Detection of antibodies to Epstein-Barr virus antigens by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) is described for the serologic analysis of antibodies to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), viral capsid antigen (VCA), and early antigen (EA). The specificity of each of the ELISAs was demonstrated by the use of well-characterized human sera shown by immunofluorescence assay to be variously reactive for antibodies to one or more of the three viral antigens studied. The ELISA for EBNA was four to 256 times more sensitive than immunofluorescence assays with all 33 EBNA-positive sera tested. The ELISAs for VCA and EA were also more sensitive than immunofluorescence assays: approximately 50% of the sera tested showed higher antibody titers. Sera that were negative for all three antigens by immunofluorescence assay were also negative by ELISA for each antigen. These ELISAs for EBV are rapid, sensitive, and objective and thus provide new and valuable methods for the detection of antibodies to EBV-related antigens.     

Detection of circulating antibodies in cutaneous leishmaniasis by enzyme-linked immunosorbent assay (ELISA)

An immunoenzymatic diagnostic technique applicable to cutaneous leishmaniasis is described. The antigen used (Leishmania tropica major) was equally useful in diagnosing visceral and mucocutaneous forms of the disease. The criteria for positivity were defined by using groups of negative controls, and the specificity of the reaction was evaluated by using sera from patients with various diseases. Among these, sera from patients with lepromatous leprosy, tuberculosis, or African trypanosomiasis strongly cross-reacted with leishmania antigen. Examining serial dilutions of the sera facilitated the interpretation of the results and eliminated a significant percentage of false positives.     

Detection and quantification of anti-ki antibodies by enzyme-linked immunosorbent assay using recombinant ki antigen

Objective. To establish an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Ki antibody, using a bovine recombinant Ki antigen, and studying its specificity. Methods. Sera from 220 patients with various connective tissue diseases were screened, and a prospective study of fluctuations in anti-Ki antibody and clinical course of a woman with systemic lupus erythematosus (SLE) was analyzed, by ELISA. Results. Anti-Ki antibodies were present in 18.9% of patients with SLE. The titer of anti-Ki antibody in the woman with SLE rose before the onset of pericarditis and pleuritis in this longitudinal study. Conclusion. ELISA using a recombinant Ki antigen is useful for the diagnosis of SLE, and it might be useful in estimating disease activity in patients with SLE.     

Detection and quantification of anti-Ki antibodies by enzyme-linked immunosorbent assay using recombinant Ki antigen.

OBJECTIVE. To establish an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Ki antibody, using a bovine recombinant Ki antigen, and studying its specificity. METHODS. Sera from 220 patients with various connective tissue diseases were screened, and a prospective study of fluctuations in anti-Ki antibody and clinical course of a woman with systemic lupus erythematosus (SLE) was analyzed, by ELISA. RESULTS. Anti-Ki antibodies were present in 18.9% of patients with SLE. The titer of anti-Ki antibody in the woman with SLE rose before the onset of pericarditis and pleuritis in this longitudinal study. CONCLUSION. ELISA using a recombinant Ki antigen is useful for the diagnosis of SLE, and it might be useful in estimating disease activity in patients with SLE.     

Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies

From a panel of mouse monoclonal antibodies reactive with a repeating epitope of the schistosome circulating anodic antigen, an IgG1 monoclonal antibody was selected. This monoclonal antibody was applied in a sandwich enzyme-linked immunosorbent assay as capture antibody and as alkaline phosphatase labeled conjugate. This assay allowed a sensitive quantitation of circulating anodic antigen in serum samples of infected individuals, detecting less than 1 ng antigen/ml serum. In Schistosoma mansoni infected individuals from Zaire, the level of antigen in serum correlated with fecal egg output. The lower detection level of the immunoassay corresponded to a level of about 10 eggs/gm feces.     

Detection of mycobacterial antigens in cerebrospinal fluid by enzyme-linked immunosorbent assay

By use of commonly available antibodies against Mycobacterium bovis BCG, Mycobacterium tuberculosis antigens can be detected by a rapid and sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The ELISA was dose-dependent and capable of detecting as little as 4 ng of antigens. Absorbance for 5 patients with confirmed tuberculous meningitis ranged from 0.150 to 0.600 with a mean value of 0.271 +/- 0.190. For 134 non-meningitis control patients and 6 treated tuberculous meningitis patients, optical densities were 0.032 +/- 0.009 and 0.029 +/- 0.010, respectively. Specificity was demonstrated by the negative results (0.028 +/- 0.006) with bacterial and cryptococcal antigens. Maximum cross-reactivity with non-tuberculous mycobacterial antigens was less than 7%.     

Evaluation of the micro enzyme-linked immunosorbent assay for antibodies to Trypanosoma cruzi

A micro enzyme-linked immunosorbent assay (ELISA) for antibodies to Trypanosoma cruzi was evaluated and the results obtained by ELISA were compared with those obtained by the complement fixation test (CF) and indirect fluorescent antibody test (IFA). Fifty sera collected from residents of the southeastern United States all had reciprocal ELISA titers less than or equal to 320. Similarly, serum samples from 17 patients with T. cruzi infection proven by xenodiagnosis had reciprocal ELISA titers of greater than or equal to 1,280. Specimens from 302 El Salvador Army recruits were tested by ELISA, IFA, and CF. Excellent correlation was observed between results obtained by the three serologic tests; 62.9% of the samples were negative by each of the three tests and 24.5% were positive by all. Overall, 29.5% of the sera were positive for antibodies to T. cruzi by ELISA, 29.5% by IFA, and 31.5% by CF. The data suggest that the micro ELISA is a promising serologic test for measuring antibodies to T. cruzi in individuals and in populations.     

Detection of murine typhus infected fleas with an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.     

A new solid-phase enzyme-linked immunosorbent assay for specific antibodies to measles virus

A convenient and flexible enzyme-linked immunosorbent assay system for the detection of specific antibodies to measles virus has been developed. In this system infected cells are desiccated on 96-well microtiter plates and stored at room temperature. After incubation of samples to be tested in the cell-coated plates and subsequent washing, bound antibodies are detected with a peroxidase-conjugated staphylococcal protein A probe. After another washing and the addition of the appropriate substrate, the amount of bound probe is estimated by colorimetric analysis. This technique offers several advantages. The need for a purified viral antigen source is obviated. The plates are easily prepared and can be stored for months at room temperature. Major viral epitopes, including surface glycoproteins as well as cytoplasmic viral antigens, are preserved despite desiccation. The method is more sensitive than the conventional means of virus-specific antibody detection.     

Detection of anticapsular antibodies to Bacteroides asaccharolyticus in serum from rabbits and humans by use of an enzyme-linked immunosorbent assay.

A sensitive serologic test, the enzyme-linked immunosorbent assay (ELISA), was used to detect serum IgG antibodies directed specifically to a capsular antigen of Bacteroides asaccharolyticus (previously known as Bacteroides melaninogenicus subspecies asaccharolyticus). Anticapsular IgG was measured in 30 specimens of rabbit serum after the animals were immunized with whole B. asaccharolyticus, the two subspecies of B. melaninogenicus, and several other bacterial species. Species-specific anticapsular IgG was demonstrated (P less than 0.001). Levels of anticapsular IgG greater than control levels were likewise detected in serum from two humans, including one patient who had periodontitis and from whom B. asaccharolyticus was isolated, and a laboratory worker who had extensive exposure over a two-year period to B. asaccharolyticus. The ELISA was found to be a relatively simple, sensitive tool for measurement of serum IgG. Its application to detection of immunoglobulins of other classes, including secretory IgA, is anticipated, provided adequate standardization methods are used.     

多克隆抗BCG IgM抗体在结核性胸膜炎的诊断价值

目的研究胸水多克隆抗卡介苗(BCG)IgM抗体的浓度是否有助于结核性胸膜炎的诊断。方法序贯纳入胸腔积液患者102例,进入研究的患者于第一次胸穿时留取胸水待检,以胸膜活检病理检查为金标准,综合其他临床资料,剔除诊断未明者,分为结核性胸膜炎组(n=40)及对照组(n=24)。建立以BCG全菌细胞包被作为固相载体的间接法ELISA反应体系,检测胸水多克隆抗BCG抗体的浓度。评价其在结核性胸膜炎诊断方面的效能。结果病例组胸水抗BCGIgM抗体浓度显著高于对照组(P0.001)。以对照组抗体水平平均值加2倍标准差为诊断界值,阳性预测值可高达96.0%。结论胸水多克隆抗卡介苗(BCG)IgM抗体的浓度对结核性胸膜炎的诊断有一定价值。     

Detection of circulating parasite antigen in murine fascioliasis by two-site enzyme-linked immunosorbent assays

A 2-site enzyme immunoassay was developed for the detection of Fasciola hepatica antigen in the serum of fascioliasis infected mice. The assay utilizes high titer rabbit immunoglobulins to parasite excretory/secretory antigens (FhES) as capture antibody, and also as detection antibody when linked to horseradish peroxidase (HRP) or to biotin for reaction with avidin-peroxidase. The assays were compared with a conventional (antibody detection) ELISA to determine diagnostic utility. Using mean rates of detection of fascioliasis, the HRP-based antigen capture assay diagnosed the infection at 1 week postinfection and showed that circulating antigen levels are maximal 3 weeks after infection. The earliest mean diagnosis for the antibody detection and the biotin-based antigen capture ELISAs were 2 and 3 weeks postinfection, respectively. The addition of known quantities of FhES antigens to normal mouse serum gave estimates of lower limits of detectability for the HRP- and biotin-based assays of 25 ng and 0.25 ng, respectively. Routine use of the biotin-avidin system in the antigen capture test resulted in high background activity making this method insensitive.     

Anti-dextran antibodies of carp detected by the enzyme-linked immunosorbent assay (ELISA)

Carp produce anti-alpha (1-6) dextran antibodies after immunization with a vaccine of Leuconostoc mesenteroides B512. These antibodies can be determined by passive haemagglutination, quantitative precipitation or by ELISA. In our hands the ELISA has proved to be 100 times more sensitive than the passive haemagglutination test. We could not found any correlation between the anti-dextran activity estimated with the passive haemagglutination on the one and the ELISA on the other hand.     

Adult T-cell leukemia with anti-HTLV-I antibody but no HTLV-I DNA in tumor cells.

Adult T-cell leukemia (ATL) is usually defined as a malignant disease of T cells infected by human T-lymphotropic virus type I (HTLV-I). In the present study, we describe a 49-year-old woman with an acute type ATL, whose leukemic cells do not contain the HTLV-I genome. Laboratory tests revealed an increase in abnormal lymphocytes with convoluted nuclei, elevated serum lactate dehydrogenase levels, increased thymidine kinase activity and soluble interleukin-2 receptor-alpha levels. Serum examination demonstrated positive anti-HTLV-I antibody, but Southern blot analysis using the whole HTLV-I genome as a probe did not detect any integration of the viral genome. In contrast, PCR detected the HTLV-I pX region in the same DNA samples as used for Southern blot analysis. These findings suggest two possibilities. One possibility is that ATL in this patient is generated by other pathogens than HTLV-I virus. She is also an HTLV-I carrier. The other possibility is that her leukemic T cell clone derived its malignant phenotype from HTLV-I infection, and once this malignant phenotype was obtained, partial deletions of viral genome repeated until the whole viral genome was deleted. Although there is no direct evidence, the former possibility is more likely in the present case.     

Detection of IgM antibodies to Neisseria meningitidis group A polysaccharide in meningitis patients by direct and antibody capture enzyme-linked immunosorbent assays.

Serum specimens obtained from culture-positive group A meningococcal meningitis patients in Cairo, Egypt were tested for immunoglobulin M (IgM) antibodies to Neisseria meningitidis group A polysaccharide by direct and IgM capture enzyme-linked immunosorbent assays (ELISAs). Sera from patients with meningitis caused by other bacteria were used as negative control specimens. The IgM antibodies to this antigen were detected by direct ELISA in 93% of 58 specimens obtained from patients with group A meningococcal disease three or more days after hospital admission, and by IgM capture ELISA in 83% of 60 such specimens. Sixteen percent of 25 specimens obtained three or more days after admission from negative control patients were positive by direct ELISA, and 4% were positive by IgM capture ELISA. The correlation coefficient of the results with the two assays was 0.85.     

Diffusion-in-gel enzyme-linked immunosorbent assay, ELISA and IFA test in the detection of malarial antibodies

Malarial antibodies in 80 patients were measured using the diffusion-in-gel enzyme linked immunosorbent assay (DIG-ELISA), enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody (IFA) test. Good correlations were obtained between all three tests in terms of sensitivity and reliability. DIG-ELISA has the advantage of being a rapid diagnostic tool for the detection of malarial antibodies.     

New enzyme-linked immunosorbent assay methods for measurement of serum erythropoietin levels and erythropoietin antibodies

For clinical studies with erythropoietin (EPO), enzyme-linked immunosorbent assays for the determination of EPO and EPO antibodies were developed. Using polyclonal and monoclonal EPO antibodies in a sandwich technique, serum EPO levels greater than 10 pg/ml (corresponding to 1 mU/ml, calibrated with the 2nd WHO IRP EPO) can be determined. In 103 healthy blood donors, a mean (+/- SD) value of 36 +/- 19 pg EPO/ml was found. Very high EPO concentrations were found in patients suffering from myelodysplastic syndrome and aplastic anemia; elevated levels were associated with rheumatoid arthritis and myelomatosis. No EPO antibodies were detectable in EPO-treated patients.     

Detection of serum albumin receptor in hepatitis B virus carriers by enzyme-linked immunosorbent assay

The relationship between glutaraldehyde-treated polymeryzed human serum albumin (pHSA) and HBe antigen (HBeAg)-positive serum was examined by the use of a new enzyme-linked immunosorbent assay (ELISA). The author succeeded in measuring the pHSA binding activity (pHSA-BA) of HB surface antigen (HBsAg) particles in the present ELISA method using horseradish peroxidase-labelled pHSA after fixation of HBsAg on an anti-HBs-coated well of polystyrene microplates. In HBeAg-positive group, the pHSA-BA of sera of 40 asymptomatic carriers and 2 chronic persistent hepatitis (CPH) patients were higher than those of 8 chronic active hepatitis (CAH) (p less than 0.01) and 8 liver cirrhosis sera (p less than 0.05). On the contrary, in the anti-HBe-positive group the pHSA-BA of 17 asymptomatic carriers and 3 CPH sera were lower than those of 8 CAH (p less than 0.005) and 10 liver cirrhosis patient sera (p less than 0.005). In the both-negative group the pHSA-BA of 8 asymptomatic carrier and 3 CPH sera were also lower than that of 8 CAH (p less than 0.05). In acute exacerbation of HBsAg-positive CAH the pHSA-BA elevated one to two months before the peak of S-GPT level, being correlated with the DNA-polymerase activity. Because of its apparent reproducibility, it is concluded that low cost and some advantages may have clinical utility in the same setting as the HBeAg is now used.