Enzyme immunoassay with synthetic peptides to detect anti-HTLV-I antibodies.

We developed an enzyme immunoassay (EIA) system to detect antibodies to human T-lymphotropic virus type I (HTLV-I). This system uses chemically synthesized oligopeptides to capture anti-HTLV-I antibodies in serum. The two epitopes of HTLV-I proteins exhibiting the most specific antigen-antibody reaction reside within amino acids 100-130 of p19, a core protein encoded by gag, and amino acids 175-199 of gp46, an envelope glycoprotein encoded by env. This new assay uses synthetic peptides corresponding to these two regions modified by adding two lysine residues at the amino terminal of each peptide to facilitate the binding to the surface of the microtiter plate wells. We compared the performance of our EIA with gelatin-particle-agglutination (PA) and indirect-immunofluorescence (IF) assays, both of which use viral proteins purified from virus-carrying cell cultures. Mass screening by EIA with various synthetic peptides was more accurate than the current confirmatory IF assay.

Linear antigenic regions of the structural proteins of human T-cell lymphotropic virus type I detected by enzyme-linked immunosorbent assays using synthetic peptides as antigens.

We synthesized 46 sequential peptides 21 to 39 amino acids long over the structural protein of human T-cell leukemia virus type I (HTLV-I; the p19 and p24 gag protein and the gp46 and p20E env proteins) and tested their reactivities against antibodies in sera from HTLV-I healthy carriers and patients diagnosed as having human T-cell leukemia-lymphoma (ATLL) and myelopathy (HAM) by using an enzyme-linked immunosorbent assay. Of the 46 synthetic peptides, 18 peptides (2 corresponding to the p19 gag protein, 2 corresponding to the p24 gag protein, 8 corresponding to the gp46 env protein, and 6 corresponding to the p20E env protein) reacted with antibodies in the sera from HTLV-I healthy carriers. In particular, the peptides comprising amino acids 100 to 119 and 119 to 130 of the gag and 175 to 199, 213 to 236, 253 to 282, and 288 to 317 of the env proteins reacted with antibodies in sera from more than 30% of HTLV-I healthy carriers. These peptides also showed high reactivities to the antibodies in the sera from patients with ATLL and HAM. The results indicate that the predominant antigenic regions of the structural protein of HTLV-I were located at the C-terminal end of the p19 gag protein and the C-terminal half of the gp46 env protein, and the corresponding peptides proved to be useful antigens in detecting antibodies in the sera from individuals infected with HTLV-I.

Elevated antibodies to synthetic peptides of HTLV-1 envelope transmembrane glycoproteins in patients with HAM/TSP.

We studied the antibody response to various kinds of well-characterized synthetic peptides of human T lymphotropic virus type 1 (HTLV-1) envelope glycoproteins in patients with HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis (TSP) and non-HAM/TSP HTLV-1 carriers. The serum antibody titers to most of the synthetic peptides were significantly higher in patients with HAM/TSP than those in non-HAM/TSP HTLV-1 carriers. However, the degree of the increase of antibody titers to the synthetic peptides corresponding to the transmembrane portions of HTLV-1 envelope glycoproteins (env-p20E), such as p20E 332-352, 374-392, 426-448 and 458-488, was greater than those to synthetic peptides of exterior portions of HTLV-1 envelope glycoproteins (env-gp46) in sera from patients with HAM/TSP. Antibodies to env-p20E 332-352 and 374-392 were elevated in the cerebrospinal fluid (CSF) only from patients with HAM/TSP but not from non-HAM/TSP HTLV-1 carriers. These data indicate that the increase of antibody titers to transmembrane portions of HTLV-1 envelope glycoproteins in sera and CSF is a characteristic feature of antibody response in patients with HAM/TSP and may be closely associated with the development of HAM/TSP from non-HAM/TSP HTLV-1 carriers.

Serological discrimination between HTLV-I and HTLV-II antibodies by ELISA using synthetic peptides as antigens.

Using the peptides from amino acids 100-130 of the HTLV-I gag protein, 175-199 of the HTLV-I env protein and the corresponding peptides of HTLV-II (amino acids 106 to 135 of the gag protein and 171 to 196 of the env protein), we tested for reactivity against antibodies by enzyme immunoassay in sera from HTLV-I and HTLV-II carriers. The peptides derived from the env proteins have high specificity for antibody binding. The peptide based on amino acids 175-199 of HTLV-I reacted with antibodies in sera from all HTLV-I carriers, and the peptide composed of amino acids 171-196 of HTLV-II reacted with antibodies in sera from all HTLV-II carriers. For the peptides derived from the gag proteins, we observed some cross-reactivity in sera from persons with anti-HTLV-I and anti-HTLV-II, due to antibody binding to the peptide corresponding to 12 amino acids from the C-terminal end of the gag protein. Separate enzyme immunoassays that used the four synthetic peptides as antigens clearly distinguished between serum with antibodies to HTLV-I or HTLV-II in various individuals and excluded false positive results using the particle agglutination assay that used a whole-virus lysate of HTLV-I as antigen.

Detection of antibodies to human T-lymphotropic virus type I by using synthetic peptides.

We describe an enzyme-linked immunosorbent assay (ELISA) for antibodies to HTLV-I using synthetic peptides corresponding to antigenic regions of HTLV-I structural protein. We observed that the peptides amino acid 100-119, 100-130, 131-160 and 295-314 of the gag protein, and 89-115, 175-199, 350-386 and 458-488 on the env protein correspond to the antigenic regions of HTLV-I structural protein. In particular, the peptide corresponding to amino acid 100-130 of the gag protein reacted with antibodies in all sera from HTLV-I carriers diagnosed by particle agglutination assay (PA) and indirect immunofluorescence assay (IF), but did not react with sera from healthy volunteers judged to be negative by PA and IF. Our results indicate that the ELISA using the peptide 100-130 of the gag protein can be used for donor screening of HTLV-I antibodies.

Prevention of acquired defects in platelet function during blood processing.

Two kinds of platelet concentrates were prepared in this study: one as pelleted platelets from platelet-rich plasma (PCs), and the other as a platelet suspension from the buffy coat fraction (nonpelleted PCs). The characteristics of both platelet concentrates were studied. White cell contamination in the nonpelleted PCs was below the threshold of detection by a microcell counter (less than 300 cells/microliter of concentrate); in the pelleted PCs, it was 3 +/- 1 x 10(7) cells per concentrate. The difference between the white cell contamination in pelleted PCs and nonpelleted PCs was significant (p less than 0.001). The degree of aggregation induced in pelleted PCs by ADP (9 microM) and collagen decreased to 33 and 55 percent, respectively, of that in platelet-rich plasma. The secondary wave of platelet aggregation induced by ADP and epinephrine was absent in pelleted PCs, and ATP secretion from these platelets by ADP stimulation also decreased remarkably. However, the degrees of aggregation and ATP secretion in nonpelleted PCs were similar to those in platelet-rich plasma. Furthermore, it was found that the amount of platelet factor 4 release seen in pelleted PCs was large in comparison to that in nonpelleted PCs and platelet-rich plasma. These results suggested that the deterioration of platelet function might have resulted from the platelet activation induced by pellet formation during the preparation of PCs from platelet-rich plasma.