Limitations of the BP26 Protein-Based Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis

Brucellosis is a serious zoonosis that occurs worldwide, and its diagnosis is typically based on the detection of antibodies against Brucella lipopolysaccharide (LPS). However, the specificity of the LPS-based test is compromised by cross-reactivity with Escherichia coli O157:H7 and Yersinia enterocolitica O:9. Also, diagnosis based on the LPS test cannot differentiate between vaccinated and infected individuals. The detection of the 26-kDa cytosoluble protein (BP26) antibody is considered an alternative that circumvents these drawbacks because it is exclusively expressed by infectious Brucella. A BP26-based enzyme-linked immunosorbent assay (ELISA) has been tried for the diagnosis of Brucella-infected animals and humans, but a few results showed that BP26 couldn''t react with all Brucella-positive sera. In order to explore whether different animals could produce antibodies against BP26 after being infected with various Brucella species, we infected sheep, goats, and beef cattle with common virulent reference Brucella species. All sera were collected from the experimental animals and tested using both LPS-based ELISAs and BP26-based ELISAs. The results showed that all Brucella-infected individuals could produce high levels of antibodies against LPS, but only B. melitensis 16M- and B. melitensis M28-infected sheep and B. melitensis 16M- and B. abortus 2308-infected goats could produce antibodies against BP26. Therefore, we concluded that the BP26-based indirect ELISA (i-ELISA) showed both Brucella species and host specificity, which obviously limits its reliability as a substitute for the traditional LPS-based ELISA for the detection of brucellosis.     

Validation of the Indirect MAP1-B Enzyme-Linked Immunosorbent Assay for Diagnosis of Experimental Cowdria ruminantium Infection in Small Ruminants

The major antigenic protein 1 fragment B (MAP1-B) enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Cowdria ruminantium infections was validated to determine cutoff values and evaluate its diagnostic performance with sheep and goat sera. Cowdria-infected populations consisted of 48 sheep and 44 goats, while the noninfected populations consisted of 64 sheep and 107 goats. Cutoff values were determined by two-graph receiver-operating characteristic (TG-ROC) curves. The cutoff value was set at 31 and 26.6% of the positive control reference samples for sheep and goat sera, respectively. The test’s diagnostic performance was evaluated with measurements of the area under the concentration-time curve (AUC) of the ROC curves and by the valid range proportion (VRP). The AUCs were 0.978 for sheep sera and 0.989 for goat sera. The VRP for both sheep and goat sera was approximately 1.0. The intermediate range (IR), which defines results that are neither positive nor negative, was 0 for goat sera and 2.81 for sheep sera. In an ideal test, the AUC and VRP would be 1.0 and the IR would be 0. In this study these parameters were close to those of an ideal test. It is concluded that the MAP1-B ELISA is a useful test for the diagnosis of C. ruminantium infection in small ruminants.     

Development of an Indirect Tams1 Enzyme-Linked Immunosorbent Assay for Diagnosis of Theileria annulata Infection in Cattle

An enzyme-linked immunosorbent assay (ELISA) was developed based on a recombinant major Theileria annulata merozoite surface antigen, Tams1. Four different recombinant proteins derived from two different Tams1 alleles, both in two different truncated forms, were tested for their performance in the ELISA. Furthermore, antigen concentration, various buffers, washing protocol, and the choice of anti-total-immunoglobulin G (IgG), anti-IgG1, or anti-IgG2 as second antibody were evaluated. The performance of the resulting ELISA was analyzed by measuring the coefficient of variation (CV). A total of 22 sera were analyzed over the measurement range, resulting in a CV of ca. 10%, whereas 30% variation is the maximum acceptable. The cutoff value was determined by the two-graph receiver operating characteristic (TG-ROC), using the indirect fluorescent antibody test (IFAT) as a reference. It was shown that up to 3 months postinfection (p.i.) IFAT is more sensitive and specific, whereas beyond 3 months p.i. ELISA performed as well as IFAT. The cutoff was determined at maximal sensitivity, based on the TG-ROC after 3 months p.i. Nine calves experimentally infected with four different T. annulata stocks remained positive in the ELISA for at least 1 year p.i. Finally, limited cross-reaction was found only with T. parva antisera, but not with any other Theileria or Babesia species. Since the T. parva endemic area hardly overlaps with T. annulata, the Tams1 ELISA has the potential to become a useful tool in the epidemiology of tropical theileriosis.     

Development of an Indirect Enzyme-Linked Immunosorbent Assay for the Organophosphorus Pesticide Paraoxon-Methyl

In the present study, the synthesis of hapten for the organophosphorus (OP) pesticide paraoxon-methyl was developed, with a spacer arm (aminocarboxylic acid) attached at the aromatic ring. It was conjugated to bovine serum albumin (BSA) for use as an immunogen and to ovalbumin (OVA) for coating antigen for ELISA testing. Rabbits were immunized with the immunogen and two polyclonal antisera were produced and screened against the coating antigen using competitive indirect enzyme-linked immunosorbent assay (ELISA). For application to textile samples, the influence of several factors such as organic solvent, ionic strength, and pH on the ELISA results were studied. Under optimized conditions, the quantitative working range was 0.012–1.158 μg/mL with a limit of detection (LOD) of 0.005 μg/mL and the IC₅₀ was 0.115 μg/mL.There was negligible cross reactivity (CR) with other OP pesticides. The recoveries obtained by standard paraoxon-methyl addition to the different textile samples such as cotton, wool and muslin delaine were all from 86.0% to 108.0%. Therefore, the optimized ELISA may become a new convenient and economical analytical tool for monitoring paraoxon-methyl residues in textile samples.     

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.     

Serodiagnosis of Borreliosis: Indirect Immunofluorescence Assay,Enzyme-Linked Immunosorbent Assay and Immunoblotting

Lyme disease is an infectious, multi-system, tick-borne disease caused by genospecies of Borrelia burgdorferi bacteria sensu lato, characterized by remarkable heterogeneity. In this situation choosing an optimal antigen array for diagnostic tests seems problematic. The serological tests for borrelia routinely done in laboratories often produce ambiguous results, which makes a proper diagnosis rather complicated and thus delays the implementation of an appropriate treatment regimen. Thirty-seven outpatients and eight inpatients with suspected borreliosis diagnosis hospitalized at the Clinics of the Pomeranian Medical University (Szczecin, Poland), participated in the study. In order to detect the antibodies against Borrelia sensu lato three kinds of serological tests were used: indirect immunofluorescence assay (IIFA), enzyme-linked immunosorbent assay (ELISA), and immunoblot. The IIFA and immunoblot tests conducted on 45 patients (100%) produced positive results for both the IgM and IgG antibody types. In the case of ELISA, positive or borderline results were observed in only 24 patients (53.3%). The immunoblot test for IgM most frequently detected antibodies against the outer surface protein C (OspC) antigen (p25), and, in the case of IgG, against the recombinant variable surface antigen (VlsE). The IIFA screening test used for diagnosing Lyme borreliosis produced the highest percentage of positive results, which were then confirmed by immunoblot, but not by ELISA. Therefore using only ELISA as a screening test or for diagnosing Lyme borreliosis seems debatable.     

Development of an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Ringworm Infection in Cattle

The aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms of Trichophyton rubrum dipeptidyl peptidase V (TruDppV) and T. rubrum leucin aminopeptidase 2 (TruLap2), which are 98% identical to Trichophyton verrucosum orthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P < 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.     

Evaluation of an IgG Enzyme-Linked Immunosorbent Assay as a Serological Assay for Detection of Mycoplasma bovis Infection in Feedlot Cattle

Mycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots.     

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 interprating DF serology.     

rK39 Enzyme-Linked Immunosorbent Assay for Diagnosis of Leishmania donovani Infection

The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months after treatment. For patients with post-kala-azar dermal leishmaniasis and individuals with subclinical infection, rK39 ELISA performed as well as DAT but could detect infection 6 months earlier in ~40% of patients. Conversion in DAT and rK39 ELISA also occurred in leishmanin skin test (LST)-positive individuals, suggesting active parasite replication (rK39 is an amastigote antigen) in these presumably immune individuals. In contrast to DAT, rK39 ELISA also detected infection in randomly selected LST-positive individuals (in four of six) and endemicity (LST-negative) controls (in one of five). rK39 ELISA appears more sensitive than DAT and may prove an important tool in epidemiological studies.     

Rapid Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Peste des Petits Ruminants Virus

Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be <50 on the basis of the mean PI plus 3 standard deviations for sera from PPRV-negative ruminants. The relative specificity and sensitivity of the rapid c-ELISA were 98.5% (722 of 733 serum samples) and 93.4% (234 of 249 serum samples), respectively. The rapid c-ELISA sensitively detected PPRV antibodies in hyperimmune sera (virus neutralization test [VNT] titer, >512), even at dilutions ≥512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was ≤128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field.     

Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection

To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.     

Evaluation of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for the Diagnosis of West Nile Virus Infections in Humans

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.The most medically important flaviviruses include dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV), and Saint Louis encephalitis virus (SLEV) (16, 31, 38). Flaviviruses are positive-strand RNA viruses with genomes of approximately 11 kb that encode three structural and seven nonstructural (NS) proteins in the gene order C (capsid), M (membrane), E (envelope), NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. WNV is a member of the JEV serocomplex within the genus Flavivirus, family Flaviviridae. The virus has been isolated in Africa, Australia, Eastern Europe, the Middle East, North America, and South America (7, 20, 24). WNV was first detected in the United States in July 1999 and spread rapidly throughout the country, causing large numbers of infections in humans, horses, and birds (19, 31).Prior to 1999, flavivirus infections in humans in the United States were infrequent, and most were attributed to sporadic cases of SLEV and travel-associated cases of DENV (41). In Thailand, all four DENV serotypes and JEV circulate (39), resulting in very high flavivirus transmission and seroprevalence rates. In the Yucatán Peninsula of Mexico, all four DENV serotypes circulate and seroprevalence rates are very high (8). Serological diagnosis of WNV infections is complicated by the high rates of both primary DENV infections and secondary DENV infections in inhabitants of Thailand and Yucatan, Mexico, with seroprevalence rates of >85% in Thailand (1) and 72% in the Yucatán in 1985 (12, 28). WNV introduction into the Yucatán in 2002 was revealed by detection of antibodies in horses (29) and then later in migratory and resident birds (10) and in zoo animals (11). However, no WNV infections of humans have been diagnosed in the Yucatán.The immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) is the preferred test used for diagnosis of WNV in humans in the United States (32). The test is used to detect antibodies to WNV in serum and/or cerebrospinal fluid. The plaque reduction neutralization test (PRNT) is the gold standard for serodiagnosis of flavivirus infections and for identifying the infecting agent (2). However, both of these tests can be confounded if patients have had previous flavivirus infections. Indeed, diagnosis of flavivirus infections in humans is very difficult in geographic areas where multiple flaviviruses are circulating and cause sequential infections. Because of “original antigenic sin” the highest antibody titer may be due to a previous flavivirus infection rather than to the etiologic agent (18, 26). Serological diagnosis of WNV, SLEV, and YFV infections is extremely difficult in patients from areas where DENV is hyperendemic.Previously, we exploited an epitope-blocking ELISA (b-ELISA) to detect antibodies to WNV in diverse species of birds and domestic mammals (3, 4). The WNV b-ELISA measures the ability of antibodies present in sera to block the binding of a monoclonal antibody (MAb) to a WNV-specific epitope on the NS1 protein (17). The WNV b-ELISA had not been previously evaluated for use in humans. In this study, a WNV-specific and a flavivirus broadly reactive b-ELISAs were evaluated for their abilities to detect antibodies against WNV in human serum specimens from countries with differing levels of flavivirus endemicity: the United States, Thailand, and Mexico. The objectives of this study were (i) to determine the ability of the b-ELISA to detect antibodies to WNV in human serum samples and (ii) to determine the effects of previous flavivirus infections of patients (e.g., DENV and JEV) on the diagnostic efficacy of the WNV b-ELISA.     

Development and Evaluation of an In-House Enzyme-Linked Immunosorbent Assay for Early Diagnosis and Monitoring of Human Pythiosis

Human pythiosis is an emerging, fatal, infectious disease caused by Pythium insidiosum and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against P. insidiosum was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to P. insidiosum. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis.     

Indirect Sandwich Enzyme-Linked Immunosorbent Assay for Rapid Detection of Haemophilus influenzae Type b Infection

We report the development and testing of an enzyme-linked immunosorbent assay with excellent sensitivity for the detection of Haemophilus influenzae type b (HI(b)) antigen in clinical specimens from patients with HI(b) meningitis. The assay, an indirect sandwich technique, uses polystyrene balls as a solid phase and an alkaline phosphatase-labeled goat anti-rabbit globulin conjugate. Specimens are incubated with polystyrene balls armed with burro anti-HI(b) antiserum, and recognition antibody is visualized by addition of alkaline phosphatase-labeled anti-globulin, together with the enzyme substrate p-nitrophenyl phosphate. Concentrations of antigen are determined from standard curves prepared by using purified HI(b) capsular antigen polyribophosphate. The assay reproducibly detects polyribophosphate at concentrations between 1 and 5 ng/ml. Cross-reactions have not as yet been encountered in simulated and authentic clinical specimens containing other species including Escherichia coli, Klebsiella pneumoniae, group B Streptococcus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria meningitidis, and Listeria monocytogenes. In preliminary tests with 11 spinal fluid specimens, 2 serum specimens, and 5 urine specimens from patients with culture-proved HI(b) meningitis, antigen was detected in all specimens in concentrations ranging from 1 to 7,000 ng/ml. Antigen was not detected in any of 62 clinical specimens which were culture negative for HI(b), including 11 spinal fluid specimens from patients with bacterial meningitis caused by microorganisms other than HI(b). The enzyme-linked immunosorbent assay technique described here is considerably simpler than radioimmunoassay and, based on concurrent tests with 14 positive clinical specimens, may be more sensitive than counterimmunoelectrophoresis. It seems, therefore, to hold considerable promise for clinical use in rapid detection of systemic HI(b) infections.     

Immunoglobulin M Enzyme-Linked Immunosorbent Assay Using Recombinant Polypeptides for Diagnosis of Dengue

We demonstrate that a mixture of four recombinant dengue virus E polypeptides corresponding to the N-terminal region of the envelope protein from all serotypes substitutes for standard antigens in two immunoglobulin M enzyme-linked immunosorbent assay formats with 100% concordance, making these polypeptides a useful and accessible reagent for serological diagnosis of dengue in endemic countries.     

An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection and Quantitation of Antisperm Antibodies

ABSTRACT: Several recent comparative investigations using various assays to detect and quantitate levels of antibody to human spermatozoa have produced widely varying results. In an attempt to reduce test variability, an indirect enzyme-linked immunosorbent assay (ELISA) was devised to measure antisperm antibodies. A standardized protocol was adapted employing sperm adsorption to polystyrene microtiter plates, at a density of 10⁵ sperm per well, serum and enzyme-conjugate incubation conditions at 37°C for 60 min, and three ten-minute washes between incubations using phosphate-buffered saline containing Tween-20. Using antihuman sperm antisera generated in rabbits, the ELISA was shown to yield significantly detectable antibody at dilutions of 1/16,384. The ELISA demonstrated approximately 89% reproducibility (ie, 100% minus the coefficient of variation) for an “intraassay” study wherein 300 determinations were performed on the same day on sperm from ten donors. However, when sperm from one donor were used in 30 determinations during ten assays over a six-month period, “interassay” reproducibility was approximately 51%. The ELISA was compared with macroagglutination, microagglutination, and immobilization tests, using rabbit antisperm serum and human sera from vasectomized males. Results of this study indicated that the ELISA was more sensitive, less subjective, and easier to perform than these other commonly used techniques.