosidase activity, and the sensitivities for antibody IgGs to p17, p24, and RT using 100 μl of serum samples were 21–22‐fold, 5.5–6.3‐fold, and 5.3–6.0‐fold, respectively, higher. When each period of time for the formation, trapping, and transferring of the immune complexes was prolonged to up to 4 hr, the sensitivities for antibody IgGs to p17, p24, and RT using 100 μl of serum samples were improved 88–93‐fold, 15–17‐fold and 20–24‐fold, respectively, as compared with those of the previous ones. J. Clin. Lab. Anal. 12:98–107, 1998. © 1998 Wiley‐Liss, Inc.