Quantitative estimation by a standardized enzyme-linked immunosorbent assay of human T-cell lymphotropic virus type I antibodies in adult T-cell leukemia and acquired immune deficiency syndrome.

Sera from patients with adult T-cell leukemia and asymptomatic carriers of human T-cell lymphotropic virus type I (HTLV-I) from widely separated areas of the world reacted strongly in a standardized quantitative enzyme-linked immunosorbent assay procedure with HTLV-I viral antigen prepared from a strain isolated in the United States. There was a sharp differentiation of the values seen in the patients as compared with a normal population. Of the 35 acquired immune deficiency syndrome patients with Kaposi's sarcoma, only 2 were positive for HTLV-I antibodies in this test, and the distribution of the negative assay values in the other acquired immune deficiency syndrome patient sera was similar to that seen in the normal sera. Sera which contained extremely high levels of antibodies to other unrelated viruses (rubella virus, cytomegalovirus, and herpes simplex virus) all showed negative anti-HTLV-I results, in a pattern similar to the normal sera. Sera from patients with several autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, thyroiditis) as well as those with infectious mononucleosis or myeloma all also showed the normal distribution of negative results, in spite of the presence of very high levels of the autoantibodies, etc., associated with their illnesses.     

Synthetic-peptide-based enzyme-linked immunosorbent assay for screening human serum or plasma for antibodies to human immunodeficiency virus type 1 and type 2.

A synthetic-peptide-based enzyme-linked immunosorbent assay (EIA) capable of screening for antibodies to both human immunodeficiency virus type 1 (HIV-1) and HIV-2 has been developed for use in blood banks and diagnostic laboratories. Microtiter wells are coated with two synthetic peptides, one corresponding to the highly conserved envelope region of HIV-1 and another corresponding to the conserved envelope region of HIV-2. Overall, sensitivity was 100% in 303 individuals diagnosed with AIDS and 96 individuals diagnosed with AIDS-related complex, 14.8% in a study of 500 high-risk group members, 99.9% in 600 EIA repeatedly reactive (RR)-HIV-1 Western blot (WB)-positive repository specimens, and 100% for 222 geographically diverse HIV-1 specimens and 216 confirmed HIV-2-positive specimens evaluated. The specificity was determined to be 99.72% for a total of 13,004 serum and plasma samples from random volunteer donors evaluated across five blood banks. Forty donors who were found to be EIA RR-WB indeterminate but nonreactive on the United Biomedical, Inc., test (UBI HIV 1/2 EIA) were prospectively followed as an additional measure of specificity. None of the 40 low-risk cases evolved into a positive WB pattern at follow-up. The sensitivity and specificity of this new assay are comparable to those of other Food and Drug Administration-licensed HIV-1 and HIV-1-HIV-2 assays that are currently available in the United States. The UBI HIV 1/2 EIA affords laboratories another choice in the detection of antibodies for HIV-1 and HIV-2 with a test based on an alternative antigen format.     

Evaluation of commercially available third-generation anti-hepatitis C virus enzyme-linked immunosorbent assay in patients on haemodialysis

Restricted antibody reactivity to hepatitis C virus (HCV) synthetic peptides has been observed in HCV-infected patients on haemodialysis (HD). The aim of this study was to evaluate third-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) test systems containing either synthetic peptide HCV antigens or recombinant HCV antigens or a combination of synthetic and recombinant antigens in screening of 69 chronic renal failure patients on HD for HCV infection. Seven patients were detected to have antibodies to HCV by the 'recombinant HCV antigens'-containing kits, of which the recombinant immunoblot assay for HCV confirmed four cases. The recombinant kits had a sensitivity of 100% and a specificity of 66%. However, the ELISA kits with only synthetic HCV antigens failed to detect antibodies in any of the cases (zero sensitivity). Hence a recombinant protein containing ELISA test system is ideal for screening of HCV infection in patients on hemodialysis.     

Antibodies to human T-cell lymphotropic virus type I (HTLV-I) by particle agglutination (PA) test in Korean blood donors.

HTLV-I infection is a recently recognized disease entity that is common in some tropical and subtropical areas, including the southwestern district of Japan. Despite the geographical proximity and frequent cultural exchanges between Korea and Japan, it is understood that Korea is not an endemic area and HTLV-I-associated illnesses are very rare in Korea. This study was designed to evaluate the positive rate of anti-HTLV-I antibodies in Korean blood donors and its regional distribution. Sera were obtained from blood donors from various districts around Korea. Anti-HTLV-I antibodies were detected by using the microtiter particle agglutination test employing an indirect agglutination technique. A total of 9,281 donors were tested and 12 donors (0.13%) were positive for anti-HTLV-I antibodies, 10 (0.11%) out of 8,845 males and 2 (0.46%) out of 436 females, with relative female predominance. A relatively high incidence of anti-HTLV-I positive donors was observed in Cheju Island (0.80%), Kyungnam (0.31%), and Chonnam (0.15%). In conclusion, the positive rate of anti-HTLV-I antibodies seemed to be very low in Korea, but the highest positive rate of anti-HTLV-I antibodies was noticed on Cheju Island, warranting further research for confirmation.     

Development of enzyme-linked immunosorbent assay for detecting antibodies to replication-competent murine leukemia virus

A method for detecting the antibodies to replication-competent retrovirus (RCR) was developed. Specific fragments of murine leukemia virus (MLV) Gag or Env protein were cloned and expressed in Escherichia coli, and used subsequently to develop the ELISA system. It was found that CA of Gag and SU of Env, but not the transmembrane portion of Env, could be used in ELISA. ELISA conditions such as coating buffer and blocking solution were optimized using sera obtained from mice immunized with amphotropic MLV particles. In an optimized ELISA system, serum samples from normal healthy individuals provided very low absorbance values. ELISA was performed using serum samples from patients who had received skin fibroblasts engineered with MLV-based retroviral vector. Experimental samples presented absorbance values comparable to those found with control serum samples from normal, healthy individuals, showing no evidence of RCR infection.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for Giardia lamblia antigen in stool.

The lack of a quick, simple, and inexpensive diagnostic test has limited the ability of public health officials to rapidly assess and control outbreaks of Giardia lamblia in child day-care centers. We evaluated the performance of a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of a G. lamblia-associated antigen in stool. Stool specimens were collected from the diapers of 426 children attending 20 day-care centers, fixed in 10% Formalin and polyvinyl alcohol, and examined by microscopy by Formalin concentration and trichrome staining techniques. Specimens were also tested visually and spectrophotometrically by ELISA. Of 99 tests positive by microscopy, 93 were visually positive by ELISA (sensitivity, 93.9%). Of 534 tests negative for G. lamblia by microscopy, 32 (6.0%) were ELISA positive. However, on the basis of examination of multiple specimens from the same child, none of these could be considered false-positive ELISAs; the specificity of the ELISA was therefore 100%. The sensitivity of both microscopy and ELISA improved as the number of specimens per child increased. An optical density value of greater than 0.040 was 98.0% sensitive and 100% specific for G. lamblia. This ELISA, which appeared to be more sensitive for G. lamblia than did microscopic examination of stool, should be useful as an epidemiologic tool, particularly in day-care settings, and may also have a role in confirming clinical diagnoses of giardiasis.     

Microplate enzyme-linked immunosorbent assay for bovine leukemia virus antibody.

A microplate enzyme-linked immunosorbent assay method was developed for the measurement of bovine immunoglobulin G antibody specific to the envelope antigen (glycoprotein 60) of bovine leukemia virus. The test was then performed on 440 serum samples from dairy cows belonging to herds in which bovine leukemia was suspected or which were leukemia free, and the results were compared with those obtained with the gel-diffusion technique.     

Comparative assessment of the gelatin particle agglutination test and an enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis

The performances of the gelatin particle agglutination test (GPAT) and enzyme-linked immunosorbent assay (ELISA) for the diagnosis of strongyloidiasis with reference to the results of the agar plate culture technique (APCT) were evaluated with samples from 459 individuals from communities in northeast Thailand where strongyloidiasis is endemic. The prevalence of strongyloidiasis in five sample groups determined by GPAT varied between 29.3 and 61.5% (mean, 38.8%). ELISA and APCT, employed concurrently, gave lower prevalence rates of 27.5% (range, 21.6 to 42.1%) and 22.7% (range, 12.7 to 53.8%), respectively. By using APCT as the standard method, the sensitivity of GPAT was generally higher than that of ELISA (81 versus 73%). The specificity of GPAT was slightly lower than that of ELISA (74 versus 86%). The resulting GPAT titers exhibited positive linear relationships with the ELISA values (optical density at 490 nm) (P < 0.05), which suggests that the GPAT titer also reflects the levels of specific antibody comparable to those reflected by the ELISA values. Based on the relative ease and simplicity of use of the technique as well as the acceptable rates of sensitivity and specificity of the test, GPAT is more practical for screening for strongyloidiasis than the conventional ELISA.     

Latex agglutination and enzyme-linked immunosorbent assays for cytomegalovirus serologic screening of transplant donors and recipients.

The effectiveness of three serologic assays (two enzyme-linked immunosorbent assays [ELISAs] and latex agglutination) for cytomegalovirus (CMV) serologic matching of donors and recipients was assessed over a 2-year period in a major organ transplant program. Sera with equivocal test results were investigated by repeat testing of serum samples and additional specimens from the individuals involved and monitoring of CMV infection in recipients. An in-house ELISA identified all CMV-infective donors as seropositive. Of 63 ELISA-positive donors, 5 were negative by latex agglutination; recipients from 3 of these donors developed primary CMV infection posttransplant. The in-house ELISA and a commercial ELISA (Abbott enzyme immunoassay; Abbott Laboratories, North Chicago, Ill.) had a 93% concordance of results; follow-up testing indicated that the Abbott assay was sensitive but had a false-positive rate of about 11%. One recipient with a false-positive result developed symptomatic primary CMV infection after receiving a seropositive organ. Thus, performance characteristics of currently used screening assays affect recipient outcome.     

Evaluation of a commercially available recombinant-protein enzyme-linked immunosorbent assay for detection of antibodies produced in scrub typhus rickettsial infections

The 56-kDa major outer membrane protein antigen of Orientia tsutsugamuchi is the immunodominant antigen in human scrub typhus (ST) infections. An enzyme-linked immunosorbent assay (ELISA) using a recombinant 56-kDa protein (r56) to detect specific immunoglobulin M (IgM) produced in ST infections was developed, and its performance was evaluated using sera from patients with active ST (n = 59), spotted fever (SF) (n = 31), and murine typhus (MT) (n = 6) and from those without rickettsial infection (n = 52). The r56 ELISA was compared to an ELISA using native whole cell lysate of O. tsutsugamushi Karp or O. tsutsugamushi Gilliam as antigens. The performance of the assays using r56 was similar to that of those using native antigens. Using indirect immunoperoxidase (IIP) as the reference test, sensitivities were 86, 88, and 88% while specificities were 84, 90, and 87% in the three assays. Furthermore, cross-reactivity in confirmed cases of SF and MT was low (5.4, 2.7, and 2.7% respectively). The additional use of IgG in the r56 ELISA gave improved performance (sensitivity, 80%; specificity, 96%; cross-reactivity in SF and MT, 2.7%). The detection of high levels of IgG in some IgM-negative patients illustrates the importance of including a test for IgG in the detection of secondary or reactivated infections, since many of these patients were from regions in Thailand where these infections are endemic.     

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.     

Comparative studies with an enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect IgG class-specific antibodies to avian infectious bronchitis virus (IBV) in chickens. The technique detected specific antibody with a high degree of sensitivity and was more sensitive than neutralisation or haemagglutination-inhibition methods of antibody assay. The possible application of the technique for diagnosis and research on IBV is discussed.     

Comparison of immunofluorescence, particle agglutination, and enzyme immunoassays for detection of human T-cell leukemia virus type I antibody in African sera.

The effectiveness of four screening tests for detecting antibody to human T-cell leukemia virus type I (HTLV-I) was determined by using 2,700 African serum specimens. The tests studied were indirect immunofluorescence, particle agglutination from Fujirebio, and two enzyme immunoassays, one from Abbott Laboratories that used virus lysate from HUT 102 cells and the other from Cambridge BioScience Corp. that used an env recombinant protein. Positive and doubtful sera were confirmed by Western immunoblot and radioimmunoprecipitation assay with Food and Drug Administration seropositivity criteria. The best results were obtained with the two enzyme immunoassays, which were more sensitive (100 and 98.6% [Abbott and Cambridge, respectively]) and more specific (98.7 and 96.5%). Indirect immunofluorescence exhibited difficulties for reading and interpretation. With particle agglutination, prozone was observed for 9 of 78 HTLV-I-positive serum specimens. False-positives in any of the tests were not linked to cross-reactions with human immunodeficiency viruses. However, confirmation tests remain necessary for HTLV-I screening.     

Simplified enzyme-linked immunosorbent assay for specific antibodies to respiratory syncytial virus.

A simplified and reliable enzyme-linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against respiratory syncytial virus (RSV). RSV-infected cells were fixed and dried on 96-well microtiter plates and kept at 4 degrees C. The titers of reference sera were determined by endpoint dilution. A linear relation was found between the titers and the logarithm of absorbance values of sera diluted to 1:1,000 (r = 0.93, P less than 0.001). Measurement of RSV antibodies was done by using a single serum dilution (1:1,000) in conjunction with a standard curve. A strong correlation was found between complement fixation and ELISA results (r = 0.89, P less than 0.001). In addition, the ELISA method exhibited higher titers and a greater sensitivity than did complement fixation, although the applicability of the assay is limited with positive serum samples of low titer.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to bovine leukemia virus in serum and milk.

The results of examination of 375 bovine serum and 150 bovine milk samples for detection of bovine leukemia virus infection by the immunodiffusion technique were compared with those of an enzyme-linked immunosorbent assay (ELISA). It was concluded that the ELISA is another useful method for the detection of antibodies to bovine leukemia virus in serum and milk. The ELISA provides a quantitative result and has the advantage of being more sensitive and less time-consuming than the conventional immunodiffusion technique.     

Large-scale serological screening for cytomegalovirus antibodies in homosexual males by enzyme-linked immunosorbent assay.

We compared an enzyme-linked immunosorbent assay (ELISA) with complement fixation and radioimmunoassay in determining the presence of immunoglobulin antibodies to cytomegalovirus. Of an initial 93 serum samples tested, the correlation between ELISA and radioimmunoassay was 98.9% and that between ELISA and complement fixation was 96.1%. ELISA was used to screen 1,123 homosexual men in San Francisco. Of 479 men attending a homosexual health fair, 35 (7%) lacked cytomegalovirus antibodies by ELISA. Only 15 (2%) of 644 homosexual men attending a municipal sexually transmitted disease clinic were found to be seronegative. All but one of the seronegatives detected by ELISA were also seronegative by radioimmunoassay and complement fixation. We conclude that ELISA can be used to reliably perform large-scale screening for the presence of immunoglobulin G antibodies to cytomegalovirus.     

Rapid enzyme-linked immunosorbent assay of whole blood for detection of antibodies to japanese encephalitis virus

The application of the rapid system of enzyme-linked immunosorbent assay (ELISA) was studied to quantify antibodies to Japanese encephalitis virus in large-scale epidemiological surveys, especially by testing under field conditions. The assay system, with 15 min for the first reaction and 30 min each for the second and the third reactions, was highly reproducible (coefficients of variation with swine positive sera were less than 5.8%) and was significantly correlated with the routine assay system with 1 h for each reaction (correlation coefficient was 0.960). Compared with the haemagglutination inhibition test, the rapid system gave a correlation coefficient of 0.916 and qualitative agreeement of 96.1%. The substitution of whole blood for serum in the first reaction was also examined not only to avoid serum separation but also to apply this system to antibody quantification in animals from which sufficient amounts of sera cannot be easily obtained: only 2 μl were needed for the test. The results obtained with 51-fold diluted whole blood had a linear relationship to those obtained with 100-fold diluted sera in swine and humans.     

Simple standardized enzyme-linked immunosorbent assay for human antibodies to Entamoeba histolytica.

A simple solid-phase enzyme-linked immunosorbent assay procedure for the detection of human antibodies to Entamoeba histolytica was developed which showed a high degree of correlation with the agar gel diffusion, counterelectrophoresis, and indirect hemagglutination methods, as well as with clinical data. The enzyme-linked immunosorbent assay is rapid (1 h 15 min, total incubation time), and the reported values are referenced to a positive control so that they correlate with levels of antibody sufficient to be detected by the gel diffusion methods. The enzyme-linked immunosorbent assay is highly reproducible, specific, and sensitive; it can be used qualitatively or quantitatively.     

Cytogenetic studies of human T-cell leukemia virus type I carriers. A family study

Recently, the chromosome 14q11 anomaly has been reported to be specific to adult T-cell leukemia (ATL), and this anomaly has also been confirmed in the preleukemic state of adult T-cell leukemia (pre-ATL) patients. Because the cytogenetic abnormality at the stage of human T-cell leukemia virus type I (HTLV-I) carrier remains uncertain, we performed cytogenetic studies of lymphocytes stimulated with phytohemagglutinin in three HTLV-I carriers and three non-HTLV-I carriers in an ATL family. As a result, in three HTLV-I carriers, four of 311 cells examined (1.3%) had chromosome 14q11 anomaly. However, in three non-HTLV-I carriers, none of 260 cells examined had chromosome 14q11 anomaly. These results suggest that chromosome 14q11 anomaly is already present at the stage of HTLV-I carrier and seems to be an important cytogenetic clue to the pathogenesis of ATL.