| Project/Area Number |
05557015
|
|---|
| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Pathological medical chemistry
|
|---|
| Research Institution | Miyazaki Medical College |
|---|
Principal Investigator |
ISHIKAWA Eiji Miyazaki Medical College Faculty of Medicine Professor, 医学部, 教授 (40029939)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MURAKAMI Yoshiaki Sumitomo Pharmaceutical Co., Ltd.Chief Director, 研究開発推進部, 部長
村上 嘉昭 住友製薬(株), 診断薬開発部, 部長
|
|---|
| Project Period (FY) |
1993 – 1994
|
| Project Status |
Completed (Fiscal Year 1994)
|
| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1994: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1993: ¥4,100,000 (Direct Cost: ¥4,100,000)
|
|---|
| Keywords | Human T-cell leukemia virus type I / HTLV-I / Antibody IgG / Enzyme immunoassay / beta-D-Galactosidase / Microplate / Fluororeader / 抗HTLV-I IgG / 成人T細胞白血病ウイルス / EIA / ELISA / HTLV-I感染 / 合成ペプチド抗原 |
|---|
| Research Abstract |
A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibody IgG to HTLV-I using synthetic peptides as antigens has been developed and improved in sensitivity, specificity and performance so as to be used in practical assays. The immune complex, consisting of 2,4-dinitrophenyl-bovine serum albumin-synthetic peptide conjugate, antibody IgG to HTLV-I and synthetic peptide-beta-D-galactosidase conjugate was trapped onto colored polystirene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG,was eluted with epsilonN-2,4-dinitrophenyl-L-lysine and was transferred onto white polystirene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG.beta-D-Galactosidase activity bound to the white polystirene balls was assayd by fluorometry. The syntehtic peptides selected from nine peptides tested for detecting antibody IgG to HTLV-I in serum samples of HTLV-I seropositives with high sensitivity and high specificity were Ac-Cys-env gp46 (188-224) , Ala-Cys-env gp46 (237-262) and Cys-gag p19 (100-130) . This method detected antibody IgG to HTLV-I at low levels below those detectable by Western blotting and immunofluorescence test. The specificity could be confirmed by preincubation with excess of the synthetic peptides, which were soluble in the absence of detergents, and was improved by demonstrating antibody IgG to HTLV-I not only in serum but also in urine and probably in oral fluids from HTLV-I to two or more different peptides. This method could detect antibody IgG to HTLV-I seropositives. For practical use, this assay method was simplified in processes by using microplates and a fluororeader, eliminating the use of tweezers for transfer of solid phase from test tube to test tube, which was causative of false-positivity due to carryover. Attempts are still being made to modify the form of solid phase for more practical use.
|
