A monoclonal antibody capture enzyme-linked immunosorbent assay for detecting antibodies to infectious bursal disease virus.

A monoclonal antibody capture enzyme-linked immunosorbent assay (mAb-ELISA) for antibodies to infectious bursal disease virus (IBDV) in chicken sera was developed and compared with conventional ELISA. When sera from farm chickens were tested by the two ELISAs and serum neutralization (SN), the correlation rate between SN and mAb-ELISA was 100% (49/49), and that between SN and conventional ELISA was 81.6% (40/49). In mAb-ELISA, all of the sera that were antibody-negative by SN had low absorbance values (below 0.05), and the absorbance values correlated closely with the SN titers. In the conventional ELISA, however, the sera antibody-negative by SN had various absorbance values ranging from 0.06 to 0.32. mAb-ELISA had much lower non-specific reactions than the conventional ELISA against sera from IBD-negative chickens.     

Validation of a monoclonal antibody-based capture enzyme-linked immunosorbent assay for detection of Campylobacter fetus

A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35 degrees C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus subspecies.     

A monoclonal antibody-based enzyme-linked immunosorbent assay for quantitation of plasma thrombospondin

An enzyme-linked immunosorbent assay was developed to quantitate the platelet secretory glycoprotein, thrombospondin (TSP), in plasma. Specificity of the assay was provided by using an anti-TSP monoclonal antibody (McAb) to coat 96 well polystyrene assay plates. The effective range of the standard curve for the assay was between 2.5 ng/mL and 500 ng/mL. Interassay and intraassay variations were 6.9% and 7.5%, respectively. To avoid spontaneous release of TSP from platelets after the blood was drawn, and to obtain a valid measurement of plasma TSP concentration, it was essential to place and maintain blood samples on ice immediately after blood was drawn and to separate plasma from cellular constituents within two hours. The mean plasma TSP concentration of 24 healthy adults was 64.3 +/- 18.0 ng/mL (mean +/- SD). The TSP concentration of pooled normal human serum (n = 6) was 15.9 micrograms/mL. Interference occurred when undiluted plasma or serum samples were assayed. This interfering effect could be eliminated completely by a tenfold dilution of plasma or serum samples before the assay. The assay also was applied successfully to quantitate TSP release from platelets after induction of aggregation by different platelet-aggregating agents.     

Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISAs) for the detection of anti-arboviral immunoglobulin G (IgG ELISAs) were developed for a comprehensive array of medically important arboviruses from the Alphavirus, Flavivirus, and Bunyavirus genera. Tests were optimized and standardized so that maximum homology could be maintained among working parameters for the different viral agents, enabling a wide range of viruses to be easily tested for at one time. MAbs were screened for suitability as capture vehicles for antigens from the three genera. The final test configuration utilized group-reactive MAbs eastern equine encephalitis virus 1A4B-6, dengue 2 virus 4G2, and La Crosse encephalitis virus 10G5.4 to capture the specific inactivated viral antigens. Serum IgG was detected by using alkaline phosphatase-conjugated anti-human IgG (Fc portion). A dilution of 1:400 was chosen as the universal screening serum dilution, with endpoint titrations of serum samples testing positive eliminating occasional false-positive results. IgG ELISA results correlated with those of the standard plaque-reduction neutralization assays. As expected, some test cross-reactivity was encountered within the individual genera, and tests were interpreted within the context of these reactions. The tests were standardized for laboratory diagnosis of arboviral infections, with the intent that they be used in tandem with the corresponding IgM antibody-capture ELISAs.     

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoringof infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV wasproduced and purified from Eschevichia coli as well as Sf9 (insect) cells, and used for the coating of enzymelinkedimmunosorbent assay (ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereasN protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive toanti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera toother avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the datafrom the detection of field samples and IBV strains indicated that using the recombinant protein as coatingantigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as asource of antigen@). ELISAs based on recombinant proteins are safe (no live virus), clean (only virus antigensare present), specific (single proteins can be used) and rapid (to respond to new viral strains and strains thatcannot necessarily be easily cultured).     

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for mitozantrone

A mouse monoclonal antibody (NO-1) with specificity for the anti-cancer drug mitozantrone (MZ) (Novantrone) was produced by immunization of a BALB/c mouse with mitozantrone-keyhole limpet haemocyanin (MZ-KLH) conjugate. When used in an indirect competitive enzyme-linked immunosorbent assay (ELISA), NO-1 permitted the accurate and reproducible detection of between 0.25-50 ng/ml of MZ in pooled human serum, the standard curve obtained within this range being virtually linear. The assay demonstrated good reproducibility with intra-assay coefficients of variation (CV) of between 1.41% and 7.02% and an inter-assay CV of 3.45%. Regression analysis of levels of MZ detected by ELISA vs. the actual amounts added to pooled human serum gave a very good correlation coefficient of r = 0.995. NO-1 showed no cross-reactivity with either bisantrene or daunorubicin. A simple pharmacokinetic study was undertaken in rabbits given MZ intravenously at a dose of 0.5 mg/kg of body weight. Levels of MZ in rabbit serum measured with the assay ranged between 82 and 170 ng/ml for rabbits 1 and 2, respectively at 15 min falling to 1.25 ng/ml by 48 h for rabbit 1 and falling to undetectable levels by 120 h for rabbit 2.     

A monoclonal antibody-based competitive enzyme-linked immunosorbent assay for detecting antibody production against avian reovirus protein sigmaA

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based c-ELISA) for detection of antibody against avian reovirus protein sigmaA in chicken is described. After the conditions for MAb-based c-ELISA had been optimized, sera collected from birds that received live and inactivated avian reovirus vaccines in different combinations were tested for antibody response against virus protein sigmaA. The results show a high level of antibody against sigmaA was in both vaccinated specific pathogenic free (SPF) and vaccinated commercially reared birds as long as one of the vaccines administered was in an inactivated form. The high level of antibody production is indicated by a high percentage inhibition (PI) values in the sera of the birds; but no antibody production was found in birds which received live vaccine only, as indicated by the low PI values. In serum samples from SPF birds receiving vaccines that include an inactivated form of the vaccine, there is a good correlation between the PI values and serum neutralizing antibody (SN) titers. Again, this correlation was not observed in birds that received only live vaccine. The PI values of commercially reared birds receiving inactivated vaccine were significantly different from those of the mock-treated birds, but this was not the case when the birds received only live vaccine. Taken together, the results suggest that MAb-based c-ELISA may provide an alternative choice for determining the immune status of a vaccinated chicken flock as long as one of the vaccines used was inactivated, and thus would allow a more precise way to predict the appropriate time for vaccination.     

Monoclonal antibody-based sensitive enzyme-linked immunosorbent assay for murine serum amyloid A

Enzyme-linked immunosorbent assay (ELISA) methods for measuring murine serum amyloid A (SAA), a representative acute phase reactant, were developed utilizing a newly produced monoclonal antibody. Two site-ELISA, in which the monoclonal antibody was used as the captured antibody, was sensitive enough to determine the SAA concentration in mice at the steady state. Direct binding ELISA, in which the sample SAA bound to the plastic wells was detected by the antibody, was simple and suitable for measuring the elevated SAA, but could not analyze the resting level of SAA because of the need for high dilution in plasma samples. Plasma SAA concentrations were measured in ten ICR mice on the day of purchase and at the end of seven days of ordinary rearing. The SAA concentration of one animal decreased from 1.6 to 0.5 mg/l during a week, while the others had no obvious changes. The plasma SAA of the ten animals after one week of rearing ranged from 0.3 to 0.8 mg/l with a mean of 0.47. These mice, two days after 10 microg lipopolysaccharide were given, had increased SAA values up to a mean of 300 mg/l, though with variations between animals.     

Comparative studies with an enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect IgG class-specific antibodies to avian infectious bronchitis virus (IBV) in chickens. The technique detected specific antibody with a high degree of sensitivity and was more sensitive than neutralisation or haemagglutination-inhibition methods of antibody assay. The possible application of the technique for diagnosis and research on IBV is discussed.     

Detection of antibodies to avian infectious bronchitis virus by a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay

The recombinant antigen obtained by cloning and expressing two IBV nucleocapsid protein fragments (143-414 aa, 281-414 aa) in Escherichia coli was used for the detection of avian infectious bronchitis virus (IBV) specific antibodies in chicken sera by the indirect ELISA (rNpIBV-ELISA). As a result of testing 1524 serum samples the diagnostic sensitivity and specificity of rNpIBV-ELISA when comparing those of the routine whole IBV ELISA have been shown to be 93.81% and 87.36%, respectively. The agreement value was 91.5%.     

Evaluation of a West Nile virus immunoglobulin A capture enzyme-linked immunosorbent assay

An in-house-developed enzyme-linked immunosorbent assay detected West Nile virus (WNV) immunoglobulin A (IgA) in 65 of 68 sera from WNV-infected patients; 40 of 63 WNV IgM-positive, IgG-negative serum or plasma specimens; 65 of 67 WNV IgM-positive, IgG-positive specimens; 0 of 70 WNV IgM-negative, IgG-negative specimens; and 0 of 64 archived blood donation sera. WNV IgA is thus highly prevalent among WNV-infected patients and typically appears after WNV IgM but before WNV IgG.     

Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples.

A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs.     

Method for detection of simian immunodeficiency virus neutralizing antibodies using a noncommercial antigen capture enzyme-linked immunosorbent assay.

A neutralization test (NT) using a noncommercial antigen capture enzyme-linked immunosorbent assay (ELISA) to detect simian immunodeficiency virus (SIV) growth in vitro was developed. The capture antibody was a mixture of purified macaque anti-SIV immunoglobulin G (IgG) and a monoclonal antibody to SIV p27. Captured antigens were detected by using purified macaque anti-SIV IgG conjugated to horseradish peroxidase. The NT reliably and sensitively detected differences when various amounts of SIV were used with positive and negative control macaque sera. Dilutions of sequential sera from a macaque (Macaca nemestrina) that had been experimentally infected with SIV were tested for neutralizing antibody with 300 50% tissue culture infective doses of SIV. In this macaque, neutralizing activity and anti-SIV IgG levels in serum (detected by ELISA) increased with time after SIV inoculation, and high IgG titers were required in serum before neutralization occurred in vitro. This simple NT, which detects the presence of SIV serum neutralizing antibodies at a low cost, will be useful for investigating the role of neutralizing antibodies in the SIV-infected macaque model for AIDS.     

Rapid serotyping of infectious pancreatic necrosis virus by one-step enzyme-linked immunosorbent assay using monoclonal antibodies.

A one-step ELISA has been developed for detection and serotyping of infectious pancreatic necrosis virus (IPNV) in infected cell cultures using monoclonal antibodies (mAb) raised against strains representing the Sp, Ab and VR 299 serotypes of IPNV. This assay uses a serotype-specific mAb as capture and a mAb directed against a common epitope as detector antibody. Avidin-peroxidase was employed for amplification. The assay was performed in 90 min by simultaneous incubation of the samples, the biotin labelled mAb and the avidin-peroxidase, and detected 37 ng/ml of purified virus. Serotyping of 34 isolates from different areas of Europe, Asia and America showed a total concordance with the results obtained by the neutralization assay.     

Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of Cryptosporidium oocysts.

The diagnosis of the small (4- to 6-microns) Cryptosporidium oocysts is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The primary purpose of this study was to evaluate the clinical utility of an antigen capture enzyme-linked immunosorbent assay (ELISA) (LMD Laboratories, Carlsbad, Calif.) in detecting Cryptosporidium oocysts in human stools. A total of 591 specimens (76 diarrheal, 515 control) obtained from 213 inhabitants of an urban slum in northeastern Brazil were examined by both ELISA and conventional microscopic examination (CME) of formalin-ethyl acetate-concentrated stool samples stained with modified acid-fast and auramine stains. Forty-eight diarrheal stools (63.2%) were positive for Cryptosporidium oocysts by CME, with 40 of these positive by ELISA. Thirty-five control stools (6.8%) had Cryptosporidium oocysts detected by CME, with 15 of these also positive by ELISA. All of the 480 nondiarrheal stools and all but one of the diarrheal stools negative by CME were negative by ELISA. The test had an overall sensitivity of 66.3% and a specificity of 99.8% (positive predictive value, 98.2%; negative predictive value, 94.8%). In the evaluation of human diarrheal stool samples, the test sensitivity increased to 83.3%, with a specificity of 96.4%, and, in analysis of samples from individual patients with diarrhea, the sensitivity was 87.9%, with a specificity of 100%. These results indicate that this stool ELISA is sensitive and specific for the detection of Cryptosporidium oocysts in human diarrheal stool specimens but has limited use in epidemiologic studies for the diagnosis of asymptomatic Cryptosporidium infection.     

A capture enzyme-linked immunosorbent assay for species-specific detection of Bothrops venoms

A direct sandwich enzyme-linked immunosorbent assay (ELISA), employing affinity purified antivenom antibodies specifically recognizing the homologous venom, was developed for species-specific detection of bothropic venom. The method is based on a two-step affinity purification of the specific antibodies. A species monovalent antivenom is adsorbed onto a venom adsorbent containing heterologous venoms from the Bothrops, Crotalus and Lachesis genera. The species-specific antibodies obtained, are then adsorbed onto a second venom adsorbent containing only the homologous venom for the removal of non antivenom antibodies. Venom concentrations of 0.1 and 1,000 ng/ml were specifically identified for Bothrops jararacussu and B. alternatus venom respectively.     

Antigen capture enzyme-linked immunosorbent assay for specific detection of Reston Ebola virus nucleoprotein

Antigen capture enzyme-linked immunosorbent assay (ELISA) is one of the most useful methods to detect Ebola virus rapidly. We previously developed an antigen capture ELISA using a monoclonal antibody (MAb), 3-3D, which reacted not only to the nucleoprotein (NP) of Zaire Ebola virus (EBO-Z) but also to the NPs of Sudan (EBO-S) and Reston Ebola (EBO-R) viruses. In this study, we developed antigen capture ELISAs using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques.     

Novel haptens synthesis and development of a monoclonal antibody-based enzyme-linked immunosorbent assay for leuco-malachite green in fish

To produce specific antibodies against leuco-malachite green (LMG), 15 haptens were synthesized and characterized, and conjugated to carrier protein for immunization. One antigen with excellent reactogenicity and immunogenicity was discovered. Specific monoclonal antibody with high sensitivity for LMG in competitive indirect enzyme-linked immunosorbent assay (ciELISA) was screened and selected. After the screening of a series of heterologous coating antigen and optimization of working conditions, the proposed ciELISA showed the 50% inhibition value (IC₅₀) of 1.16?ng?mL^?1 and the limit of detection of 0.06?ng?mL^?1 for LMG. The average recoveries of LMG from spiked fish samples ranged from 78.0% to 101.0%, with coefficients of variation below 15%. Good correlation (R²?=?0.9977) was obtained between the results of ciELISA analysis and those of standard liquid chromatography–tandem mass spectrometry analysis. The proposed ciELISA is ideal for the rapid and sensitive detection of LMG with a low cost and high throughput.     

Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.     

Enzyme-linked immunosorbent assay for identification of infectious pancreatic necrosis virus.

The sensitivity, reproducibility, and specificity of an enzyme-linked immunosorbent assay for the identification of infectious pancreatic necrosis virus was determined. It was shown that 10(5.5) tissue culture infectious doses could be assayed by this method. The assay provided significant advantages in terms of time and ease of performance over the routinely employed serological tests. When an antiserum against a single isolate was used, it was possible to identify nine of ten North American isolates, four of six European isolates, and one isolate from Taiwan.