Evaluation of quantitative anti-F1 IgG and anti-V IgG ELISAs for use as an in vitro-based potency assay of plague vaccine in mice.

Quantitative anti-F1 and anti-V IgG enzyme-linked immunosorbent assays (ELISAs) were developed to measure the serological response of female Swiss Webster mice after vaccination with the recombinant fusion protein, rF1-V, which is being developed as a plague vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, and stability. Experimental results suggested that a potency assay based upon the serological response of female Swiss Webster mice, as measured by quantitative anti-F1 IgG and anti-V IgG ELISAs, might be used to evaluate the rF1-V fusion protein vaccine.     

Development of ELISAs for quantification of HMFG1-specific human anti-mouse IgG and IgM antibodies

The aim of this study was to develop and validate ELISAs for quantification of HAMA-IgM and HAMA-IgG in serum of patients with ovarian cancer who enrolled in a large international randomized phase III trial of intraperitoneal Yttrium-90-labeled HMFG1 murine monoclonal antibody therapy. The capture antibody of these 2 assays was the murine antibody HMFG1, while mouse anti-human IgM-HRP or mouse anti-human IgG(Fc)-HRP served as tracer antibodies. A pool of HAMA-positive serum samples was used to prepare a series of assay standards and another pool served as reference preparation. The analytical sensitivity of the HAMA-IgM assay was 2.5 arbitrary units per mL (AU/mL) and 4.7 AU/mL for the HAMA-IgG ELISA. Diluted serum samples showed good parallelism with the HAMA-IgM and HAMA-IgG standard dose-response curves. Within-assay coefficient of variation was 7.5% for HAMA-IgM and 6.5% for HAMA-IgG. Between-assay variation was 14.2% for HAMA-IgM and 15.3% for HAMA-IgG. The developed HAMA-IgM and HAMA-IgG ELISAs show satisfactory reliability criteria (sensitivity, parallelism and precision) and are suitable for monitoring of HAMA-IgM and HAMA-IgG responses in ovarian cancer patients. These ELISAs will be used to monitor the development of HAMAs in patients who received radioimmunotherapy with murine HMFG1.     

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.     

Development and validation of sandwich ELISA microarrays with minimal assay interference

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA's ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of a multiplexed 24-assay system. We find that nonspecific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a "purified antigen". We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals than within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.     

Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas)

Measurement of vitellogenin (VTG) concentrations in the fathead minnow (Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp (Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17alpha-ethynylestradiol (EE(2); 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17beta-estradiol (E(2); 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE(2)-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.     

A comparison of three salmonella antigen-capture ELISAs and culture for veterinary diagnostic specimens

Three sandwich-ELISAs, two of which are commercially available (Tecra and Locate), and one developed at the Veterinary Sciences Division, Stormont and a 3-step culture protocol, were compared for the detection of salmonella in 1000 animal specimens. Eight hundred and fifty of these were new submissions and the remainder were frozen portions from specimens previously shown to contain salmonellas by culture. The incidence of ELISA false-negative and false-positive results was highest for the Stormont and Locate kits respectively although the differences in sensitivity and specificity between the three ELISAs was not statistically significant. On 16 occasions all ELISA methods indicated the presence of salmonellas when none were isolated by initial culture, eight of these specimens contained salmonellas when reinvestigated by culture.     

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity.

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.     

Serological reactivity of baculovirus-expressed Ebola virus VP35 and nucleoproteins

Ebola virus (EBOV) is a member of the family Filoviridae and is classified as a biosafety level 4 virus. This classification makes the preparation of antigen and performance of diagnostic assays time-consuming and complicated. The objective of this study was to evaluate the value of EBOV immunoassays based on recombinant nucleoprotein (r-NP) and recombinant VP35 (r-VP35) using large serum panels of African origin and from primates. Furthermore, we investigated whether the results obtained with EBOV r-VP35 enzyme-linked immunosorbent assay (ELISA) could improve on the findings obtained with the EBOV r-NP ELISA. The full-length EBOV NP and VP35 of the EBOV subtype Zaire were expressed as histidine-tagged recombinant proteins in the baculovirus expression system. The antigenic reactivity and specificity of these recombinant proteins were determined by Western blotting and ELISA using EBOV specific monoclonal antibodies. The results obtained with the r-NP and r-VP35 ELISAs were compared with the results obtained in an indirect immunofluorescence assay based on native EBOV subtype Zaire. EBOV specific monoclonal antibodies reacted specifically with the respective proteins in both Western blot and ELISA. Five hundred and twenty six samples from humans and primates were tested with r-NP and r-VP35 ELISAs. Monkey serum samples positive for EBOV subtype Reston and Zaire were both positive in the EBOV r-NP ELISA, whereas only the EBOV Zaire infected monkeys were positive in the r-VP35 ELISA. The sensitivity and specificity values of the EBOV recombinants' ELISAs compared to those of the immunofluorescence assay were 92% and 99% for r-NP and 44% and 100% for r-VP35. r-NP ELISA proved to be a sensitive and specific assay for EBOV diagnosis and for epidemiological studies for both EBOV subtypes Reston and Zaire. The use of r-VP35 in an ELISA format has no additional value for EBOV serodiagnosis.     

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.     

Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.     

Sandwich ELISAs for quantification of Xenopus laevis vitellogenin and albumin and their application to measurement of estradiol-17 beta effects on whole animals and primary-cultured hepatocytes

Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for quantification of vitellogenin (VTG) and albumin (ALB) in Xenopus laevis. Working ranges of the ELISAs were 2-1000 ng/ml for VTG and 1-300 ng/ml for ALB. Recoveries of plasma VTG by ELISA were over 90% in dilutions of more than 200 times. The VTG-inducing activity of estradiol-17beta (E2) was measured in whole animals and primary cultured hepatocytes. Immersion of mature male animals in more than 1 nM E2 induced a detectable amount of plasma VTG. VTG induction in younger animals was less potent than in the mature animals but the youngest animals (1.5-3 g body mass) was applicable to the exposure test, irrespective of sex. In vitro exposure of hepatocytes to more than 0.1 nM E2 dose-dependently induced secretion of VTG into the culture medium, while ALB secretion was not significantly affected by E2 treatment. When the VTG-induction levels were normalized by use of a concentration ratio of VTG to ALB, the values obtained from three independent experiments were mutually comparable irrespective of differences in cell density and hepatocyte preparation. Thus, this ratio is thought to be useful for large-scale in vitro screening of estrogenic activities of chemical substances.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78 ng/ml to 77 pg/ml (or 74-0.1 fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.     

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep

Caseous lymphadenitis (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false-negative results. This study aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.     

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.     

Elevation of angiotensin converting enzyme in bovine endothelial cells quantitated by an ELISA.

Levels of angiotensin converting enzyme (ACE) in cultured bovine pulmonary artery endothelial cells treated with dexamethasone, aldosterone, 3,3',5'-triiodo-L-thyronine, Ca2+ ionophore, 3-isobutyl-1-methylxanthine, dibutyryl cAMP and forskolin were quantitated by an enzyme linked immunosorbent assay (ELISA). The configuration for the ELISA consisted of purified bovine lung ACE adsorbed to a solid phase competing with endothelial cellular ACE for a limited amount of anti-ACE immunoglobulin. ACE-IgG complex on the solid phase was detected by goat anti-rabbit IgG-alkaline phosphatase conjugate with enzymatic activity measured by p-nitrophenylphosphate as substrate. This ELISA detected ACE with a sensitivity of 32 ng/ml cellular ACE. Elevation in cellular ACE catalytic activity as measured by fluorescent assay of detergent extracts from bovine endothelial cells corresponded well with an increase in ACE protein as determined by the ELISA. These results provide direct evidence that increases in catalytic activity of ACE produced in endothelial cells by a variety of agents result from enhancement of the synthesis of ACE protein.     

Molecular ratio between borna disease viral-p40 and -p24 proteins in infected cells determined by quantitative antigen capture ELISA

We developed the antigen capture enzyme-linked immunosorbent assay (ELISA) systems for quantification of Borna disease virus (BDV) major antigens, p40 and p24. Using these ELISAs, we quantified the two proteins in various BDV-infected materials, including the cell lysates and culture supernatants as well as the homogenates of experimental animal brains. The ELISAs were also applied to measure the infectious titer of BDV in persistently infected cell lines. Quantitative analysis with these ELISAs allowed us to measure the molecular ratio between the p40 and p24 in infected samples. Interestingly, the ratio of p24 to p40 in persistently infected cells was much higher than that observed in acutely infected cells although the ratios in the supernatants from both cell lines were quite similar. BDV-inoculated gerbil brain cells showed a relatively high ratio of p24 to p40 as compared with acutely infected cells. These observations suggested that the molecular ratio between the proteins strongly depended on the infectious status of BDV in the host cells. The ELISA system developed here could be a convenient method for the quantification of BDV infection and may also be beneficial for understanding viral replication and infectious status in the host cells.     

Validation of an enzyme-linked immunosorbent assay kit using herpesvirus papio 2 (HVP2) antigen for detection of herpesvirus simiae (B virus) infection in rhesus monkeys

Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is nonpathogenic to humans and is genetically and antigenically more closely related to BV than is human herpes simplex virus 1. This paper describes the results of our in-house laboratory that compared a BV antigen-based enzyme-linked immunosorbent assay (ELISA) by commercial testing laboratory and an HVP2-based ELISA in our laboratory by using 447 sera from 290 rhesus monkeys. The HVP2-based ELISA identified as positive 99.11% of the sera identified as BV-positive by the BV ELISA. The BV antigen-based ELISA identified as positive 98.21% of the sera identified as BV-positive by the HVP2-based ELISA. The HVP2 ELISA also identified two BV-negative and six BV-equivocal sera as positive. Both ELISAs identified the same 85 negative and three equivocal samples as negative and equivocal, respectively. The high degree of correlation (weighted kappa coefficient, 0.94) between the two tests indicates that the HVP2 ELISA is a sensitive and reliable assay for in-house testing of the BV status of rhesus monkeys.     

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater.

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.     

Clinical usefulness of urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori: a collaborative study in nine medical institutions in Japan

Background. A urine-based enzyme-linked immunosorbent assay (ELISA) kit for detection of antibody to Helicobacter pylori has been developed in Japan. Urine samples can be obtained noninvasively and are easier and safer to handle than are serum samples. The aim of this study was to examine the clinical usefulness of this urine-based ELISA kit.
Materials and Methods. A pair of random, single-void urine and serum samples was collected from each of 1,061 subjects, including 238 patients with gastroduodenal disease. The sensitivity and specificity of the urine-based ELISA was compared with those of three commercially available serum-based ELISA kits. For those patients with gastroduodenal disease, the urine- and serum-based ELISA results were also compared with those for other diagnostic methods using endoscopic biopsy specimens, such as culture, histology, and rapid urease tests.
Results. Based on the three serum-based ELISA results, the sensitivity, specificity, and accuracy of the urine-based ELISA were 97.7%, 95.6%, and 96.8%, respectively. On the basis of the biopsy test results, the sensitivity (96.2%), specificity (78.9%), and accuracy (91.0%) of the urine-based ELISA were almost equivalent or superior to all three serum-based ELISAs tested. In addition, 10 of the 12 false-positive cases for urine-based ELISA were confirmed to be true positives for antibodies to H. pylori by Western blot analysis and inhibition ELISA.
Conclusions. The urine-based ELISA (URINELISA H. pylori Antibody) is very accurate and should be useful as an alternative to serum-based ELISAs for screening of H. pylori infection.