Avidin-biotin ELISA Research Articles

Seroprevalence of Infectious Bovine Rhinotracheitis (IBR) in Indian Buffaloes

Background: Infectious bovine rhinotracheitis (IBR) is an infectious disease of bovines causing respiratory problem, abortions and reduced milk yield leading to huge economic losses. IBR is endemic in India. India ranks first in buffalo population. Reports on seroprevalence of IBR in cattle in India are available but not in buffaloes. The study was aimed at conducting a seroprevalence of IBR in Indian buffaloes. Methods: A total of 1000 buffalo serum samples collected randomly from top 10 milk producing states of India and were tested for IBR antibodies using Avidin-Biotin (AB) ELISA. Result: Cumulative seropositivity was found to be 43.30%. Punjab and Andhra Pradesh states showed highest (58%) and lowest (27%) seropositivity respectively and were part of north and south zone of the country. India has highest number of buffalo population. Presently, India do not vaccinate bovines against IBR and should take call on vaccination of dairy buffalo after having witnessed a high seroprevalence of IBR. This is the first report of such kind of number of buffalo samples tested for IBR antibodies.

Potential diagnostic application of the baculovirus-expressed recombinant truncated nucleocapsid protein of peste des petits ruminants virus in ELISA

The study describes the expression of recombinant truncated nucleocapsid protein (NP) of peste des petits ruminants (PPR) virus in the baculovirus system (PPRV-rBNP) and its potential application as a diagnostic antigen in ELISA for diagnosis of PPR in sheep and goats. The PPRV N-terminal immunogenic region (1–266 aa) of the NP coding sequence was amplified and cloned into the pFastBac HT A vector. The PPRV-rBNP with a molecular weight of ∼30 kDa was expressed in an insect cell system using generated recombinant baculovirus through Bac-to-Bac® Baculovirus Expression System. The crude PPRV-rBNP or Ni-NTA affinity-purified NP was characterized by SDS-PAGE and immunoblot using standard PPRV-specific sera. The PPRV-rBNP reacted well with PPRV anti-N specific monoclonal and polyclonal antibodies and PPRV-specific antiserum, suggesting that the expressed PPRV-rBNP is in its native form. The crude PPRV-rBNP as a diagnostic antigen was evaluated either as a coating antigen or standard positive control antigen in the Avidin-Biotin ELISA using the known standard panel reagents. The results showed that the expressed PPRV-rBNP can be an alternative diagnostic antigen to E. coli expressed recombinant PPRV-NPN and the utility of PPRV-rBNP avoids the need to use live PPRV antigen in the diagnostic ELISA. Hence, this allows scope in the future for large-scale field application of the recombinant antigen-based assays for diagnosis/surveillance and monitoring of PPR at the eradication as well as post-eradication phases in endemic countries or PPR non-endemic countries.

SEROPREVALENCE OF INFECTIOUS BOVINE RHINOTRACHEITIS (IBR) IN NORTH EASTERN (NE) STATES OF INDIA

Infectious bovine rhinotracheitis (IBR) is an infectious disease caused by BoHV-1 and belongs to the Herpesviridae family. IBR is endemic in India including north eastern states of the country. Hence the study was undertaken to understand the seroprevalence of IBR in north eastern parts of the country. A total of 3125 cattle (Holstein Friesian crossbred) serum samples from 35 districts of five north eastern states (Assam, Manipur, Meghalaya, Mizoram, and Sikkim) of India were screened for infectious bovine rhinotracheitis (IBR) virus antibodies using Avidin biotin ELISA. A two-stage random sampling methodology was followed for the collection of samples. Results from the present study revealed that the overall seropositivity was reported around 29.50% while the highest and lowest seropositivity of 43.39% and 16.66% were reported in the states of Sikkim and Assam respectively, followed by Mizoram (42.16%), Manipur (29.86%) and Meghalaya (27.40%). Cattle of higher age groups showed the highest seropositivity compared to younger ones. A higher percent of IBR antibodies in cattle of NE states is a cause of concern and a detailed study on IBR prevalence comprising of a large number of the bovine population need to be undertaken.

Preparation of a Specific ssDNA Aptamer for Brevetoxin-2 Using SELEX.

The existing assays for detecting brevetoxin (BTX) depend on expensive equipment with a professional operator or on an antibody with limited stability, which requires complex processes, a high cost, and a considerable amount of time. The development of an alternative detection probe is another promising research direction. This paper reports the use of aptamers binding to BTX-2 in an analytical assay using the systematic evolution of ligands by exponential enrichment (SELEX). After 12 rounds of selection, the secondary structures of 25 sequences were predicted. Compared to other aptamers, Bap5 has relatively high affinity with the lowest dissociation constant of 4.83 μM, and IC50 is 73.81 ng mL−1. A good linear regression formula of y = 30.688x − 7.329 with a coefficient correlation of R 2 = 0.9798 was obtained using a biotin-avidin ELISA. Moreover, there is no cross-reaction with the detected marine toxins, except for BTX-2. Thus, Bap5 has potential to detect BTX-2 in shellfish in the future as a substitute for the recognition probe.

Seroprevalence of infectious bovine rhinotracheitis in organized dairy farms of India

In this study, a systematic sero-surveillance of IBR was undertaken from 11 dairy farms located in 4 different regions of India. A total of 1000 cattle serum samples were tested for the presence of antibodies against IBR using Avidin Biotin ELISA. The results revealed that IBR antibodies were widely prevalent in all regions of the country ranging from 36.5% in Central region to 84.5% in Northern region with an overall prevalence of 61.6%. The prevalence of IBR antibodies was different between various age groups being 22.3%, 62.1%, 59.3%, 76.1% and 66.78 % in the age groups less than 1 year, 1-2 years, 2-3 years, 3-4 years and more than 4 years old respectively. Based upon the medical history of the herd, it was found that 83% abortion cases, 76% metritis cases, 83% repeat breeding cases and 65% retention of placenta cases were seropositive for IBR.

Sero-epidemiology of infectious bovine rhinotracheitis and brucellosis in organised dairy farms in southern India

The present study was conducted to know the seroprevalence of infectious bovine rhinotracheitis (IBR) and burcellosis and its epidemiology in organised dairy farms in southern India. Sera samples (559) 398 cattle and 161 buffaloes) were collected from 6 organised dairy farms in southern India. Samples were screened for IBR by avidin biotin ELISA and brucellosis by RBPT and indirect ELISA. The overall apparent prevalence were 61.54%, 10.20% and 11.63% for IBR, Brucella by RBPT and iELISA respectively. The state-wise seroprevalence showed highest in Andhra Pradesh for IBR and Karnataka for both IBR and brucellosis; lowest in Tamil Nadu for both the diseases. There was no significant difference in male and female in seroprevalence of these diseases. Crossbred cattle showed high seroprevalence for IBR and Brucella antibodies when compared to indigenous cattle breeds. Buffaloes showed increased seroprevalence for IBR and Brucella when compared to cattle. The IBR seropositive animals showed positive relationship with increase in age. Animals with history of abortions showed seroprevalence of 100% for IBR and 40-50% for brucellosis. The animals with history of reproductive problems showed increased seroprevalence when compared to apparently healthy bovines. The seropositivity for both IBR and brucellosis were 2.76% and 29.19% in cattle and buffaloes, respectively and with overall seropositivity of 10.38%. Thus IBR and brucellosis seroprevalence has increased over the years and there is a need to tackle these diseases effectively by zoosanitary measures and control programmes in organised dairy farms which would benefit the dairying in Southern India.

A comparative evaluation of avidin-biotin ELISA and micro SNT for detection of antibodies to infectious bovine rhinotracheitis in cattle population of Odisha, India

AbstractAim:MaterialsandMethods:Results:Conclusion:Keywords The present study was undertaken to serologically d etect Infectious Bovine Rhinotracheitis (IBR) in the cattlepopulation of Odisha, India using (micro SNT) and(ABELISA)andfindingouttheircomparativeefficacytoserveasasuitablediagnostictoolinfieldcondition.Thestudywascarriedoutusingserumsamples(n=180)collectedrandomlyfromcattlepopulationsof nine districts of Odisha. Similarly vaginal swabs (n=26) from cattle having history of repeat breeding, abortion, vulvo-vaginitis and nasal swabs (n=8) from calves with respiratory symptoms and nasal discharge were collected aseptically, toascertainthecirculationofvirusamongthecattlepopulation.Virus isolation by cell culture and subsequent confirmation by polymerase chain reaction confirmed four isolates.Screening of serum samples revealed 9.44% and 12.22% samples positive for IBR antibodies in micro SNT andAB ELISArespectively.ThesensitivityandspecificityofABELISA testwasfoundtobe88.23%and95.70%respectivelytakingmicroSNTasgoldstandardandthekappavaluebetweenthetwotestswas0.75.Screeningofserumsamplesrevealed9.44%and12.22%samplespositiveforIBRantibodiesinmicroSNTandAB ELISA respectively, thus highlighting the circulation of virus among the livestock population of Odisha and that ABELISA could be more efficiently applied for the sero-diagnosis of IBR virus infections at field conditions, with demand formorestudyonfaster,efficientandlargescalescreeningoftheinfectedanimals.: infectious bovine rhinotracheitis, serumneutralizationtest,Odisha.avidin-biotin ELISA, bovine herpes virus -1, cattle, isolation,micro-Serum neutralization test Avidin-Biotin Enzyme linked immunosorbentassay

Cloning of partial glycoprotein B gene and molecular epidemiological studies of bovine herpes virus -1 isolates

The study describes genetic grouping and molecular epidemiology of bovine herpes virus-1 (BoHV-1) through cloning of partial glycoprotein B gene of BoHV-1 isolates, with special reference to isolates recovered from cattle breeding stations. Samples were collected from 212 animals (91 bulls and 121 female cattle). Avidin-biotin ELISA employed on serum samples found 74 animals as seropositive for BoHV-1. On inoculation of 212 semen/swab samples to MDBK cell line for virus isolation, samples of 4 seropositive and 5 seronegative animals yielded cytopathic changes characteristic of BoHV-1. Partial gB gene of these isolates were cloned in pGEM T vector, nucleotide sequences were deduced and phylogenetic tree was constructed. Sequence analysis grouped 5 of these isolates under BoHV-1.1 cluster having highest sequence identities with previously described Indian, European and Brazilian isolates of BoHV-1.1. The other 4 isolates were clustered as BoHV-1.2 subtypes having 100% sequence identity with European strain of BoHV-1.2. We found that, apparently healthy, seronegative animals can be sources of BoHV-1, attributable to the unique pathogenesis/ latency of BoHV-1. This finding necessitates mandatory culling of breeding animals which are positive either in antigen detection or by serology, especially in countries which do not practice vaccination but report high seroprevalance of BoHV-1.

A novel site-II directed glycoprotein estimation ELISA to aid rabies vaccine manufacture for veterinary and human use

Although the World Health Organization recommends the use of in vitro techniques to qualify rabies vaccine lot release, very limited proposals have been made to arrive at a harmonized approach for wide scale usage. The present study proposed and evaluated the use of a novel avidin–biotin ELISA as an alternative to these in vivo tests in rabies vaccine manufacture. This assay utilized a neutralizing pan reactive monoclonal antibody (mAb) reactive with the conserved site-II of the natively folded rabies glycoprotein. Linear regression analysis of the in vitro glycoprotein estimates with the in vivo potency values, showed a good correlation (r2=0.8) with veterinary vaccines, but a poor correlation (r2=0.2) with human vaccines. However, we could qualitatively arrive at cut-off glycoprotein estimates from the ELISA, above which all the vaccines were declared to be protective by mouse challenge studies (>2.5IU/dose).

Development of a probe based real time PCR assay for detection of bovine herpes virus-1 in semen and other clinical samples.

This study describes development of a TaqMan probe based real time PCR assay that can detect BoHV-1 of as low as 0.001 TCID50/0.1ml in clinical samples, its comparative evaluation with indirect ELISA and virus isolation for detection of Bovine herpes virus-1 (BoHV-1) in semen and swab clinical samples. For this study, we collected samples from 212 animals (cattle and buffaloes) comprising 91 bulls and 121 females. Avidin-biotin ELISA employed on serum samples from 212 animals revealed 74 as seropositive for BoHV-1. On inoculation of semen/swabs on MDBK cell line, nine samples yielded cytopathic changes characteristic of herpes viruses. The isolates were confirmed by VNT and a conventional PCR. A real time PCR assay was standardised by designing a new set of TaqMan probe and primers targeting a 71bp region on gB gene of the virus. The assay detected viral antigen in 21 seropositive and 14 seronegative animals, emphasizing the relevance of serology in BoHV-1 diagnosis, particularly in breeding stations. Further, real time PCR assay was 100% sensitive and 87.19% specific compared to virus isolation in detection of the BoHV-1 in clinical samples. The assay was validated at reputed national laboratories, with a sensitivity of ≥99%.

Clinical application of avidin-biotin ELISA to detect serum hepatoma-specific gamma-glutamyltransferase in patients with primary hepatic cancer

To detect serum hepatoma-specific datura stramonium lectin-tightly binding gamma-glutamyl transferase (DSA-GGT) in patients with primary hepatic cancer (PHC) by avidin-biotin ELISA method which was established in our laboratory, and carry on a study of its clinical application. To detect serum DSA-GGT in 45 healthy control subjects, 58 PHC patients and 203 non-PHC patients (including 36 patients with other tumors and 167 patients with benign liver diseases) with the method was established; meanwhile, AFP was detected by ELISA method. 38 individuals were DSA-GGT positive in 58 PHC patients, the sensitivity was 65.5%. 18 individuals were DSA-GGT positive in 203 patients without PHC, the specificity was 91.1%. The sensitivity and specificity of AFP in diagnosis of PHC patients was 69.0% and 90.6%, respectively. The sensitivity and specificity of combination of DSA-GGT and AFP was 93.1% and 85.7%, respectively. The average intra-CV and inter-CV of DSA-GGT ELISA was 8.9% and 11.5%, respectively. The sensitivity and specificity of DSA-GGT ELISA method established in our lab is similar with that of AFP assay and the accuracy is good. Combination of DSA-GGT and AFP may improve the diagnostic sensitivity. The method should be potentially as a new way to improve diagnosis of PHC.

A highly sensitive enzyme-linked immunosorbent assay for measurement of leptin secretion in human adipocytes

To establish a highly sensitive and specific ELISA method for measurement of leptin and further to study the secretion of leptin during human preadipocytes differentiation and effects of troglitazone. Rabbits Balb/c mice were immunized by recombinant human leptin and Balb/c mice were immunized by human leptin so as to produce rabbit anti-human leptin polyclonal antibodies (PAb) and mouse anti-human leptin monoclonal antibodies (MAb). Combination of the PAb as coating antibody, with a carefully paired biotinylated MAb as detector, and the avidin-horseradish peroxidase as the amplifier of detecting signals, a sandwich method, biotin-avidin ELISA (BA-ELISA) was established. Human omental preadipocytes were cultured, introduced to differentiate, and treated with 10 micromol/L troglitazone; the leptin secretion in the supernatant was detected by BA-ELISA. Peripheral blood samples were collected from 114 healthy persons and the serum leptin was detected by BA-ELISA. The sensitivity of BA-ELISA was 0.03 ng/ml with a working range of 0.05 - 5 ng/ml and a exogenous leptin recovery rate of 97.8%, and the intra- and interassay coefficients of variation (CVs) were less than 7.4% and 9.3% respectively. The assay detected only a single free leptin peak in gel chromatographic fractions from the mixed human sera or adipocytes culture media. The leptin secretion level detected by BA-ELISA showed that the leptin secretion of the preadipocytes increased strongly when the cells differentiated into mature adipocytes. The peak leptin secretion level of the troglitazone treated group was 2 times as that of the control group. The leptin concentration of women was than 7.6 ng/ml, significantly higher than that of the men (3.2 ng/ml, P < 0.001), and the serum leptin level was significantly correlated with body mass index both for men (r = 0.67, P < 0.001) and for women (r = 0.61, P < 0.001). A highly sensitive BA-ELISA specific for free leptin has been developed that is especially suited for the accurate measurement of the rather low leptin levels of clinical blood specimens and for basic research use.

Seroprevalence of brucellosis in yaks (Poephagus grunniens) in India and evaluation of protective immunity to S19 vaccine

The present study was carried out to explore the seroprevalence of brucellosis in yaks of North-Eastern hilly yak tracts of Arunachal Pradesh, India. Of 374 animals tested, 23.79, 21.11 and 18.98% were found positive for brucellosis using avidin-biotin ELISA (AB-ELISA), Rose-Bengal plate test (RBPT) and standard tube-agglutination test (STAT), respectively. The relative sensitivity and specificity for STAT were 79.77 and 100%, respectively and the same for RBPT were 88.76 and 100%, respectively in comparison to AB-ELISA. The alarming prevalence as recorded was highest among the yak cows (31.42%) followed by heifers (23.85%) and bulls (8.88%). The immune response in yaks following standard dose of calfhood vaccination with Brucella abortus strain 19 vaccine showed that protective antibody level persisted up to 210 days. This is the first report from India on prevalence of brucellosis and immunization with B abortus strain 19 vaccine in yaks. The present investigation would be a valuable guideline for future control measure and eradication programme of brucellosis in yaks.

Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression.

Introducing avidin-biotin complex ELISA for anti-DNA antibody, the mechanism of in vitro production of anti-ssDNA antibody as well as of polyclonal immunoglobulin mediated by an IL-6-IL-6R loop was studied in patients with systemic lupus erythematosus (SLE). Regardless of the presence or absence of T cells, B cells from SLE patients could produce IgG anti-ssDNA antibody as well as total IgG without any stimulation. Low density B cells obtained by Percoll gradient density centrifugation responded to rIL-6 to produce IgG and IgG anti-ssDNA antibody. rIL-2 and rIL-4 had lesser effects on the differentiation of low density B cells. In fact, IL-6R was preferentially expressed on low density B cells from active SLE patients, as detected by anti-IL-6R MoAb, MT18, which did not inhibit IL-6 binding. SLE B cells, especially high density B cells, produced greater amounts of IL-6 in culture supernatants than did T cells, regardless of whether disease was active or inactive. Normal T cells and B cells did not produce significant amounts of IL-6. Thus, endogenous IL-6 produced by high density B cells bound to the IL-6R preferentially expressed on the low density B cells, and drove them into terminal differentiation, especially in active SLE patients. Further, addition of polyclonal anti-IL-6 or anti-IL-6R MoAb (PM1), which inhibited IL-6 binding, both inhibited IgG anti-ssDNA antibody as well as total IgG production by SLE B cells in a dose-dependent manner. These results suggest that interruption of the autocrine IL-6 loop would be of therapeutic value in SLE.

IL-4 down-regulates the surface expression of CD5 on B cells and inhibits spontaneous immunoglobulin and IgM-rheumatoid factor production in patients with rheumatoid arthritis.

There is evidence to suggest that CD5+ B cells may be associated with autoimmunity, e.g. they are increased in patients with rheumatoid arthritis (RA). In this study, we found that the expression of CD5 on RA B cells increased spontaneously, following culture for up to 4 days in vitro in the absence of T cells, supporting the idea that the CD5+ B cell possesses distinctive features. The spontaneous increase of CD5 expression was down-regulated by recombinant IL-4 (rIL-4). Other cytokines studied (rIL-1 alpha, rIL-2, rIL-5, rIL-6) did not alter CD5 expression. Studies of antibody production showed that rIL-4 could reduce spontaneous production of total IgG and IgM in non-stimulated RA T plus B cell cultures. Spontaneous production of IgM rheumatoid factor (IgM-RF), measured by a newly developed avidin-biotin complex ELISA, was also reduced by rIL-4. Furthermore, rIL-4 reduced the increase in IgM-RF production observed on stimulation with Staphylococcus aureus Cowan I (SAC) or pokeweed mitogen (PWM). Thus, IL-4 might act as a regulator of the development of abnormal B cell differentiation in patients with RA.

Determination of natural olive fruit fly pheromone in insect samples by enzyme linked immunoassays

The olive fruit fly pheromone avidin–biotin ELISA immunoassay, based on the use of polyclonal G antibodies derived from rabbits (reported previously) and a newer assay, based on the use of polyclonal Y antibodies isolated from the eggs of laying hens (reported in this paper), were applied successfully for the analysis of natural pheromone in virgin adult female olive fruit flies. According to the results obtained, the pheromone content in the glands of adult female olive fruit flies increases from the third to the ninth day of their age. During the calling period, the female olive fruit flies seem to emit ∼1.1 μg pheromone/insect/day at least. The immunoassay, based on the Y antibodies, is slightly more sensitive (detection limit 40 ng/mL) than the assay based on polyclonal anti-pheromone rabbit antiserum (detection limit 80 ng/mL). As revealed by thorough cross-reactivity studies, including 14 structurally similar to the olive fruit fly pheromone molecules, the newer immunoassay is less selective than the previous one and seems to cross react with few molecules bearing the spiroketal moiety.

A severe episode of wildlife-derived scabies in domestic goats in Italy

A scabies outbreak, with unusually high mortality and morbidity, occurred in a poorly managed goat flock in north-eastern Italy. The source of infection was traced back to contact with scabietic chamois ( Rupicapra rupicapra) within a recognised sylvatic focus of the disease. Spill-over of scabies from chamois to goats is a matter of great concern to resource managers. Currently scabies-free populations of wild Caprinae in the central and western Alps may eventually be exposed to infected goats transported over long distances. Sanitation of the studied flock was obtained following three applications of pour-on moxidectin at an approximate dose of 500 mcg/kg. Follow-up included titration of circulating anti- Sarcoptes antibodies in a labelled avidin–biotin ELISA test. Two operators developed pseudoscabies after handling the infested flock.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specific Proteolysis in Barley Chloroplasts During Dark Induced Senescence

Intact chloroplasts were isolated from dark-senescing primary barley (Hordeum vulgare L.) leaves in order to study selective ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) degradation by the stromal and membrane fractions. RuBPCO specific degradation was estimated and characterised applying sensitive avidin-biotin ELISA method with non-modified or oxidatively modified biotinylated RuBPCO (BR) as substrates. Distinct proteolytic activities were detected. They differed in ATP and divalent metal ion dependence, protease inhibitory profile, and dynamics in the time-course of dark-induced senescence. The results supported involvement of ATP- and metal ion-dependent serine type proteolytic activity against non-modified BR early in induced senescence and appearance of ATP-independent activity at later stage. Active oxygen-modified BR was degraded by ATP-independent serine-type protease probably containing essential SH-groups and requiring divalent metal ions.

Study of serum Helicobacter pylori soluble antigen

Objective: to explore a new serological method for detectingHelicobacter pylori (H. pylori) infection. Methods: Serum soluble antigen ofH. pylori was detected by using avidin-biotin ELISA technique to evaluate the status ofH. pylori infection and for comparison with rapid urease test (RUT), histologic examination and serology. Results: The sensitivity, specificity, positive predictive value and negative predictive value were 77.46%, 91.07%, 91.67% and 76.12%, respectively. The prevalence rate of serumH. pylori soluble antigen in 138 patients undergoing endoscopy was similar to the rate obtained by14C-UBT methods (P>0.05). Conclusions: The detection of serumH. pylori soluble antigen(HpSAg) could be used as a new serological method which is accurate, and convenient, not affected by the memorizing reaction of serum antibody; is more sensitive, more specific and suitable for clinical diagnosis, and evaluation of eradication and for follow-up ofH. pylori as well as for detection in children and pregnant women.

Seroconversion and seroreactivity patterns of dairy goats naturally exposed to caprine arthritis-encephalitis virus in Brazil

A labelled avidin-biotin ELISA (lab-ELISA) using repeated serum samples of goats showed a progressive seroconversion with higher seroconversion rate at the period going from the beginning of the breeding up to the last half of lactation (35.0%), compared to that recorded at the beginning of breeding (17.8%)(p&lt;0.05). Furthermore, the seroreactivity pattern, evaluated by a lab-ELISA standard-curve with serum samples collected at 30-40 days intervals during 12 months, was caracterized by high individual variability. No seroreversion was observed and there were higher titers in the group of animals which delivered kids and established a lactation period (n=6; mean titre=913.4 units) compared to the group of goats that failed to conceived (n=4; mean titre=261.2 units) (p&lt;0.01).