Fractions containing the conjugate were stored at 4C after addition of bovine serum albumin (0.1 g/liter) and NaN3(1 g/liter) until use. == (ii) 2,4-DinitrophenylSerSerrp66 conjugate. transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG Bnip3 to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (logY= 0.99 logX+ 0.23;r= Lanopepden 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1. Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a heterodimer of p66 and p51 (p66 devoid of 120 C-terminal amino acids) (6,28), has been reported to be highly reactive with serum samples from HIV-1-seropositive subjects. By conventional enzyme-linked immunosorbent assay (ELISA) (5) and Western blotting (3), the seropositivity rate obtained with recombinant RT (rRT) as antigen was higher than those obtained withgagproteins (p17 and p24) and regulatory proteins (nef,vif, and so on) as antigens and was as high as that obtained withenvgp41 as antigen. By a sandwich enzyme immunoassay using rRT-coated microplates and rRT-alkaline phosphatase conjugate, seroconversion was detected as early as by the conventional ELISA using recombinant proteins (gp120, gp41, p24, p17, and p15) as antigens and the gelatin particle agglutination test using a lysate of HIV-1 as antigen (25). In addition, the specificity of immunoassays using rRT as antigen appears to be fairly high. In a sandwich enzyme immunoassay using rRT-coated microplates and rRT-alkaline phosphatase for anti-HIV-1 antibodies, no reaction has been observed with sera from subjects infected with either HIV-2 or hepatitis B virus (25). RT of HIV has been reported to be antigenically distinct from those of human T-lymphotropic virus type I (HTLV-I) and HTLV-II (4), and no significant reaction has been observed with serum samples from HTLV-I-infected subjects by the immune complex transfer enzyme immunoassay (13). Recently, an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibody immunoglobulin G (IgG) to HIV-1 using rRT as antigen and -d-galactosidase fromEscherichia colias label has been developed (13). Antibody IgG to RT was allowed to react simultaneously with 2,4-dinitrophenylbovine serum albuminrRT conjugate and rRT-d-galactosidase conjugate, and the Lanopepden immune complex of the three components formed was trapped on polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was transferred to polystyrene beads coated with (anti-human IgG -chain) IgG in the presence of 2,4-dinitrophenyl-l-lysine. By this enzyme immunoassay, which was 300- to 1 1,000-fold more sensitive than Western blotting for p66 band, diagnosis and confirmation of HIV-1 infection using urine (7,8,13), whole saliva Lanopepden (19,20), and serum (9) have become more reliable than by previous methods. Notably, diagnosis of HIV-1 infection was possible using even 1 l of whole saliva (19). However, the following disadvantages were noted in the use of rRT as antigen. rRT had to be produced using a rather long (3,012-bp) DNA fragment of the whole HIV-1polgene (1,26) and was not efficiently produced in widely used strains ofE. coli. In addition, it was impossible to produce fusion proteins of RT for simple purification since rRT is a heterodimer of p66 and p51, as described above. This report describes the preparation of recombinant HIV-1 p51 and its use as antigen in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 in comparison with those of recombinant HIV-1 p66 and rRT. == MATERIALS AND METHODS == == Enzymes and competentE. colicells. == RecombinantTaqDNA polymerase (TaKaRaTaq) and ligase were obtained from Takara Shuzo Co., Kyoto, Japan. Restriction enzymes were obtained from.