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PubMed1995

Application of ELISA for IgM, IgA and antigen detection for rapid diagnosis of respiratory syncytial virus infections: a comparative study.

BACKGROUND: Infection with Respiratory Syncytial Virus (RSV) can be rapidly diagnosed by detection of viral antigen in nasopharyngeal secretions (NPS) or serologically by detecting IgM and IgA antibodies. OBJECTIVES: To evaluate the above methods for reliability and rapidity, and compare them with the complement fixation (CF) test and virus isolation. STUDY DESIGN: Viral antigen was tested in 145 NPS samples by ELISA in parallel with tissue culture isolation. The geometric mean titer (GMT) of complement fixing antibodies in various age groups was determined from 92 individual serum samples by CF test. The diagnostic methods, CF test, ELISA-IgM and ELISA-IgA, were evaluated using 21 pairs of acute and convalescent sera. Appearance of IgM and IgA in serum samples with low or negative CF titers was studied in two age groups: 0-10 months (n = 82), and 11 months-9 years (n = 47). RESULTS: From the 145 NPS samples, 20 samples were positive by both ELISA and virus isolation, 9 were positive only by ELISA and 5 were positive only by virus isolation. The GMT by age group for 92 sera evaluated by CF test were 40 (0-10 months), 195 (11-24 months), 269 (2-4 years), 173 (5-12 years) and 132 (adults). Among the 21 paired sera examined, CF test detected 13 RSV infections by antibody rise or seroconversion while the ELISA-IgM/IgA tests identified all 21 infections, 7 of them in the first sample. The presence of IgM alone or IgM and IgA antibodies was demonstrated in both age groups examined; however, IgA alone was found only in the age group 11 months and older. CONCLUSIONS: ELISA for antigen detection is better than virus isolation because it is faster (6-20 h vs. 3-20 days, respectively) and more sensitive (29/34 vs. 25/34 positives, respectively). ELISA-IgM and ELISA-IgA was more sensitive and reliable than the CF test, particularly for the 0-10 month age group. Thus, when necessary, serological diagnosis of RSV infection can be based on the presence of IgM and/or IgA antibodies in serum samples obtained early after onset of symptoms.

Journal Article↗
PubMed1996

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): report of an EORTC/BIOMED-1 workshop.

The urokinase-type plasminogen activator (uPA) is considered to play a key role in the process of invasion and metastasis. In several independent studies, in a variety of cancer types (e.g. of the breast, colon, stomach, lung, ovary), high antigen levels of uPA in tumour extracts have been associated with rapid disease progression. In these studies, different sets of antibodies and standards (often as commercially available uPA ELISA kits) have been used. The standards provided with the different uPA ELISA kits are different from each other in both composition and source. In addition, the different uPA ELISA kits use antibodies which differ in specificity and affinity for the various forms of uPA including pro-uPA, HMW-uPA, LMW-uPA, the aminoterminal fragment (ATF) and complexes with inhibitors (PAI-1 and PAI-2) and the receptor (uPAR). Further, the composition of tumour tissue extraction buffers differ significantly among the published studies. Thus, it is not surprising that the ranges of cytosolic uPA levels reported differ considerably even when measured within the same tumour type. These discrepancies led the EORTC Receptor and Biomarker Study Group, in conjunction with the BIOMED-1 consortium on 'Clinical Relevance of Proteases in Tumour Invasion and Metastasis', to organise a workshop to study the characteristics associated with six different uPA immunoassays (ELISA) used in clinical studies reported in the literature. Although the absolute uPA antigen values measured with the respective uPA ELISA kits differed, high correlations were obtained for any two of the four uPA ELISA kits finally applied to sets of breast cancer cytosol preparations. The preparations used at present as standards in the various uPA ELISA kits are not representative of actual human breast cancer cytosols. Thus absolute standardisation is only possible by using a common reference sample (breast cancer cytosol) and similarly composed ELISA uPA kits. Then it will be possible to generate comparable data on clinical tissue as well as to check for batch-to-batch variations within particular ELISA kits.

Biomarkers, Tumor↗
PubMed2004

Evaluation of three serum i-ELISAs using monoclonal antibodies and protein G as peroxidase conjugate for the diagnosis of bovine brucellosis.

Three i-ELISAs using LPS, the immunodominant component of Brucella abortus, were developed with three different conjugates: monoclonal antibodies 1C8 (anti-bovine IgG(1)) and 3H3 (mainly specific for bovine IgG(2) but also reacting with IgG(1)) and protein G (reacts with both bovine IgG subclasses). Using a cut-off value of 2.5U/ml, the i-ELISA with 3H3 as conjugate had a specificity (95% CI: 98.32-99.63%) that was significantly higher than the same assay with 1C8 (95% CI: 96.08-98.26%) or PG (95% CI: 95.83-98.09%). In areas where false positive serological reactions (FPSR) were common, the specificity of the i-ELISAs decreased significantly. The specificity of the i-ELISAs increased with the age of the animals tested, irrespective of the conjugate. The specificity of the i-ELISAs and traditional tests was also examined using sera from animals infected per os with bacteria bearing LPS similar to the Brucella LPS. It appeared that Yersinia enterocolitica O:9, Xanthomonas maltophilia and Salmonella urbana infections induced FPSR both in the i-ELISAs and in the traditional tests, but the 3H3 assay was significantly less prone to produce false positive reactions than the 1C8 and PG assays. The i-ELISAs were more sensitive, allowed earlier detection, and were more persistent than the traditional serological tests both in experimentally and naturally Brucella-infected animals. Weekly i-ELISA monitoring of experimentally infected pregnant heifers (previously vaccinated or not) allowed a prediction of abortion. Furthermore, the 1C8 assay showed significantly higher titres irrespective of day post-infection and vaccination status. The accuracy of the assay could be improved by making use of additional information (e.g. animal age or conjugate) and by selecting appropriate cut-off points on the basis of the prevailing epidemiological situation. The i-ELISAs appear an appropriate choice in order to maintain an official brucellosis-free status because of their sensitivity, early detection and long persistence and, for the same reasons, seem especially valuable for the detection of latent carriers (i.e. animals classified negative by classical serological tests) among imported animals.

Agglutination Tests↗
PubMed2006

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk.

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.

Animals↗
PubMed2001

Assessment of the epidemiological status of Echinococcus multilocularis in foxes in France using ELISA coprotests on fox faeces collected in the field.

The aim of this study was to estimate the relevance of Echinococcus multilocularis coproantigen detection in fox faeces collected in the field to identify different levels of endemicity for Echinococcus multilocularis on a large scale (n x 10 km(2)). Six study sites were selected in a high endemicity area and two study sites in a low endemicity area in eastern France on the basis of landscape composition. Sampling was undertaken in the winters of 1996-97, 1997-98 and 1998-99. At each site, (i) necropsy and intestine examination was undertaken on a sample of shot foxes (total number of foxes, 222), and (ii) fox faeces were collected in the field along road verges, and scored for degradation status (total number of faeces, 625). Fox faeces were also sampled in a control area (n=30) in western France in the summer of 1998. Intestines were examined according to the sedimentation method. Echinococcus multilocularis coproantigens were detected by using two ELISA tests: EM-ELISA and EmA9-ELISA. The necropsy prevalence in high and low endemicity areas was 63.3% and 19.4%, respectively, and the distribution of adult worms in the fox population was highly overdispersed (75.5% of the total biomass was harboured by 11.6% of foxes). Using the two ELISA tests, there was no difference in the detection of E. multilocularis coproantigens in field faeces, regardless of the degradation status. The medians of EM- and EmA9-ELISA OD values of field faeces in high endemicity area were significantly higher than in low endemicity area (P<0.001 for both ELISA). The distribution of EM-ELISA OD values in low endemicity area was significantly higher (P=0.002) than in the control area. Moreover, for the two ELISA, the observed ELISA OD value distributions in high endemicity area, low endemicity area and control area seemed representative of the distribution of adult worms in fox populations. These results indicate that E. multilocularis coproantigen detection in field faeces could serve for large-scale surveillance, as an alternative to necropsy.

Animals↗
PubMed1998

Validation of a monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus.

A simple, rapid and sensitive competitive monoclonal antibody-based ELISA for the detection of antibodies directed against swine vesicular disease virus (SVDV) was developed. The ELISA was validated using field sera originating from SVDV-infected and non-infected Dutch pig herds, reference sera obtained from the Community Reference Laboratory for Swine Vesicular Disease at the Institute for Animal Health, Pirbright Laboratory, UK, and sera from animals infected experimentally. When testing 4277 sera originating from non-infected Dutch pig herds and collected as part of the national screening program, this ELISA had only 0.6% false positive results, whereas approximately 2% of false positive results were obtained with a conventional blocking ELISA used until recently. A sensitivity relative to the virus neutralisation test of > 97% was achieved when testing sera collected from Dutch pig farms where an outbreak of SVDV had occurred. All international reference sera scored consistently correct. Sera collected sequentially from pigs experimentally infected with SVDV isolates representing all currently recognized antigenic groups, were scored positive slightly earlier by the ELISA compared to the virus neutralisation test. This monoclonal antibody-based competitive ELISA for SVDV antibodies designated the Ceditest ELISA for SVDV-Ab, is as sensitive but more specific than the ELISA used until recently. Because sera are tested at a single dilution (1:5), incubations are carried out at room temperature and test results are available within 3 h, this ELISA is simple, easy to automate and therefore very suitable for screening large numbers of serum samples.

Animals↗
PubMed2003

Immunodetection of pneumolysin in human urine by ELISA.

An ELISA test has been employed for the detection of pneumolysin (PLY) in urine from 14 pneumococcal pneumonia patients and from 11 healthy adult volunteers. The urines of all the 11 healthy adult volunteers developed signals around the mean of the blanks, whereas all the pneumococcal pneumonia patient urines rendered signals at least five times this mean. Chemiluminescent Western blot analyses of these urines, carried out with the PLY-specific rabbit polyclonal IgG preparation used in ELISA, were negative. The 30-kDa filtrates of three high-signal urines were ELISA negative, suggesting that this ELISA test mainly detected high molecular weight forms in urine rather than free PLY-derived antigenic fragments. The urine sample, which rendered the highest ELISA signal, was then concentrated by filtration through a 10-kDa filter. When this concentrate was subjected to Western blot with the ELISA-capture monoclonal antibody, a major band was developed. Its relative molecular mass was similar to that of recombinant PLY and its peptide mass fingerprinting showed peptides corresponding to amino acid stretches from the four domains of the PLY molecule. When the pool of PLY-negative urines was sham-contaminated with purified recombinant pneumolysin, a conspicuous matrix effect was observed; nevertheless, this ELISA test was still reproducible and highly sensitive, detecting pneumolysin in the order of picograms per milliliter. A comparison was also made between this PLY-ELISA and the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in analysing bacterial isolates. On the basis of the minimum number of pneumococci examined, both tests were shown to have similar potency, but strain-dependent discrepancies were observed. This ELISA could provide an alternative to the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in the diagnosis of pneumococcal pneumonia.

Adult↗
PubMed2000

Validation of ELISAs for the detection of antibodies to Sarcoptes scabiei in pigs.

An Enzyme-linked ImmunoSorbent Assay (ELISA) was developed for the detection of antibodies to Sarcoptes scabiei. This 'Animal Health Service'-ELISA (AHS-ELISA) was compared with a commercial test (Checkit(R) Sarcoptest) using experimental and field sera. The experimental study was a contact infestation experiment. Eighty piglets were randomly divided between the experimental and control group. After introduction of three Sarcoptes scabiei var. suis infested pigs in the experimental group, both groups were monitored by determining scratching indices, taking ear scrapings and blood samples in Weeks 0, 2, 4, 6, 8, 12 and 16. Four pigs in the control group were immunised with either Dermatophagoides pteronyssinus (Dp) antigens (n=2), or Acarus siro (As) antigens (n=2). In the control group all (non-immunised) pigs were negative in all tests. In the experimental group only slightly elevated scratching indices were observed, with a maximum in Week 8. After 2 weeks for the first time an ear scraping was positive (2.5%). In Week 8 the highest number of positive ear scrapings were found (25.0%). Positive results in the Sarcoptest were first obtained in Week 12 (10.5% positive), while eventually 29.0% of the finishing pigs were positive after 16 weeks. The AHS-ELISA first detected a serological response after 6 weeks (5. 0% positives), increasing until after 16 weeks a large proportion (74.2%) of the finishing pigs were seropositive, making the AHS-ELISA the most sensitive test. In the AHS-ELISA one As-immunised pig remained seronegative, but the other hyper-immunised pigs crossreacted. In the Sarcoptest, only Dp-immunised pigs had elevated Optical Densities (OD's) albeit below the cut-off level. Although hyper-immunisation is not a representation of field conditions, it cannot be excluded that the AHS-ELISA is not 100% specific.Field samples were taken from 20 sows in 30 herds, classified as mange-free, suspect, or infested. On a herd level there was high agreement among the ELISAs. Both serological tests were suitable to distinguish mange-free herds from infested herds. In one infested herd the decline of maternal antibody in piglets was studied by sampling 40 piglets from 20 different litters. The lowest average OD using the AHS-ELISA was found at 5 weeks of age, followed by a significant increase at 7 weeks. The average OD with the Sarcoptest was at a minimum level at 3 weeks, but no increase was found later. For screening of herds, interference of maternal antibodies is avoided by sampling at an age of 7 weeks or older.

Animals↗
PubMed2000

ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method.

The sensitivity and specificity of the ELISA and fecal culture tests for paratuberculosis in dairy cattle are examined. ELISA and fecal culture data from seven dairy herds where both fecal cultures and ELISA testing was done concurrently are included. A cohort of 954 cattle including 697 parturient adults, cultured every 6 months from 10 herds followed over 4 years served as the basis to determine fecal culture sensitivity. The fecal culture technique utilized a 2g sample with centrifugation and double incubation. Of the 954 cattle cohort of all ages (calf to adult) that were fecal sampled on the first herd visit, 79 were culture positive. An additional 131 animals were detected as culture positive over the next seven tests at 6-month intervals. The sensitivity of fecal culture to detect infected cattle on the first sampling was 38%. Of the 697 parturient cattle cohort, 67 were positive on the first fecal culture, while an additional 91 adult cattle were culture positive over the next seven tests, resulting in a sensitivity of 42% on the first culture of the total animals identified as culture positive. Animals culled from the herds prior to being detected as infected and animals always fecal culture negative with culture positive tissues at slaughter are not included in the calculations. Both groups of infected cattle will lower the apparent sensitivity of fecal culture. Infected dairy herds tested concurrently with both fecal culture and ELISA usually resulted in more than twofold positive animals by culture compared to ELISA. The classification of infected cattle by the extent of shedding of Mycobacterium paratuberculosis in the feces helps define the relative proportion of cattle in each group and therefore the likelihood of detection by the ELISA test. ELISA has a higher sensitivity in animals with a heavier bacterial load, i.e. high shedders (75%) compared to low shedders (15%). Repeated testing of infected herds identifies a higher proportion of low shedders which are more likely to be ELISA negative. Thus, the sensitivity of the ELISA test decreases with repeated herd testing over time, since heavy shedders will be culled first from the herds.

Animals↗
PubMed2000

A quantitative enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of neurocysticercosis using a purified fraction from Taenia solium cysticerci.

A quantitative enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of neurocysticercosis is described. The ELISA was standardized using a purified Taenia solium cysticerci fraction (PCF) obtained by ion exchange chromatography. The ELISA using PCF (PCF-ELISA) and a qualitative ELISA using a whole extract from T. solium cysticerci (WEC-ELISA) were used to screen for cysticercus-specific IgG antibodies in cerebrospinal fluid (CSF) samples from 57 patients with neurocysticercosis and 50 patients with other neurologic disorders. The sensitivity of both assays was 95%, whereas the specificities of PCF-ELISA and WEC-ELISA were 100% and 92%, respectively. The excellent sensitivity and specificity of the PCF-ELISA make this assay a potentially useful tool in screening for antibodies against T. solium cysticerci.

Animals↗
PubMed1997

Comparative evaluation of three ELISA techniques and an indirect immunofluorescence assay for the serological diagnosis of Epstein-Barr virus infection.

BACKGROUND: The reference method for detecting specific Epstein-Barr virus (EBV) antibodies is indirect immunofluorescence (IF) with EBV-infected cells. The availability of protein purified from infected cells and more recently of recombinant polypeptides designed to contain immunodominant epitopes, has enabled the development of commercial enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of EBV infection. OBJECTIVE: Evaluation of ELISA-based EBV serodiagnosis in comparison with indirect immunofluorescence. STUDY DESIGN: We have first compared three commercial ELISA test systems with our in house indirect immunofluorescence assay for classifying correctly a set of serum samples into clinical categories (acute infection, past infection, interfering non-EBV infection, persistent infection). Additionally a prospective analysis with the best performing ELISA test (Enzygnost) was then carried out by running the ELISA test in parallel with the indirect immunofluorescence assay on 324 consecutive clinical samples sent to our laboratory for EBV serodiagnosis. RESULTS: For the serodiagnosis of past EBV infection and acute EBV infection all three commercial ELISAs performed well in comparison with indirect immunofluorescence. When testing samples positive for cytomegalovirus (CMV), Toxoplasma or herpes simplex IgM, interference in the IgM tests was observed with the three ELISAs. In some instances we could demonstrate that the positive IgM results were due to EBV reactivation. The observed discrepancies between ELISA and IF for the serodiagnosis of chronic EBV infection or EBV reactivation, point to the difficulty for the serodiagnosis of persistent EBV infection on single serum samples. According to our prospective study the EBV IgG determination was accurate. A positive IgM result was not always indicative of an acute infection. Positive IgM results due to EBV reactivation were observed. A positive EBV nuclear antigen (EBNA) IgG result in those samples precluded acute infection. CONCLUSIONS: 90-95% of samples could be classified correctly into clinical categories by a two parameter ELISA system detecting IgG and IgM against a standardized mixture of EBV antigens, allowing standardization and automation of EBV-specific serology. The absence of EBNA IgG was useful as a second line confirmatory assay for acute EBV infection.

Antibodies, Viral↗
PubMed1999

A labelled avidin-biotin ELISA to detect antibodies to caprine arthritis-encephalitis virus in goats' sera.

A labelled avidin-biotin ELISA (lab-ELISA) was developed and compared with indirect ELISA (i-ELISA) and agar-gel immunodiffusion assay (AGID) for its efficacy in detecting antibodies against caprine arthritis-encephalitis virus (CAEV) in goat sera. The enzyme immunoassays were standardized using 113 sera from CAEV-negative goat flocks. The tests were compared using the results from 339 serum samples. The lab-ELISA showed the greatest number of positive results (94/339) as compared with AGID (51) and i-ELISA (64). The comparison of the other two tests with the lab-ELISA showed an agreement of 87.3% with AGID and 90.6% with i-ELISA. The lab-ELISA may be useful for screening large populations for CAEV antibodies, in epidemiological surveys and in the control of caprine arthritis-encephalitis.

Animals↗
PubMed2003

Application of ELISA in determining the fate of tetracyclines in land-applied livestock wastes.

The potential use of a class-specific enzyme-linked immunosorbent assay (ELISA) in studying the occurrence and fate of tetracyclines in the environment was evaluated. Several manure samples collected from hog lagoons and cattle feedlots were screened for the presence of tetracycline residues using ELISA. The levels varied from less than the detection limit (0.5 parts per billion) to 200 parts per million. The degradation of tetracyclines in soil-applied manure was followed using ELISA to measure the decline in tetracycline concentrations. Low levels of tetracyclines remained detectable in soil for up to 28 days. The ELISA procedure also proved useful in determining the leaching potential of tetracyclines in undisturbed soil columns and in the analysis of total tetracyclines in manure, soil, and water. Based on the cross-reactivity of the antibodies employed, this ELISA method can be an important screening tool for the presence of other tetracycline compounds, such as chlortetracycline and oxytetracycline. The ELISA method also detects the epimers of tetracyclines and the corresponding dehydration by-products, anhydrotetracyclines. Analysis of selected manure extracts by liquid chromatography with mass spectrometry (LC-MS) showed lower concentrations of total tetracyclines compared to the values obtained by ELISA, indicating the presence of other structurally related compounds or transformation products of tetracyclines being detected by ELISA in the samples. Because analysis of manure and soil samples by LC-MS requires extensive clean-up procedures, ELISA provides an alternative method for conducting environmental fate and transport studies of antibiotics.

Animals↗
PubMed2002

Der p 2 isoallergens have different allergenicity, and quantification with 2-site ELISA using monoclonal antibodies is influenced by the isoallergens.

BACKGROUND: Der p 2 isoallergens have been reported and the possibility of different allergenicity has also been suggested. In addition, the quantification with 2-site ELISA may be affected by the isoallergens. OBJECTIVES: Two different recombinant Der p 2 (rDer p 2) isoallergens were compared in terms of human IgE responses and the reliability of quantification of them with two-site ELISA kits which use monoclonal antibodies (mAbs) as capture and detection of Der p 2. METHODS: Seven different Der p 2 cDNA from the cultured Dermatophagoides pteronyssinus (DP) were cloned and polymorphism in nine amino acid residues was found. Two different recombinant isoallergens (rDer p 2A and rDer p 2B) were expressed and compared to their human IgE immune responses by ELISA and the ELISA inhibition test with 23 sera of DP-allergic patients. The reliability of quantification of two different available 2-site ELISA kits, which used mAbs for capture and detection of Der p 2, was evaluated. RESULTS: The ELISA optical density of rDer p 2B-specific IgE (sIgE) was higher than that of rDer p 2A (P < 0.001). The ELISA inhibition curve of rDer p 2B sIgE in pool I sera (n = 5; high sIgE both to rDer p 2A and rDer p 2B) did not show any differences in the 50% inhibition concentration and maximum inhibitory percentage of rDer p 2A and rDer p 2B sIgE. However, with pool II sera (n = 5; markedly higher sIgE to rDer p 2B than rDer p 2A), the 50% inhibitory concentrations (10 microg/mL vs. 40 ng/mL) and maximum inhibitory percentage (61% vs. 99%) of rDer p 2B sIgE with the two recombinant isoallergens were quite different. rDer p 2B could be quantified with two different 2-site ELISA kits, but rDer p 2A was detected by only one kit. CONCLUSION: We conclude that isoallergens of Der p 2 may have different IgE immune responses. Quantification of Der p 2 with 2-site ELISA kits that adopted mAbs, might be affected by the prevalent form of the isoallergens in reservoir dust.

Adult↗
PubMed1992

Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.

An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.

Acute Disease↗
PubMed2002

Choice of an ELISA assay for screening postmortem blood for amphetamine and/or methamphetamine.

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for the screening of postmortem blood for amphetamine and methamphetamine and to choose the more appropriate assay for screening. Forty-seven postmortem whole blood specimens were obtained from drug-involved deaths, which had been screened and confirmed positive for methamphetamine and/or amphetamine. Eighty-five negative specimens were obtained from non-amphetamines-involved deaths, 17 of which involved decomposition. Specimens were tested using the Neogen Amphetamine Ultra and Neogen Methamphetamine/MDMA microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays, and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of amphetamines concentrations encountered in medical examiner specimens. True positives, true negatives, false positives, and false negatives were determined relative to gas chromatography-mass spectrometry (GC-MS) and graphed for the ELISA. From these graphs and the receiver operating curves (ROC), the optimal cut-off for the Neogen Methamphetamine/MDMA ELISA was 50 ng/mL methamphetamine equivalents and the optimum cut-off for the Neogen Amphetamine Ultra ELISA was 100 ng/mL amphetamine equivalents. The Neogen Methamphetamine ELISA had a sensitivity of 93.6% +/- 3.5% and a specificity of 77.6% +/- 4.5% versus GC-MS at the cut-off of 50-ng/mL methamphetamine equivalents. The Neogen Amphetamine Ultra ELISA had a sensitivity of 95.7% +/- 3.0% and a specificity of 72.9% +/- 5.2% versus GC-MS at the 100-ng/mL amphetamine equivalents cut-off. The areas under the ROCs were equivalent for the two ELISA assays.

Amphetamine↗
PubMed2005

Heparin-based ELISA reduces background reactivity in virus-like particle-based papillomavirus serology.

The interaction between human papillomavirus (HPV) particles and cell surface heparan sulfate requires intact conformation of the HPV particles. Type-specific HPV serology is currently based on virus-like particles (VLPs) with intact conformation. Presence of incorrectly folded VLPs in VLP preparations is recognized as an important cause of cross-reactivity in HPV serology. Heparin-coated microtitre plates were evaluated for capturing conformationally correct VLPs and improving the type specificity of HPV serology. Hybrid VLPs between HPV16 and HPV11, which had been found to have significant reactivity with children's sera and a batch of HPV18 VLPs that had failed the quality control because of significant reactivity with sera from virginal women, were tested in parallel with heparin ELISA, ordinary ELISA and type-specific mAb capture ELISA. Control sera from children that had detectable reactivity with HPV16/11 hybrid VLPs in ordinary ELISA did not react in heparin-based ELISA, but some hybrid VLPs also had background reactivity in capture ELISAs. Control sera from virginal women that had some reactivity with a poor quality HPV18 VLP preparation in ordinary ELISA had no reactivity in heparin or capture ELISA, suggesting that certain VLP preparations expose cross-reactive epitopes that are not exposed on VLPs with heparin-binding ability. As the sensitivity was similar or only marginally affected by the use of heparin plates, use of heparin-coated plates may improve the type specificity of VLP-based ELISAs and reduce interassay variability attributable to variable quality of different VLP batches.

Adolescent↗
PubMed1995

Comparison of two second-generation anti-hepatitis C virus ELISA on 21431 US blood donor samples.

We have compared two different second-generation (2.0) enzyme-linked immunosorbent assays (ELISA) for the presence of antibodies to hepatitis C virus (anti-HCV) in blood from volunteer, unpaid donors. At two separate blood centres, a total of 21,431 donor samples were tested with Abbott Anti-HCV 2.0 ELISA and Ortho Anti-HCV 2.0 ELISA. Samples found to be repeatedly reactive were tested by supplemental/investigational assays. MATRIX HCV (Abbott) and anti-HCV RIBA II (Ortho/Chiron), to 'confirm' the presence of anti-HCV. Discordant ELISA samples were additionally tested by the polymerase chain reaction (PCR) for the presence of HCV RNA. The Abbott anti-HCV assay had a repeatedly reactive rate of 0.59% (127/21,431) and the Ortho anti-HCV assay 0.51% (110/21,431). Overall agreement between assays was 99.76%, 72/127 (56.7%) of Abbott repeatedly reactive samples confirmed on MATRIX and 61/127 (48.0%) on RIBAII; 70/110 (63.6%) of Ortho repeatedly reactivate samples confirmed on MATRIX and 61/110 (55.5%) on RIBA II. Discordant ELISA samples tested by PCR yielded negative results. Hence the two ELISA had equal sensitivity, as defined by detection of true positive samples; the slightly lower specificity of the Abbott Anti-HCV 2.0 ELISA may be owing to culling of donors with a false positive test by Ortho's Anti-HCV 1.0 and 2.0 ELISA tests (the routine tests in place at each blood centre). A sample found to be repeatedly reactive by two different ELISA tests for anti-HCV is likely to be a true positive and may not require further 'confirmatory' testing.

Blood Donors↗