Immunoglobulin A-specific capture enzyme-linked immunosorbent assay for diagnosis of dengue fever

Dengue fever (DF) is usually diagnosed by testing for dengue virus immunoglobulin M (IgM) by a capture enzyme-linked immunosorbent assay (ELISA) (MAC-ELISA). However, IgM can last for months, and its presence might reflect a previous infection. We have tested the use of anti-dengue virus IgA capture ELISA (AAC-ELISA) for the diagnosis of DF by comparing the results of MAC-ELISAs and AAC-ELISAs for 178 serum samples taken from patients with confirmed cases of DF. IgM appears more rapidly (mean delay of positivity, 3.8 days after the onset of DF) than IgA (4.6 days) but lasts longer; the peak IgA titer is obtained on day 8. The specificity and the positive predictive value of AAC-ELISA are 100%; its sensitivity and negative predictive value (NPV) are also 100% between days 6 and 25 after the onset of DF, but they decrease drastically when data for tests conducted with specimens from the first days of infection are included, because the IgA titers, like the IgM titers, have not yet risen. AAC-ELISA is a simple method that can be performed together with MAC-ELISA and that can help in interpreting DF serology.     

Evaluation of an enzyme linked immunosorbent assay (ELISA) for determination of insulin in dogs

Between 1970 and 1983, 345 patients with ovarian cancer clinical stage I, II, and III were irradiated postoperatively. Five-year NED survival was achieved in 41.7% of patients. The most important prognostic factors were histological grade and clinical stage of cancer. Postoperative external beam radiotherapy appeared to be highly efficient for the patients with microscopic residual disease, giving 70% 5-year survival, and moderately efficient for patients with small, i.e. < or = 3 cm in diameter residual disease, giving 40% 5-year survival. The optimal technique of irradiation appeared to be the irradiation given to the entire abdominal cavity with additional irradiation coned down to the pelvis. External beam radiotherapy was ineffective in patients with gross residual disease, i.e. > 3 cm in diameter, and useless as palliative treatment given to patients with inoperable cancer of the ovary.     

Evaluation of enzyme-linked immunosorbent assay for antinuclear antibodies

Antinuclear antibodies (ANAs) are clinically important indicators of collagen diseases. As corresponding antigens for ANAs vary considerably, patients with collagen diseases usually demonstrate several ANAs coincidentally, making difficult to detect the full spectrum of ANAs in each patient's serum. To design an efficient system for measuring ANAs, an enzyme-linked immunosorbent assay (ELISA) which adsorbs eight kinds of recombinant or purified antigens in each well of a multiwell plate was used and results were compared to those obtained with conventional assays by the fluorescent antinuclear antibodies (FANA), and double immunodiffusion (DID) methods. The positivity rates of 106 sera from patients with collagen diseases and 286 sera from healthy subjects were 92.5% and 5.5%, respectively. Sixty-one of 65 positive sera (93.8%) in the corresponding ANAs positive sera by DID or other conventional assay methods were positive by ELISA. Anti-SSA/Ro antibody could be detected with higher sensitivity by this assay method than with the FANA and DID method, but the sensitivities for anti-Scl-70 antibody and anti-centromere antibody were lower. Application of this ELISA method for measuring ANAs along with the FANA test may be beneficial for diagnosis of collagen diseases.     

Evaluation of the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs

A workshop was convened, at which seven enzyme-linked immunosorbent assays (ELISAs) were compared with virus isolation for the detection of viraemia in serial blood samples collected from six pigs at up to fourteen days after inoculation with classical swine fever virus. All ELISAs were of the double antibody sandwich type, using monoclonal and/or polyclonal antibodies to detect a variety of viral proteins in leukocytes, or in anti-coagulated blood or serum. Compared to virus isolation, specificity of the ELISA was good: only one sample found negative by virus isolation yielded a positive result in a single ELISA. Some false-negative results occurred with samples collected at up to eight days after inoculation, but all tests found samples collected between nine and fourteen days post-inoculation to be positive. The ELISAs require less-specialised facilities and can be performed much more rapidly than virus isolation. They are therefore extremely promising tools for screening large numbers of live pigs.     

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection

The PanBio Leptospira immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is a commercially available screening test for the diagnosis of acute leptospiral infection. The ability of the test to diagnose early or recent Leptospira interrogans infection was assessed by testing sera with known microagglutination test (MAT) titers to serovars pomona, hardjo, copenhageni, and australis. The IgM ELISA detected all 41 cases of early or recent leptospiral infection (sensitivity, 100%), with a positive ELISA result seen in many cases before MAT antibody titers reached 1:50. Thirty-eight of 41 patients showed seroconversion (fourfold or greater increase in titer by MAT, 2 of 41 patients had a single sample with elevated titer, and 1 patient from whom leptospires were isolated from a blood sample failed to show MAT titers, despite a seroconversion (negative to positive result) in the ELISA. Follow-up sera obtained from 8 of 12 patients (67%) for 3 to 48 months after the acute stage of illness showed persisting IgM antibody. However, the range of levels detected in these samples (maximum ELISA ratio, 2.0) was lower than the range seen when infection was recent. Reactivity in the IgM ELISA was observed for only 1 of 59 serum samples from asymptomatic donors (specificity, 98%) and 16 of 233 serum samples from patients with Ross River virus, brucella, Epstein-Barr virus, cytomegalovirus, mycoplasma, Q-fever, toxoplasma, hepatitis A virus, Treponema pallidum, or Borrelia burgdorferi infection (specificity, 93%), with the majority of these patients showing lower levels of IgM in comparison to those in patients with leptospiral infection. We conclude that this ELISA is sufficiently sensitive for use as an initial screen for leptospiral infections, with subsequent confirmation of positive test results by MAT.     

PCR enzyme-linked immunosorbent assay for diagnosis of leishmaniasis in human immunodeficiency virus-infected patients

A PCR enzyme-linked immunosorbent assay (ELISA) involving the use of bone marrow aspirates (BMA) and blood samples (BS) for the diagnosis of visceral leishmaniasis (VL) in human immunodeficiency virus-infected patients was developed with primers selected from the sequence of the small-subunit rRNA gene and compared with direct examination and in vitro cultivation. The PCR was optimized for routine diagnosis: processing of samples with lysis of erythrocytes without isolation of leukocytes, enzymatic prevention of contamination, internal control of the reaction, and ELISA testing in a microtitration plate hybridization. Of 79 samples (33 BMA and 46 BS) from 77 patients without VL, all the results were negative. Fifty-three samples (9 BMA and 44 BS) were obtained from 13 patients with VL: 6 samples drawn during anti-Leishmania treatment were negative whatever the technique used, and 47 samples (9 BMA and 38 BS) were positive with at least one technique. The sensitivities were 51% (24 of 47), 81% (38 of 47), and 98% (46 of 47) for direct examination, culture, and PCR, respectively. Thus, PCR ELISA is reliable for diagnosing VL in human immunodeficiency virus-infected patients, and blood sampling should be sufficient for the follow-up.     

Application of double sandwich enzyme-linked immunosorbent assay for the diagnosis of foot-and-mouth disease in Saudi Arabia

A case of Hemangiopericytoma at the thigh is reported. The Hemangiopericytoma is a rare tumour made with pericyties. This neoplasia is usually benign and it is located in the soft tissues. Tumour is profusely irrigated. The most common locations of the Hemangiopericytoma are the lower limbs and the abdominal cavity. The Hemangiopericytoma is difficult to be recognized by histological criteria. The high number of relapses and metastasis involve to an extent surgical ablation of the tumour and its borders. Present literature is reviewed and the different diagnostic and therapeutic options are discussed.     

Evaluation of three commercial enzyme-linked immunosorbent assays for diagnosis of Chagas' disease

Chagas' disease is a common cause of morbidity in Latin American countries. In Brazil, naturally occurring transmission of its etiologic agent, Trypanosoma cruzi, has been almost completely abolished through effective control programs aimed at the triatomid insect vector. Thus, transfusion of blood from infected donors has become the major route for contracting Chagas' disease due to the socioeconomically motivated migration of residents from areas where the disease is endemic to the larger urban centers. Therefore, the employment of screening tests is mandatory for all blood banks throughout the country. We compared the diagnostic performances of three commercially available screening assays used in routine testing in Brazilian blood banks: the Abbott Chagas antibody enzyme immunoassay (Abbott Laboratórios do Brasil, S?o Paulo), the BIOELISACRUZI kit (Biolab-Mérieux, Rio de Janeiro, Brazil), and the BIOZIMA Chagas kit (Polychaco S.A.I.C., Buenos Aires, Argentina). The evaluation was performed with sera obtained from chagasic patients and healthy residents of four different areas in Brazil where Chagas' disease is either endemic or emergent and where clinical manifestations of the disease and circulating parasite strains vary. The results obtained with each kit were compared to matched in-house enzyme-linked immunosorbent assay and immunofluorescence assay data obtained for each sample. Depending on the area under investigation, the three commercial kits produced specificity values between 93.3 and 100.0%, sensitivity values between 97.7 and 100%, and accuracies ranging from 93.6 to 100.0%.     

Evaluation of enzyme-linked immunosorbent assay for the diagnosis of Helicobacter pylori infection in children from different age groups with and without duodenal ulcer

BACKGROUND: Myocardial ischemia induced by 5-fluorouracil (5-FU) is a relatively rare, but potentially serious, occurrence. Some case reports have indicated that recurrent ischemia may be prevented if 5-FU is resumed after pretreatment with antianginal therapy. METHODS: A 54-year old woman was diagnosed with stage IIA squamous cell carcinoma of the anus. Treatment with concurrent radiation and chemotherapy (mitomycin-C and 5-FU) was initiated with curative intent. RESULTS: The patient had no evidence of underlying coronary artery disease based on history, physical examination or ECG. Approximately 48 h after initiation of 5-FU infusion the patient developed anginal pain associated with ECG changes compatible with ischemia. After resolution of ischemia and ruling out of myocardial infarction, coronary arteriography demonstrated normal coronary arteries. In an attempt to prevent myocardial ischemia, calcium channel blocker and nitrate therapy was started, but anginal pain with ECG change recurred when 5-FU was resumed. This necessitated selection of an alternative chemotherapy regimen. CONCLUSIONS: Even in the presence of normal coronary arteries, antianginal therapy may not preclude the occurrence of potentially serious 5-FU induced myocardial ischemia. For patients who experience 5-FU-induced myocardial ischemia, development of alternative chemotherapy regimens should be considered.     

Serological diagnosis of influenza B virus infection: comparison of an enzyme-linked immunosorbent assay and the hemagglutination inhibition test

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to influenza B virus was compared with that of the hemagglutination inhibition test on acute- and convalescence-phase sera obtained from adults and children infected with influenza B virus. Two whole virus, tissue culture-grown antigen preparations were used in the ELISA, influenza B/West Virginia/81 and influenza B/Hong Kong/72. Four antigens were used in the hemagglutination inhibition test. These included the tissue culture-grown whole virus antigens that were used in the ELISA. In addition the standard egg-grown antigens, influenza B/Singapore/79 and influenza B/Hong Kong/72, were included for comparison. The ELISA antibody titer was significantly correlated to the hemagglutination inhibition antibody titer, and 10 of 10 adults and 17 of 21 children infected with influenza B had fourfold antibody increases as detected by ELISA with either antigenic type of tissue culture-grown whole virus. Increases in geometric mean antibody titers of 16- to 71-fold were detected by ELISA. Increases in geometric mean antibody titers of 3- to 10-fold were detected by hemagglutination inhibition depending on the type of antigen utilized. We found that ELISA with whole virus antigens was a sensitive and specific test for the detection of antibody to influenza B virus.     

Epitope characterization of malondialdehyde-acetaldehyde adducts using an enzyme-linked immunosorbent assay

Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.     

Analysis of the thrombin inhibitor DuP 714 by an enzyme-linked immunosorbent assay

Resident proteins of the exocytic pathway contain at least two types of information in their primary sequence for determining their subcellular location. The first type of information is found at the carboxyl terminus of soluble proteins of the endoplasmic reticulum (ER) and in the cytoplasmic domain of some ER and Golgi membrane proteins. It acts as a retrieval signal, returning proteins that have left the compartment in which they reside. The second type of information has been found in the membrane-spanning domain of several ER and Golgi proteins and, though the mechanism by which it operates is still unclear, it acts as a retention signal, keeping the protein at a particular location within the organelle. The presence of both a retrieval signal and a retention signal in a trans-Golgi network resident protein suggests that more than one mechanism operates to ensure correct localization of resident proteins along the exocytic pathway.     

Development of an enzyme-linked immunosorbent assay for human metallothionein-1 in plasma and urine

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) for human metallothionein-1 is reported. Metallothionein was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human metallothionein-1 (MT-1), and there was no significant cross-reaction with human metallothionein-2. The detection limit (sensitivity) of the assay was 5 ng/ml, and the added MT-1 could be fully recovered from plasma and urine. The normal reference range for MT-1 was 32 +/- 16 ng/ml in plasma and 10 +/- 6 ng MT-1 per micromole of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concentration of MT-1 was greatly increased between 24 and 48 hours after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.     

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis

Oral squamous cell carcinoma develops through a series of precancerous stages manifested at the microscopic level as epithelial dysplasia. Mutation of the p53 tumour suppressor gene is thought to be an important component of oral carcinogenesis. p53 regulates cell proliferation and DNA repair by inhibiting the cell cycle at G1/S; loss of p53 function may therefore lead to aberrant cell kinetics. To date, no studies have examined the relationship between p53 protein and alterations in cell kinetics in oral epithelial dysplasia from a single anatomical site. Serial sections were studied from 40 routinely processed biopsy specimens of epithelial dysplasia from the floor of the mouth. The expression of p53 protein was determined by immunohistochemistry and cell proliferation was studied by immunostaining for the cell cycle-dependent protein Ki-67. The number of positive cells per millimetre of basement membrane was determined using computer image analysis and compared with site-matched normal controls. The mean p53 labelling index (LI) in normal mucosa was low, 3.48 +/- 0.92 [mean +/- 95 per cent confidence interval (CI)], and increased sharply in the transition from mild (42.49 +/- 21.71) to moderate (104.86 +/- 51.39) epithelial dysplasia. The mean p53 LI for severe dysplasia was 119.09 +/- 56.50. Differences were also observed in the distribution of p53-positive cells between grades of dysplasia, with the development of compact p53-positive foci in severe dysplasia. Mean proliferative indices, as determined by Ki-67 expression, were significantly associated with grade of epithelial dysplasia. Furthermore, there was a significant correlation between p53 LI and Ki-67 score (r2 = 0.37, P = 0.01). It is concluded that altered p53 protein expression is probably an early event in oral carcinogenesis in the floor of the mouth and is associated with dysregulation of cell proliferation at this site.     

Evaluation of enzyme-linked immunosorbent assay for the determination of polycyclic aromatic hydrocarbons in house dust and residential soil

Two commercially available enzyme-linked immunosorbent assays (ELISA) for total polycyclic aromatic hydrocarbon (PAH) and carcinogenic PAH (C-PAH) were evaluated. The testing procedures were refined for application to screening PAH and C-PAH in house dust and soil samples for human exposure studies. The overall method precision expressed as percent relative standard deviation (%RSD) of triplicate real world dust and soil samples was within +/- 29% (12-29%) for PAH ELISA and +/- 21% (5.9-21%) for C-PAH ELISA. Spike recoveries from real world dust/soil samples were 114 +/- 30% for phenanthrene from PAH ELISA and 120 +/- 8.2% for benzo[a]pyrene from C-PAH ELISA. The overall method accuracy for PAH and C-PAH assays cannot be assessed for multiple PAH components in dust/soil samples (which represent real-world samples), because of the assays' cross reactivities with other PAH components. Over 100 dust/soil samples from 13 North Carolina homes and 22 Arizona homes were analyzed by PAH and C-PAH assays, as well as by the conventional gas chromatography/mass spectrometry (GC/MS) method. Statistical analysis showed that dust/soil PAH data from ELISA and GC/MS methods are significantly different. In general PAH ELISA responses were higher than PAH GC/MS responses. The regression analysis showed that the linear relationship between ELISA and GC/MS measurements is not strong in the combined data. The relationship became stronger for the data from the same type of dust/soil samples. The screening performance of ELISA was evaluated based on the frequency distribution of ELISA and GC/MS data. The results indicated that the ELISA PAH and C-PAH assays cannot be used as a quantitative analytical tool for determining PAH in real-world dust/soil samples. However, the ELISA is an effective screening tool for ranking PAH concentrations in similar types of real world dust/soil samples.     

Determination of captopril in human blood by an enzyme-linked immunosorbent assay

An immunoassay for the quantitation of the angiotensin-converting enzyme inhibitor, captopril in human plasma is described. Antisera very specific for captopril were produced by immunization with captopril conjugated to bovine serum albumin or porcine thyroglobulin via the drug's thiol group. The antibodies were used to develop an enzyme-linked immunosorbent assay (ELISA) with a detection limit of 0.3 ng mL-1 and intra- and inter-assay coefficients of variation of 7 and 12%, respectively. Apart from stabilizing captopril by the addition of N-ethyl maleimide, the assay was used to detect the drug in human plasma without further extraction or purification. Our immunoassay provides a very sensitive and rapid (four hours) alternative for the study of captopril pharmacokinetics.     

Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C

ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus. Subsequently, in vivo pharmacokinetic evaluation of ALX40-4C necessitated the establishment of a detection system for the measurement of ALX40-4C in subject serum. For this purpose, an indirect-competition enzyme-linked immunosorbent assay with generated rabbit anti-ALX40-4C antiserum was developed. The original assay took 12 h to complete and required many manipulations. Herein, we describe alterations to the system that resulted in the overall reduction in assay time and manipulation. We demonstrate that our alterations do not affect the specificity or sensitivity of the assay compared to that of the original system. ALX40-4C levels in spiked serum samples as well as drug levels from patient samples were used to validate the assay.     

Seroepidemiology of rinderpest in bovids in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique

Approximately 0.2% (n = 4397) of the bovids (cattle and buffalo) in Sri Lanka were sampled, from June 1992 using a multi-stage sampling procedure. Serum antibodies for the rinderpest virus were detected using the competitive enzyme-linked immunosorbent assay. The age, the agroclimatic zone, the management system practiced in the farms, and the vaccination history of the sampled bovids were studied as potential risk factors for being seropositive. The prevalence of rinderpest antibodies in non-vaccinated bovids was 3.5% (n = 4101). The prevalence was higher in the dry zone (9%; where the outbreak emerged in 1987), compared to bovids in the other zones (1%). Seropositive bovids over three years of age were approximately at fourfold higher chances of being seropositive compared to those that were < or = 3 years old. The higher prevalence in older animals is probably due to exposure to the virus during the 1987 epidemic. Bovids from the dry zone (annual rainfall 20 to 35 inches) were at higher odds of being seropositive even after controlling for the possible effects of age, agroclimatic zone, management system and vaccination. The fact that 62% of bovids from the dry zone in this study were reared under extensive management system (free grazing) which allow unrestricted contact between animals, may be the reason for the above finding. A relatively poor response to vaccination observed in vaccinated bovids (seroprevalence = 12%; n = 296) could be attributed to difficulties in maintaining the vaccine at recommended temperatures in the field. This is the first island-wide study on seroprevalence of rinderpest in Sri Lanka.     

An enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of Neospora sp. infection in cattle

A kinetic enzyme-linked immunosorbent assay (ELISA) was developed and optimized for detection of antibodies to Neospora sp. in cattle. Sonicated tachyzoites of Neospora sp. isolated from an aborted bovine fetus were used as antigen. Variability in immunoblot patterns among positive sera, and the fact that all life stages of the parasites are unknown, justified use of a multiple-antigen ELISA to allow for maximum sensitivity. Immunoblot analysis revealed negligible cross-reactions between Toxoplasma gondii antigen and Neospora sp. antisera and between Neospora sp. antigen and antisera from various apicomplexan parasites. The maximum positive-to-negative Vmax (average maximum slope of the optical density over time) ratio was obtained using 200 ng/well of sonicated tachyzoite antigen and a 1:200 serum dilution. Using logistic regression to determine the optimal cutoff point between known infected and noninfected cattle, a sample-to-positive control Vmax ratio of 0.45 was found to maximize the percent correct classification, with an estimated sensitivity of 88.6% and specificity of 96.5%. Use of Neospora caninum antigen following the same protocol demonstrated no difference in ELISA interpretation. Comparison with an existing indirect immunofluorescent antibody (IFA) test showed the ELISA to be the more sensitive and specific test for serodiagnosis of Neospora infection in cattle.