Single Dilution ELISA Research Articles

Development of recombinant capsid protein-based single serum dilution ELISA for sero-detection of porcine circovirus type 2 infection in pigs.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Glycoprotein D peptide-based diagnostic approach for the detection of avian infectious laryngotracheitis antibodies

ABSTRACTInfectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens, pheasants, and peafowl. It is caused by the alpha herpesvirus, infectious laryngotracheitis virus (ILTV). Glycoprotein D (gD) of ILTV is immunogenic and helps in its binding to the susceptible host cell receptor. In the present study, a recombinant gD protein was expressed in a prokaryotic system to develop a single serum dilution ELISA. In addition, two immunogenic peptides, corresponding to regions 77–89 and 317–328, were identified in gD protein. The peptides were synthesized using solid-phase peptide synthesis, purified using reversed-phase HPLC, and characterized using mass spectrometry. The peptides displayed a good titre and were found to be promising antigens to coat the ELISA plate to detect the ILTV antibodies in the serum sample. The developed ELISA showed 96.9% sensitivity, 87.5% specificity, and 95.3% accuracy as compared to OIE referenced standard indirect ILTV ELISA (whole viral coated). The assay may not differentiate vaccinated from infected birds when the flocks are administered with live attenuated vaccines. However, the assay could be useful to detect the disease condition in birds vaccinated with recombinant vaccine expressing glycoproteins other than gD. The developed ILTV single serum dilution ELISA could be an alternative to the existing diagnostics for the detection of ILTV antibodies.

Lentiviral-mediated delivery of classical swine fever virus Erns gene into porcine kidney-15 cells for production of recombinant ELISA diagnostic antigen.

Classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family causes contagious fatal disease in swine. Antibodies against E2, Erns and NS3 proteins of virus can be detected in infected animals. Development of an ELISA coating antigen to improve the sensitivity of detecting Erns-specific antibodies in pig sera is always desirable for diagnosis as well as for differentiation of infected from vaccinated animals. In present study, a lentivirus-based gene delivery system was used to develop a stable PK-15 cell line expressing Erns (PK-Erns) for production of diagnostic antigen. The Lenti-Erns virus was purified from the supernatant of co-transfected 293LTV cells and used to transduce PK-15 cells. The homogenous PK-Erns cell line was produced by single cell cloning by monitoring eGFP expression. The Erns gene in the genomic DNA and RNA transcripts in total RNA isolated from PK-Erns cells were detected by PCR and RT-PCR, respectively. Expression of 45kDa Erns glycoprotein was detected in western blot using CSFV-specific hyperimmune sera. The use of PK-Erns cell lysate as antigen in serial dilution and single dilution ELISAs with known positive and negative pig sera was investigated. The PK-Erns ELISA revealed sensitivity equivalent to commercial HerdChek ELISA kit. The sensitivity, specificity and accuracy of the PK-Erns ELISA was 95%, 100% and 96.66%, respectively compared to ELISA using purified CSFV as coating antigen. When field pig sera (n = 69) were tested in PK-Erns ELISA, a significant correlation between the titers from serial dilution and single dilution ELISA was observed. This indicated that PK-Erns cell line can serve as continuous source of ELISA diagnostic antigen for detection of CSFV-specific antibodies in pig sera.

Development of single dilution immunoassay to detect E2 protein specific classical swine fever virus antibody

Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in swine. The disease is endemic in different parts of the world and vaccination is the only way to protect pigs from CSFV infection. The virus surface protein E2 is the major immunogenic protein eliciting protective immunity against CSFV infection in swine. The whole virus antigen cannot differentiate CSFV from other pestiviruses as it cross reacts with border disease and bovine viral diarrhoea viruses. Commercial available ELISA is based on the whole CSFV particle and can lead to false positive results. Moreover, the available commercial ELISA is not cost effective. In the present study, a recombinant E2 protein based single serum dilution ELISA was developed which showed enhanced sensitivity, specificity and accuracy as compared to commercial CSFV detection ELISA. The recombinant E2 protein based ELISA could be an alternate to existing diagnostics against CSFV infection in pigs.

Recombinant phosphoprotein based single serum dilution ELISA for rapid serological detection of Newcastle disease virus

Newcastle disease virus (NDV) is the causative agent of a highly contagious disease in avian species. All strains of NDV belong to avian paramyxovirus serotype-1. The disease is endemic in different parts of the world and vaccination is the only way to protect birds from NDV infection. The virus non-structural phosphoprotein (P) is the second most abundant protein and a major modulator of viral replication. Although P protein shows lesser evolutionary divergence among NDV isolates, it is known to be highly divergent among different avian paramyxovirus serotypes. In the present study, a recombinant P protein based single serum dilution ELISA was developed which showed better sensitivity, specificity and accuracy as compared to conventional methods for NDV detection. The recombinant P protein based ELISA could be an alternative to existing diagnostics against NDV infection in chickens.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens

A recombinant hexon antigen based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Fowl adenovirus-4 (FAdV) causing Hydropericardium syndrome (HPS). An immunodominant partial hexon gene of 737bp was cloned into pRSET vector and expressed in Escherichia coli strain BL21 DE3 pLys S. Expression was tested by Western Blot test. The purified recombinant protein antigen was used in coating ELISA plate for FAdV-4 serology. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to Q-AGID test. Recombinant antigen was also used in Dot ELISA. In an experimental vaccination of broiler chicken at 10 days old age, the geometric mean (GM) antibody titres as measured by ELISA ranged from 5.006±0.11log10 to 4.526±0.04log10 and by Dot ELISA titre were from 2.240±0.08log10 to 0.180±0.04log10 during 5th–8th weeks of age, results were compared with Q-AGID results. Field samples were collected randomly from breeder flocks, found to have antibody titre by both ELISA and Dot ELISA at 10th and only 75% samples were positive at 14th weeks of age. After revaccination at 16th weeks of age, all sera samples were found have considerably high antibody titre on 24th week but all samples were negative at 32nd weeks. Advantages of recombinant hexon antigen based ELISA and Dot ELISA in FAdV-4 serology have been discussed.

Immunokinetics of Theileria equi specific antibodies: a comparison in serial and single dilution ELISA antibody end titres

Theileria equi, a haemoprotozoan of equids, is responsible for endemic infection/latency in tropical/subtropical parts of the world. The present study investigated the immunokinetics of T. equi infection in experimental animals and the diagnostic applicability of single dilution ELISA vis-à-vis conventional serial dilution ELISA. Sequential serum samples were collected up to 90 days postinfection (PI) from 4 donkeys experimentally infected with T. equi. Immunokinetics of T. equi antibodies end titre were determined by serial and single dilution ELISA. A significant rise in initial ELISA antibody end titre (1:800 in serial dilution ELISA and from 1:600 to 1:650 in single dilution ELISA) was observed from day 9 PI in all 4 donkeys, which plateaued between days 66 and 73 PI (end antibody titre varied between 1:6400 to 1:25,600 and 1:6050 to 1:22,100). The efficacy of these ELISAs was also determined by testing 256 field serum samples at 1:200 dilution, collected from apparently healthy animals. Sample analysis revealed overall seroprevalence of 91.01% in the horse population. The frequency distribution of antibody titre of these field samples by single dilution ELISA exhibited that most of the positive horses had high levels of specific antibodies. This study has emphasised the usefulness of single dilution ELISA as a diagnostic assay in determining the end antibody titre by testing it at a single dilution of 1:200.

Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis

A recombinant UL30 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively.

Development of rHA1-ELISA for specific and sensitive detection of H9 subtype influenza virus

A recombinant antigen-based single serum dilution ELISA was developed for simultaneous detection and subtyping of influenza viruses. Recombinant baculovirus encoding the hemagglutinin (HA 1 subunit) of H9N2 virus was generated. To evaluate the rHA1-ELISA, microplates were coated with purified HA1 protein and tested with reference control sera. Subsequently, 92 field sera collected from chickens suspected to be infected with H9N2 AIV were employed to test the efficacy of the rHA1-ELISA. The sera were tested simultaneously by HI and a commercial AIV ELISA kit. The rHA1-ELISA appeared to be highly specific and sensitive for direct detection of H9N2 antibodies in serum samples.

Recombinant haemagglutinin neuraminidase antigen-based single serum dilution ELISA for rapid serological profiling of Newcastle disease virus

A recombinant haemagglutinin neuraminidase (HN) antigen-based single serum dilution enzyme linked immuno-sorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Newcastle disease virus. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the standard haemagglutination inhibition (HI) test.

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation can be derived that allows demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).

Impact of field vaccination with a Theileria annulata schizont cell culture vaccine on the epidemiology of tropical theileriosis

Tropical theileriosis, caused by Theileria annulata, is an important tick-borne disease of cattle. A cell culture attenuated vaccine has been developed in our laboratory by long-term in vitro propagation of the schizont stage of the parasite. A longitudinal study was conducted at selected farms housing indigenous, cross-bred and exotic animals to investigate the effect of vaccination on the epidemiology of the disease. A total of 120 animals in 4 age groups were vaccinated with the vaccine before the onset of disease season. An equal number of age-matched animals were kept as controls at the same sites. Animals were monitored for 14 months at monthly intervals. The 97.5% vaccinated animals showed a rise in antibody titres 1 month post-vaccination, as determined by single dilution ELISA. The 78.3% of non-vaccinated animals became sero-positive over the period of observation. Mean antibody titres were significantly higher in vaccinated than non-vaccinated animals. Cross-bred animals showed higher antibody titres followed by exotic and indigenous animals in both the vaccinated and non-vaccinated groups. However, the antibody titres in animals of different ages were similar. The 36.7% vaccinated and 64.2% non-vaccinated animals became carriers (<0.5% piroplasms in erythrocytes) during the observation period. Clinical cases of theileriosis were recorded only in the non-vaccinated group suggesting that vaccinated animals were sufficiently immune to withstand field tick challenge for at least 14 months.

Single dilution ELISAs using soluble piroplasm, cellular schizont and soluble schizont antigens for the detection of antibodies against Theileria annulata.

Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log10 antibody titres by multi-dilution ELISA. The value of "r" was the highest at the dilution of 1:400. From the log10 antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a + bX, where Y = predicted log10 titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 + 1.35X for soluble piroplasm, Y = 2.67 + 0.547X for cellular schizont and Y = 1.817 + 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multidilution ELISAs showed non-significant differences (P < 0.01) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.

Evaluation of an immunofluorescent antibody test to detect bovine herpesvirus 1-specific IgM.

An indirect immunofluorescent antibody test (IIFAT) was developed to detect bovine herpesvirus 1 (BHV-1)-specific IgM. All sera were treated with protein-G agarose prior to testing to eliminate the possibility of false-positive results due to IgM-isotype rheumatoid factor (IgM-RF). Specific IgM was first detected 8 days after experimental infection of 3 calves free of maternally derived antibody, with peak responses occurring 2-7 days later. Seroconversion was detected in all 3 calves using a single-dilution enzyme-linked immunosorbent assay. Following reinfection at 30 days postinfection, a low-level IgM response was detected in only 1 calf. Seroconversion was detected in 2 calves. There was no evidence of activation of IgM-RF by infection or reinfection with BHV-1. When 87 acute and convalescent serum pairs collected from 21 outbreaks of respiratory disease were tested, specific IgM was detected in 58 animals (66.6%) from 19 (90.5%) outbreaks. Seroconversion was detected in 44 of these animals (50.6%) from 17 outbreaks (81.0%). The correlations between these 2 assays on a calf and outbreak basis were 79.3% and 90.5%, respectively. Specific IgM was detected in 17/20 sera (85.0%) collected from an additional outbreak. No virus was detected by virus isolation or immunofluorescent staining in nasal mucus samples collected at the same time. Detection of specific IgM by IIFAT is a useful technique for the serodiagnosis of BHV-1 infection.

Antibody responses to Pasteurella haemolytica 1:A and three of its outer membrane proteins in serum, nasal secretions, and bronchoalveolar lavage fluid from calves

Abstract Objective To determine systemic and mucosal antibody responses in calves to Pasteurella haemolytica 1:A and to 2 major outer membrane proteins (OMP) and 1 major iron-regulated OMP of P haemolytica 1:A. Animals 23 crossbred calves. Procedure 2 experiments were performed. In the first experiment, 6 calves were vaccinated and challenge exposed intranasally with an aerosol of P haemolytica 1:A and 6 calves were only challenge exposed. In the second experiment, 8 calves were vaccinated in the area of the tracheal bifurcation with an aerosol of P haemolytica 1:A and 3 calves were used as controls. Serum, nasal secretions, and bronchoalveolar lavage (BAL) samples were collected, and IgG1, IgG2, IgA, and IgM titers were determined. Nasal secretions and BAL samples were also submitted for bacterial culture. Results Serum antibody responses in the 2 groups were similar. Antibody titers in nasal secretions and BAL samples increased in calves vaccinated intranasally. In calves vaccinated in the area of the tracheal bifurcation, antibody titers increased in BAL samples but not in nasal secretions. Antibody responses did not correlate with results of bacterial culture. Conclusions Results indicated that intranasal administration of P haemolytica 1:A may be a better method for stimulating protective immune responses in the upper portion of the respiratory tract than lung administration. The single dilution ELISA provided a reliable and economical method for determining antibody titers. (Am J Vet Res 1998;59:727-732)

Development of a single dilution ELISA to detect antibody to Dermatophilus congolensis in goat and cattle sera

A solid phase immunosorbent assay to detect antibodies to Dermatophilus congolensis in ruminant sera was developed to be used as a single dilution ELISA in large epidemiological surveys. Optimal conditions for the test are described. The use of blocking proteins to reduce non specific binding was necessary. Non fat dry cow milk and fetal calf serum were the only two efficient blocking agents out of six tested. Comparison of 4 antigenic fractions obtained after sonication differential centrifugations of D. congolensis cultures showed that cell-wall (CW) or membrane (M) enriched preparations were more specific than a crude extract (CR) or a soluble (S) antigen. Whole spores and filaments performed poorly as antigens. The best sensitivity and specificity of the ELISA were obtained when the cut-off point of positivity was fixed at mean absorbance of negative sera + 2.58 sd. The specificity was then 97.6% either with M or CR antigen. The sensitivity was improved from 93.4% with CR to 98.2% with M antigen. Threshold values for a positive test varied between the 3 geographical areas tested. CW and M were also the most efficient antigens for discerning between serotypes of D. congolensis. The precision of the test was evaluated with CR antigen and expressed in residual coefficient of variation (CV). The precision was CV = 5.1% when each serum was titrated in duplicate and the antibody levels were expressed in absorbances. The expression of antibody levels in arbitrary standard units estimated from calibration curves reduced the precision (CV = 13.8%). Several methods were tested to decrease between plate variability but these did not greatly improve the reproducibility Since it was shown that the main source of variation was within the plate.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to bovine viral diarrhea virus

A single dilution enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to bovine viral diarrhea (BVD) virus in cattle sera. Viral antigen (NADL strain) was grown in a pig kidney cell line (PK15), and after removal of nuclear debris, was purified by ultracentrifugation through a potassium tartrate cushion. Antigen grown in embryonic bovine tracheal epithelial cells was also satisfactory. The test used a high salt buffer to minimize nonspecific reactivity, polyethylene glycol to enhance the reaction, and Protein G as the labelling agent. Comparative testing with the virus neutralization test (VNT) showed the ELISA results to have a high level of correlation with the VNT titers ( r = 0.83). In vaccinated animals the ELISA detected antibodies earlier than the VNT. All animals sampled from a BVD-free herd were negative for BVD antibody. The single dilution test showed close agreement ( r = 0.84) with ELISA values obtained using a serial dilution technique, and also proved to have a high level of reproducibility. The test procedures were relatively easy to carry out, and were economic in their use of materials.

Detection of Antibodies to Helicobacter pylori Cell Surface Antigens

Serum IgG antibodies of Helicobacter pylori were detected in single-dilution ELISA using glycine extracted material. Among 148 endoscopy patients 59% displayed antibodies; as expected, a higher occurrence (90%) was found in patients with positive gastric culture for H. pylori than in culture negative patients (37%). Among 68 blood donors the frequency of H. pylori antibodies was 28%. In 73 children less than 15 years of age examined for unrelated disorders the occurrence was 4%. By immunoblotting using the same extract, 3 prominent bands, 29K, 54K and 60K and several weak bands were identified. These were formed by 57%, 92%, and 65%, respectively, of the ELISA positive patient sera. Comparing culture positive and negative patients, the 3 bands occurred more often among the culture positive subjects though between 18 and 61% of the sera from culture negative patients gave either of the bands. When comparing the glycine extracts of 4 different H. pylori strains with separate haemagglutinating patterns no differences in the position of the major bands emerged. By absorption experiments no immunological cross-reactivity with components of Escherichia coli, Klebsiella pneumoniae, Campylobacter jejuni or C. fetus was found. Thus, the glycine extract seemed specific for the detection of antibodies to H. pylori.

Single dilution ELISA for detection of serum antibody to foot-and-mouth disease virus in cattle

A single dilution blocking ELISA was developed and evaluated for measuring serum antibody to foot-and-mouth disease virus (FMDV). Basic parameters of the assay were established and a positive-negative threshold determined from testing 176 specific antibody negative sera from Australian cattle. Sera collected from immunised animals in Thailand were tested by ELISA and virus-neutralisation (VN) tests and the results compared. A positive correlation between ELISA and VN titres was recorded for each of the 3 FMDV serotypes endemic in Thailand, with the overall correlation coefficient being r = 0.8990. A positive correlation for each of the serotypes was also found between ELISA titre and the degree of blocking (percentage inhibition) of each test serum at a dilution of 1:16, with the overall correlation being r = 0.8704. This simplified ELISA was sensitive, specific and gave reproducible results, and had the potential to test quickly and efficiently a considerable number of sera.