Abstract:
: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' -beta-D-galactosidase conjugate was trapped onto polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, was eluted with epsilon N-2, 4-dintrophenyl-L-lysine, and was transferred to polystyrene beads coated with streptavidin. beta-D-Galactosidase activity bound to the streptavidin-coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound beta-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 beta-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' -beta-D-galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at -20 degrees C, and this increase was prevented without prolongation of the assay time for bound beta-D-galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate for affinity-purified rabbit anti-p24 Fab'beta-D-galactosidase conjugate.
journal_name
J Clin Lab Analjournal_title
Journal of clinical laboratory analysisauthors
Hashida S,Hashinaka K,Nishikata I,Saito A,Takamizawa A,Shinagawa H,Ishikawa Edoi
10.1002/(SICI)1098-2825(1996)10:5<302::AID-JCLA11>subject
Has Abstractpub_date
1996-01-01 00:00:00pages
302-7issue
5eissn
0887-8013issn
1098-2825pii
10.1002/(SICI)1098-2825(1996)10:5<302::AID-JCLA11>journal_volume
10pub_type
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journal_title:Journal of clinical laboratory analysis
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