Specific serodiagnosis with adult Onchocerca volvulus antigen in an enzyme-linked immunosorbent assay

The cross-reactivity of the blood from onchocerciasis, loiasis, and dipetalonemiasis was tested by a micro-ELISA technique, utilizing adult Onchocerca volvulus antigen and blood samples taken on filter paper. The average ELISA values (OD at 500 nm) were as follows: 0.58 in persons with O. volvulus microfilariae (n = 81), 0.49 in microfilariae-negatives from the same endemic area (n = 39), 0.15 in dipetalonemiasis (n = 27), and 0.25 in loiasis (n = 12), while those of 65 Dipetalonema perstans-negative people were markedly low (average 0.14) and that of 22 Loa loa-negatives, 0.22, respectively. This ELISA could successfully differentiate onchocerciasis from dipetalonemiasis and loiasis.     

Serologic diagnosis of whooping cough by an enzyme-linked immunosorbent assay using fimbrial hemagglutinin as antigen

Diagnosis of whooping cough by an enzyme-linked immunosorbent assay (ELISA) that measures serum IgG, IgM, and IgA antibody to the fimbrial hemagglutinin of Bordetella pertussis was compared to isolation from nasopharyngeal swabs in a prospective study. Of 77 patients with upper respiratory tract infections of unknown etiology in which B. pertussis infection could not be excluded on clinical grounds, 26 were culture-positive, including one for Bordetella parapertussis. All 26 patients were positive by ELISA except one asymptomatic erythromycin-treated patient (ELISA sensitivity, 96%). Among culture-negative patients, 24 additional patients were positive by ELISA. Thus, only about one half of the patients with whooping cough were identified by culture under optimal conditions. Positive titers of IgM antibody and/or high titers of IgG antibody in the first serum sample, allowing for a rapid diagnosis, were found only in 26 (53%) of 49 serologically positive patients. By combining culture testing with serology, rapid diagnosis was obtained in 41 (82%) of 50 patients.     

An enzyme-linked immunosorbent assay using an arc 5 antigen for the diagnosis of cystic hydatid disease

We report on an easily standardized enzyme-linked immunosorbent assay (ELISA) for the diagnosis of cystic hydatid disease. The antigen used is commercially available, and purified to elicit an arc 5 precipitation line by immunoelectrophoresis (IEP) with sera of patients harbouring hydatid cysts. The IgG-ELISA was highly sensitive and specific, and of diagnostic value compared to total Ig-ELISA, IgM-ELISA or IgA-ELISA. When compared to the indirect hemagglutination test and to counterimmunoelectrophoresis using an arc 5 antigen, minimal cross-reactions were observed in sera of patients with a presumptive diagnosis of hydatidosis but none were observed in those harbouring intestinal helminths, schistosomes or filarial parasites. The assay is of low cost, simple to perform, and highly reproducible. The IgG-ELISA provides an unequivocal laboratory diagnosis of hydatidosis. It is eminently suited for accurate and early identification of hydatid disease patients, for instance in mass surveys, who may benefit immediately from currently available anthelmintics, thereby obviating the need for surgery later.     

Seroepidemiological study of leprosy in a highly endemic population of south India based on an ELISA using synthetic PGL-I

As part of a continuing longitudinal immuno-epidemiological study, blood samples were collected by finger prick from 4243 individuals living in a highly endemic area for leprosy in South India. The samples were tested for IgM antibodies against phenolic glycolipid-I using an ELISA. Seropositivity defined as optical density greater than or equal to 0.2000 was marginally higher in the age group 10-30 years and in females. There was no evidence for a higher level in contacts than in non-contacts. The future prospect for the large scale use of this ELISA in high-endemic populations in special epidemiological investigations or routine control programs as a serological tool to detect leprosy infection appears questionable.     

Early and specific diagnosis of seropositivity to HIVs by an enzyme-linked immunosorbent assay using env-derived synthetic peptides

We describe and evaluate the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using a 22-amino-acid peptide corresponding to the carboxy-terminal end of HIV-1 gp120 and two 30-amino-acid long cyclic peptides including the two vicinal cysteines present on HIV-1 gp41 and on HIV-2 gp36. This test was evaluated. Data obtained with the Western blot (WB) and the peptide-based ELISA on a first panel composed of sera from 547 patients attending a specialized outpatient clinic (high-risk population) are in perfect agreement; moreover, 39 samples that had falsely been found positive with a viral lysate-based ELISA were not detected by peptide-based ELISA. The second panel was composed of 309 sera which were difficult to resolve using both WB and viral lysate-based ELISA. Using the peptide-based ELISA, 134 were found clearly positive and 173 clearly negative; only two were falsely positive. Finally, sera from 16 individuals examined at the time of seroconversion gave high absorbancy readings even if they were weakly reactive by WB (weak gp160 band). This test is thus highly sensitive and specific, and capable of detecting early seroconversion. It is also instrumental in clearly defining samples that are found indeterminate in the WB, and consequently it avoids the unnecessary follow-up required when a false-positive result is obtained using viral lysate-based ELISA.     

The diagnosis of invasive aspergillosis by an enzyme-linked immunosorbent assay for circulating antigen

An inhibition enzyme-linked immunosorbent assay (ELISA) capable of detecting 10 ng of aspergillus carbohydrate antigen/ml of serum was developed. When retrospectively applied to the sera of 19 patients with invasive aspergillosis, the ELISA detected antigen in 11 patients. None of 14 healthy controls or 28 patients with a variety of other infections were positive for circulating antigen. A rabbit model of invasive aspergillosis was also developed. Daily fungal cultures of blood were negative in the rabbits, as in human disease, but antigen was detected in increasing amounts up to the time of death. This ELISA is a sensitive, specific, and easily performed assay for circulating aspergillus antigen that should facilitate early diagnosis of invasive aspergillosis, heretofore, seldom made without invasive tests.     

Identification of Plasmodium vivax sporozoites in mosquitoes using an enzyme-linked immunosorbent assay

A sandwich enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium vivax sporozoites in mosquitoes is described. Monoclonal antibodies produced against Thailand P. vivax sporozoites were used in an ELISA to detect and identify homologous sporozoites of Southeast Asian, Mexican and North Korean origin in extracts of frozen or dried infected mosquitoes. The assay was sensitive enough to detect 1 infected mosquito in a pool of 20 insects or 125-250 sporozoites per 30 microliter of mosquito extract. The use of a nonionic detergent and a single freeze-thaw to disrupt the circumsporozoite antigen significantly increased the sensitivity of the method.     

Detecting artificial anti-dengue IgM immune complexes using an enzyme-linked immunosorbent assay

A variety of methods have been employed to detect viral immune complexes (IC) in clinical specimens. However, most techniques used were not antigen-specific. We developed a simple, specific double antibody sandwich technique to detect artificial anti-dengue (DEN) IgM immune complex (IgM-IC). Positive reactivity with IgM-ICs prepared with live DEN-1, -2, and -3 viruses was found to be related to IgM titers exceeding 1:20 and to the titer of the viruses. Most IgM-ICs prepared with live DEN-4 virus did not react. In contrast, IgM-ICs prepared with hemagglutination antigens, representing all 4 serotypes, reacted positively with amounts of antigens ranging from 2 to 8 units. These IgM-ICs were not type-specific.     

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.     

Rapid diagnosis of tuberculous meningitis by using an enzyme-linked immunosorbent assay to detect mycobacterial antigen and antibody in cerebrospinal fluid

Rapid diagnostic tests for tuberculous meningitis are urgently needed because delayed treatment increases the already high mortality rate of this disease. Direct acid-fast staining of cerebrospinal fluid is the only quick method generally available, but it lacks sensitivity. Therefore, we evaluated the use of an enzyme-linked immunosorbent assay (ELISA) to mycobacterial antigen and antibody in the cerebrospinal fluid of 29 patients with proven tuberculous meningitis, 83 patients with nontuberculous central nervous system infections, and 15 normal controls. The specificity of the test was 96%; the four false-positive results all occurred in patients with bacterial meningitis. Fifteen (52%) of 29 patients with tuberculous meningitis had either a positive antigen or antibody ELISA test, which was significantly more than the number of patients testing positive by direct staining (two of 29 positive; P less than .01). We therefore recommend using an ELISA to detect antigen and antibody but caution that because of limited sensitivity a negative test result does not exclude the diagnosis of tuberculous meningitis.     

Measurement of von Willebrand factor antigen in plasma and platelets with an enzyme-linked immunosorbent assay based on two murine monoclonal antibodies.

Two murine monoclonal antibodies, raised against von Willebrand factor (vWF), were used to construct an enzyme-linked immunosorbent assay (ELISA), for quantitation of vWF antigen (vWFAg) in human plasma and platelets. This assay had a lower limit of sensitivity of 0.0001 IU/ml in buffer, and thus is one to two orders of magnitude more sensitive than other ELISA assays which have been reported. The intraassay, interassay and interdilution coefficients of variation were 4.1, 10.4 and 9.9%, respectively. In normal plasma (n = 20), the vWFAg level was 0.83 (range: 0.42-1.25) IU/ml. In normal washed platelets (n = 10), 0.35 (0.25-0.49) IU/10(9) platelets was found. In plasma obtained from various patient groups the following vWFAg levels (geometric mean and range) were observed: von Willebrand's disease (n = 19): 0.18 (0.02-0.77) IU/ml; patients with liver cirrhosis (n = 20): 3.73 (1.68-9.20) IU/ml; patients with pregnancy-induced hypertension (n = 20): 4.14 (2.28-7.44) IU/ml and patients with malignant disease (n = 10), 2.54 (1.51-5.60) IU/ml. A linear correlation was found between vWFAg levels measured with a polyclonal antibody based Laurell electroimmunoassay (r = 0.92, n = 58) or with a polyclonal antibody based ELISA (r = 0.94, n = 64). The present assay is based on stable and reproducible reagents and allows the specific measurement of vWFAg in plasma and in platelets. This assay may constitute a useful tool for the further investigation of clinical conditions associated with changes in vWFAg levels. In addition, its high sensitivity may facilitate a more detailed study of platelet vWFAg in normal and in pathological conditions.     

Characterization of the antigen reactive with anti-Scl-70 antibodies and its application in an enzyme-linked immunosorbent assay

The characteristics of the Scl-70 antigen (topoisomerase I) have been analyzed by means of autoantibodies. This antigen is a DNA-binding protein, dissociable from DNA at 0.3M NaCl and bound to a fraction of DNA that is very sensitive to nucleases. The molecular weight of the antigen is 105,000 daltons, whether dissociation conditions are used or not. Using chicken erythrocytes, and taking advantage of the strong interaction of the antigen with hydroxyapatite, we have designed a simple and fast purification protocol that allows the determination of anti-topoisomerase I antibodies by enzyme-linked immunosorbent assay.     

Immunodiagnosis of human fascioliasis by enzyme-linked immunosorbent assay using excretory-secretory products

In sera from patients with fascioliasis the enzyme-linked immunosorbent assay (ELISA) was used to detect antibody using excretory-secretory products (ES) from Fasciola hepatica adult worms. The specificity of ES-ELISA (with OD values greater than 0.38) allowed the differentiation among fascioliasis, schistosomiasis, clonorchiasis, and other human parasite infections.     

Measurement of factor VIII procoagulant antigen in normal subjects and in hemophilia A patients by an immunoradiometric assay and by an enzyme-linked immunosorbent assay

Factor VIII coagulant antigen (FVIII:Ag) and FVIII coagulant activity (FVIII:C) were measured in 102 healthy individuals, in 5 hemophilia A carriers and in 21 hemophilia A patients before and after infusion of heat-treated high-purity FVIII concentrates. Factor VIII:Ag was determined by a solid-phase micro enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and by a conventional solid-phase immunoradiometric assay (IRMA). Factor VIII:C was assessed using a one-stage assay. The micro ELISA was decidedly more precise than the IRMA. There was a close correlation between the results obtained by the three assays in the plasma of healthy subjects and in hemophilia A carriers. After transfusion of FVIII concentrates to hemophilia A patients, the FVIII:Ag recoveries were significantly lower than the FVIII:C recoveries and the biological half-life of FVIII:Ag was significantly shorter than for FVIII:C. The calculated half-life of FVIII:C was longer than in any previous study.     

Seroepidemiological study on 724 household contacts of leprosy patients in French Polynesia using disaccharide-octyl-BSA as antigen

A seroepidemiological surveillance of a contact population was started in 1984 in French Polynesia. The ELISA test was used to measure IgM anti-ND-O-BSA in the sera. Specific antibody levels were higher in healthy Polynesians than in normal individuals living in a nonendemic country. The positive threshold of the reaction was fixed according to this background activity in healthy Polynesians. Under these conditions, 100% of the multibacillary patients were detected as seropositive as compared to 5% of the paucibacillary group. In the population of 724 household contacts tested and observed for 2 years: 93 (12.8%) were seropositive, with 8 (1.1%) showing activity equivalent to multibacillary patients (1 of these 8 individuals developed a lepromatous form of leprosy); 631 (87%) were seronegative and 3 developed a paucibacillary form of the disease (2 BT, 1 I) without any antibody increase. Among those four contacts who developed leprosy, three were related to a multibacillary index case. These data suggest that this test may be useful for the prediction of multibacillary leprosy. A long-term surveillance of this high-risk population will be able to evaluate the diagnostic and prognostic value of the serological assay.     

Identification and quantification of Hb C with an enzyme-linked immunosorbent assay

A highly specific enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification and quantification of hemoglobin C in hemolysates. The procedure involves coating the surface of microtiter wells with Hb C and then addition of monospecific rabbit antibodies that recognize the unique beta 6 GLU----LYS substitution in Hb C. Next, an antibody to rabbit gamma-globulin conjugated with alkaline phosphatase is added, followed by substrate; a yellow color is formed due to the enzymatic hydrolysis of the substrate, which can be measured spectrophotometrically. For quantification purposes, a hemolysate containing Hb C is introduced just prior to the addition of the Hb C antibody. This results in blocking the attachment of the anti-Hb C to the Hb C coated to the plastic surface. Upon addition of anti-rabbit gamma-globulin conjugate and substrate, there is a consequent reduction or elimination of color formation. Since the degree of diminution of color formation is dose-dependent, standard curves can be developed for quantification of Hb C in unknowns. Of the total hemoglobin, the amounts of Hb C in heterozygotes averaged 27.3 +/- 5.7% by ELISA and 25.1 +/- 3.9% by radioimmunoassay (RIA). In SC individuals the corresponding values were 30.2 +/- 10.1% by ELISA and 24.7 +/- 10.9% by RIA. In homozygotes, Hb C values averaged 83.2 +/- 4.2% by ELISA and 85.0 +/- 6.6% by RIA. Subjects with Hb C beta(+)-thalassemia had 66.5 +/- 3.7% Hb C as measured by ELISA and 63.5 +/- 9.1% as determined by RIA. The ELISA procedure offers distinct advantages for Hb C identification and quantification over other techniques in parameters such as specificity, sensitivity, and rapidity.