Abstract
In order to perform the immune complex transfer enzyme immunoassays for HIV‐1 p24 antigen and antibody IgGs to HIV‐1 p17, reverse transcriptase and gp41 antigens as rapidly as possible, methods for rapid formation of the immune complexes on solid phase are described. HIV‐1 p24 antigen was reacted with monoclonal anti‐p24 Fab′‐β‐d‐galactosidase conjugate at a high concentration and subsequently with polystyrene beads coated successively with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG and 2,4‐dinitrophenyl‐biotinyl bovine serum albumin‐affinity‐purified anti‐p24 Fab′ conjugate. Antibody IgGs to HIV‐1 were reacted with polystyrene beads coated successively with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG and 2,4‐dinitrophenyl‐HIV‐1 antigen conjugates and subsequently with HIV‐1 antigen‐β‐D‐galactosidase conjugates. The periods of time used for the formation of the immune complexes comprising the three components on the polystyrene beads (15–30 min) were much shorter than those used in the previous immune complex transfer enzyme immunoassays (90–300 min), and the sensitivities of the present and previous immune complex transfer enzyme immunoassays were similar. The detection limit of the HIV‐1 p24 antigen by the present and previous methods were similar (3 to 10 zmol/assay). J. Clin. Lab. Anal. 12:227–237, 1998. © 1998 Wiley‐Liss, Inc.
Keywords: antibody, human immunodeficiency virus type 1, enzyme immunoassay, βd‐galactosidase
References
- 1. Hashida S, Hirota K, Hashinaka K, et al.: Detection of antibody IgG to HIV‐1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV‐1 infection. J Clin Lab Anal 7: 353–364, 1993. [DOI] [PubMed] [Google Scholar]
- 2. Hashida S, Hashinaka K, Hirota K, et al.: Detection of antibody IgG to HIV‐1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV‐1 infection. J Clin Lab Anal 8: 86–95, 1994. [DOI] [PubMed] [Google Scholar]
- 3. Hashinaka K, Hashida S, Hirota K, et al.: Detection of anti‐human immunodeficiency virus type 1 (HIV‐1) immunoglobulin G in urine by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) with recombinant reverse transcriptase as an antigen. J Clin Microbiol 32: 819–822, 1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Hashida S, Hashinaka K, Saitoh A, et al.: Diagnosis of HIV‐1 infection by detection of antibody IgG to HIV‐1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens. J Clin Lab Anal 8: 237–246, 1994. [DOI] [PubMed] [Google Scholar]
- 5. Ishikawa S, Hashida S, Hashinaka K, et al.: Diagnosis of HIV‐1 infection with whole saliva by detection of antibody IgG to HIV‐1 with ultrasensitive enzyme immunoassay using recombinant reverse transcriptase as antigen. J Acquir Immune Defic Syndr Hum Retrovirol 10: 41–47, 1995. [PubMed] [Google Scholar]
- 6. Ishikawa S, Hashida S, Hashinaka K, et al.: Whole saliva dried on filter paper for diagnosis of HIV‐1 infection by detection of antibody IgG to HIV‐1 with ultrasensitive enzyme immunoassay using recombinant reverse transcriptase as antigen. J Clin Lab Anal 10: 35–41, 1996. [DOI] [PubMed] [Google Scholar]
- 7. Hashida S, Hashinaka K, Nishikata I, et al.: Immune complex transfer enzyme immunoassay that is more sensitive and specific than Western blotting for detection of antibody immunoglobulin G to human immunodeficiency virus type 1 in serum with recombinant pol and gag proteins as antigens. Clin Diagn Lab Immunol 2: 535–541, 1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Hashida S, Hashinaka K, Nishikata I, et al.: 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. J Virol Methods 62: 43–53, 1996. [DOI] [PubMed] [Google Scholar]
- 9. Hashida S, Hashinaka K, Nishikata I, et al.: Measurement of human immunodeficiency virus type 1 p24 in serum by an ultrasensitive enzyme immunoassay, the two‐site immune complex transfer enzyme immunoassay. J Clin Microbiol 33: 298–303, 1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Hashida S, Hashinaka K, Nishikata I, et al.: 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'. J Clin Lab Anal 10: 302–307, 1996. [DOI] [PubMed] [Google Scholar]
- 11. Ishikawa S, Hashida S, Hashinaka K, et al.: More sensitive immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV‐1 with shorter incubation time for immunoreactions and larger volumes of serum samples. J Clin Lab Anal 11: 244–250, 1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Hashida S, Ishikawa S, Hashinaka K, et al.: Optimal conditions of immune complex transfer enzyme immunoassays for antibody IgGs to HIV‐1 using recombinant p17, p24 and reverse transcriptase as antigens. J Clin Lab Anal 12: 98–107, 1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Hashida S, Ishikawa S, Hashinaka K, et al.: Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV‐1. J Clin Lab Anal 12: 115–120, 1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Ishikawa S, Hashida S, Hashinaka K, Adachi A, Oka S, Ishikawa E: Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV‐1 p17 antigen. J Clin Lab Anal 12: 179–189, 1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Hashinaka K, Hashida S, Saitoh A, et al.: Conjugation of recombinant reverse transcriptase of HIV‐1 to β‐D‐galactosidase from Escherichia coli for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti‐HIV‐1 IgG. J Immunol Methods 172: 179–187, 1994. [DOI] [PubMed] [Google Scholar]