Ichiro Nishikata

Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay

Following HIV infection, there is a window period of 6-8 weeks, during which HIV antibodies are not detectable and the infection cannot be diagnosed by methods for detecting HIV antibodies. However, HIV antigens are detectable in the latter part of the window period, although the level of HIV antigens declines as the level of HIV antibodies increases. We developed an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for the simultaneous detection of both p24 antigen of HIV-1 and antibody IgGs to p17 and reverse transcriptase of HIV-1 in a single assay tube and tested 11 HIV-1 seroconversion serum panels and serum samples randomly collected from 79 HIV-1 seropositive subjects and 100 HIV-1 seronegative subjects. The simultaneous detection was shown not only to shorten the window period significantly as compared with conventional methods for HIV-1 antibody detection but also to make possible a reliable diagnosis of HIV-1 infection from the time of seroconversion until late stages of the infection.

Ultrasensitive and more specific enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV‐1 in serum using affinity‐purified rabbit anti‐p24 Fab′ and monoclonal mouse anti‐p24 Fab′

Previously, an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV‐1 was developed. The immune complex comprising 2,4‐dinitrophenyl‐biotinyl‐bovine serum albumin‐rabbit anti‐p24 Fab′ conjugate, p24 antigen, and rabbit anti‐p24 Fab′‐β‐D‐galactosidase conjugate was trapped onto polystyrene beads coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG, was eluted with ϵN‐2,4‐dinitrophenyl‐L‐lysine, and was transferred to polystyrene beads coated with streptavidin. β‐D‐Galactosidase activity bound to the streptavidin‐coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound β‐D‐galactosidase activity had to be assayed for a long time (20 h), and the nonspecific signal was observed in 5% serum samples from subjects with low risk of HIV infection. In the present study, the assay time for bound β‐D‐galactosidase activity was shortened to 2.5 h by using 2,4‐dinitrophenyl‐biotinyl‐bovine serum albumin‐affinity‐purified rabbit anti‐p24 Fab′ conjugate and affinity‐purified rabbit anti‐p24 Fab′‐β‐D‐galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at ‐20°C, and this increase was prevented without prolongation of the assay time for bound β‐D‐galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti‐p24 Fab′‐β‐D‐galactosidase conjugate for affinity‐purified rabbit anti‐p24 Fab′β‐D‐galactosidase conjugate.

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

ABSTRACT For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.

Earlier diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgGs to p17 and reverse transcriptase in serum with enzyme immunoassay.

Serum samples of four HIV‐1 seroconversion serum panels were subjected in a single assay tube simultaneously to ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) for p24 antigen of HIV‐1 and for antibody IgGs to p17 and reverse transcriptase (RT) of HIV‐1. Signals became positive 7–15 days earlier than the detection of antibodies to HIV‐1 by conventional methods and remained strongly positive even after levels of p24 antigen declined. Thus the simultaneous detection of p24 antigen and antibody IgGs to p17 and RT made possible both as early a diagnosis of HIV‐1 infection as the appearance of p24 antigen in the circulation, shortening “the window period,” and as reliable a diagnosis of the infection as that by the detection of antibodies to HIV‐1 from the time of seroconversion until late stages of the infection, since the serum level of antibody IgG to RT was high not only in asymptomatic carriers but also in patients with AIDS‐related complex and AIDS.

Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV-1.

In the immune complex transfer enzyme immunoassay for HIV‐1 p24 antigen, different preparations of anti‐p24 Fab′‐β‐D‐galactosidase conjugate, various periods of time for immunoreactions involved, and shaking for incubations with polystyrene beads were tested. On the basis of the results of these experiments, p24 antigen was measured as follows. The antigen was reacted simultaneously with 2,4‐dinitrophenyl‐biotinyl‐bovine serum albumin‐affinity‐purified rabbit anti‐p24 Fab′ conjugate and highly polymerized monoclonal mouse anti‐p24 Fab′‐β‐D‐galactosidase conjugate at 37°C for 2 hr. The immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG for 1.5 hr and was transferred to white polystyrene beads coated with streptavidin in the presence of ϵN‐2,4‐dinitrophenyl‐L‐lysine for 1.5 hr. The incubations with polystyrene beads were performed at room temperature with shaking. β‐D‐Galactosidase activity bound to the white polystyrene beads was assayed by fluorometry at 30°C for 2 hr. The detection limit of p24 antigen (0.1 amol/tube and 10 amol (0.24 pg)/ml of serum) was equal to that obtained when the formation, trapping, and transferring of the immune complex were performed for 4, 16, and 3 hr, respectively, by incubation without shaking. Namely, the period of time required for the immune complex transfer enzyme immunoassay of p24 antigen was markedly shortened (25.5–7 hr) without loss of the sensitivity. By the improved immune complex transfer enzyme immunoassay, p24 antigen was detected 12–20 days earlier than the detection of antibodies to HIV‐1, i.e., seroconversion by the conventional ELISA. J. Clin. Lab. Anal. 12:115–120, 1998.

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

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.

Immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen

The immune complex transfer enzyme immunoassay for antibody IgM to HIV‐1 p17 antigen is described. Serum samples containing antibody IgM to HIV‐1 p17 antigen were incubated simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin‐recombinant p17 (rp17) conjugate and rp17‐β‐D‐galactosidase conjugate, and the immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG. Subsequently, the immune complex was transferred to white polystyrene beads coated with monoclonal mouse (antihuman IgM) IgG in the presence of excess of ϵN‐2,4‐dinitrophenyl‐L‐lysine. The signal for antibody IgM to p17 antigen was the fluorescence intensity by fluorometric assay of β‐D‐galactosidase activity bound to the white polystyrene beads. The periods of time required for the formation, trapping, and transferring of the immune complex comprising the three components were more than 4 hr, 2 hr, and 3 hr, respectively. The immunoassay developed was shown to be specific by inhibition of transferring the immune complex in the presence of excess of nonspecific IgM but not IgG. Signals for antibody IgM to p17 antigen with serum samples of HIV‐1 seroconversion serum panels,—that is, with serum samples in early stages of the infection—tended to be higher than those with serum samples from HIV‐1 asymptomatic carriers probably long after the infection and patients with ARC and AIDS. In contrast, signals for antibody IgG to p17 antigen with serum samples of HIV‐1 seroconversion serum panels tended to be higher than signals for antibody IgM to p17 antigen but were much lower than signals for antibody IgG to p17 antigen with serum samples from HIV‐1 asymptomatic carriers and patients with ARC and AIDS. J. Clin. Lab. Anal. 12:329–336, 1998.

Recombinant p51 as antigen in an immune complex transfer enzyme immunoassay of immunoglobulin G antibody to human immunodeficiency virus type 1.

ABSTRACT An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced inE. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to β-d-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex 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 to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 logX + 0.23; r = 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.

IMMUNE COMPLEX TRANSFER ENZYME IMMUNOASSAY FOR ANTIBODY IGG TO HIV-1 GP41 ANTIGEN USING SYNTHETIC PEPTIDES AS ANTIGENS

The immune complex transfer enzyme immunoassay for antibody IgG to HIV‐1 gp41 antigen was developed using two synthetic peptides. An aliquot (10 μl) of serum samples from HIV‐1 seropositive subjects was incubated simultaneously with 2,4‐dinitrophenyl‐bovine serum albumin‐synthetic HIV‐1 gp41 peptide conjugates and synthetic HIV‐1 gp41 peptide‐β‐D‐galactosidase conjugates and subsequently with colored polystyrene beads coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG to trap the immune complexes formed comprising the three components. After washing, the colored polystyrene beads were incubated with white polystyrene beads coated with affinity‐purified (anti‐human IgG γ‐chain) IgG in the presence of ϵN‐2,4‐dinitrophenyl‐L‐lysine to transfer the immune complexes to the white polystyrene beads. β‐D‐Galactosidase activity bound to the white polystyrene beads was assayed by fluorometry. The formation, trapping and transferring of the immune complexes were completed within 0.5, 0.5 and 1.5 hr, respectively. Since each peptide appeared to react with its own specific antibody IgG, serum samples were tested by the equimolar combination of the two peptides. The lowest signals (fluorescence intensities for bound β‐D‐galactosidase activity) for serum samples from HIV‐1 asymptomatic carriers, patients with AIDS‐related complex and patients with AIDS were 1490‐, 2210‐ and 1460‐fold, respectively, higher than the highest signal for serum samples from HIV‐1 seronegative subjects. In five seroconversion serum panels, antibody IgG to HIV‐1 gp41 antigen was detected as early as antibodies to HIV‐1 detected by three currently commercially available methods. J. Clin. Lab. Anal. 12:197–204, 1998.

Preparations of recombinant HIV-1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 reverse transcriptase

Recombinant HIV‐1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV‐1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease (pol–rp66). Second, rp66 with Ser–Ser at the N‐terminus was produced as a fusion protein with maltose‐binding protein containing a factor Xa site between the two proteins (MBP–Ser–Ser–rp66) and was released from the fusion protein by factor Xa (Ser–Ser–rp66). Third, rp66 with Met–Gly at the N‐terminus was produced in transformed cells (Met–Gly–rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP–Ser–Ser–rp66 and Met–Gly–rp66 were readily purified in sufficient amounts for labeling with 2,4‐dinitrophenyl groups and β‐D‐galactosidase from E. coli, but pol–rp66 and Ser–Ser–rp66 were not for enzyme‐labeling. Ser–Ser–rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4‐dinitrophenyl groups and β‐D‐galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV‐1 RT using serum samples from 600 HIV‐1 seronegative and 30 HIV‐1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4‐dinitrophenylated MBP–Ser–Ser–rp66 and pol–rp66 with β‐D‐galactosidase‐labeled Met–Gly–rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98.0%). J. Clin. Lab. Anal. 14:169–179, 2000.