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Recombinant antigen based sero-diagnosis of Infectious Bursal Disease by indirect ELISA
Infectious bursal disease is an economically important disease of poultry causing immunosuppression and predisposing them to other infections.
Serological diagnosis is commonly performed with agar gel immuno diffusion (AGID) test, virus neutralization (VN) test or enzyme linked immunosorbent
assay (ELISA). AGID test is the simplest assay, but its sensitivity and specificity may vary from laboratory to laboratory and moreover it is time consuming.
VN has the highest specificity and VN antibody test correlates with pro tection. But as VN is labour intensive, and requires the facilities of virology laboratory
and as it provides delayed results, it is unpractical for routine use. Technology Details Recombinant antigen-based diagnostics are a new class of diagnostic tools
which could replace the conventional methods based on whole cell antigens. This ensures that there is no risk involved for both the producer and the user in handling
of live organisms. The recombinant antigen developed in this technology is specific for all the strains of infectious bursal disease virus. Moreover, it is useful to
assess the immune status of vaccinated birds and management of large flocks. The assay can monitor the efficacy of live and killed IBD vaccines. The kit is available
in the form of recombinant antigen coated ELISA plates with a shelf life of 6 months under refrigerated condition. Patent Number 277404 for the recombinant antigen that
was used to develop a single serum dilution ELISA for the serodiagnosis of IBD
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Recombinant antigen based diagnostic kit for sero-diagnosis of Newcastle disease by single serum dilution ELISA
Newcastle disease (ND) is a highly contagious viral disease caused by NDV affecting almost all species of birds of different age groups worldwide. Depending upon the pathotype involved and the susceptibility of the flock, the virus causes respiratory diseases, drop in egg production and mass mortality in endemic areas. Conventional
detection of the disease is slow, laborious and require an undesirable use of in-vivo techniques. Hence, a recombinant antigen based single serum dilution ELISA has been desig ned to measure the relative level of antibody to Newcastle disease virus (NDV) in chicken serum. Technology Details The assay is useful to assess the immune status of vaccinated birds and management of large flocks. It can monitor the efficacy of live and killed vaccines. The innovation has the following advantages: Recombinant antigen based single serum dilution ELISA reduces the reagent costs and increases the number of samples to be screened. Has excellent diagnostic specificity and sensitivity. A total of 45 sera samples in duplicate can be analyzed and their antibody titres quantified with sufficient accuracy in a single microtiter plate, whereas in serial dilution ELISA only 8 – 10 samples can be analyzed. The recombinant antigen is non-infectious and free from risk of bio-hazard. The test is very specific as it avoids cross reactivity among related proteins. The recombinant antigen is specific for all pathotypes of the virus. The kit is available in the form of recombinant antigen coated ELISA plates with a shelf life of 6 months under refrigerated condition. A patent application (No. 628/DEL/2012) has been granted.
(Patent Number :325899)
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Blue tongue sandwich ELISA kit for antigen detection
Bluetongue, caused by the bluetongue virus (BTV), is a disease of sheep and characterized by high fever, excessive salivation, swelling of the face and tongue and cyanosis of the tongue. The virus can infect all ruminants.
The virus has 27 serotypes and is transmitted by the vectors of Culicoides sp. Detection of virus is conventionally done by virus isolation, serum neutralization, electropherotyping of viral genome, demonstration of viral nucleic acid by RT-PCR and detection of viral antigen in the clinical samples. A sandwich ELISA (s-ELISA) has been developed for detection of BTV antigen in blood, tissue and culture fluid and observed advantageous over the conventional methods. It is rapid, cost effective, easy to perform and large number of the samples can be tested at the same time. The kit has immense market potential and useful for Disease Investigation Laboratories and Research organizations.
The salient feature of the kit is that it can detect BTV or group specific antigens (BTV 1, 2, 3, 9, 15, 16, 21, 23) in blood, tissue or culture supernatant in about 51/2 hours with a virus detection limit of 102.4 TCID50/ml.
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Synthetic peptide based antigen capture ELISA for detecting animal rotaviruses
Rotavirus is a leading cause of diarrhea in both humans and animals worldwide where group A rotaviruses are responsible for approximately one-fourth of global mortality in young ones annually. Indian scenario in terms of animal mortality caused by rotavirus has not been testified so far due to the non-availability of economic diagnostic tools.
The rapid and accurate diagnosis of rotavirus remains a major hurdle in rotavirus surveillance studies owing to the cost involved in commercially available diagnostic kits. Therefore, a rapid, accurate and cost-effective diagnostic assay for rotavirus to meet the present demand of the livestock industry, is in great need. This invention relates to the development of antigen c apture ELISA for rotavirus antigen detection in faecal samples of different animal species. Mono-specific antibodies against the VP6 protein of bovine rotavirus are highly specific. The novel combination of anti-VP6 antibodies as coating and anti-multiple antigenic synthetic peptide (MAP) antibodies as detector antibodies identify the rotavirus antigen with high specificity and sensitivity. Therefore, makes this method a more useful diagnostic tool for screening a large number of samples in standard immunoassay procedures. The major solutions with this invention are: An indigenously developed kit for detecting rotavirus infection in animal species is made available with this invention. It is safe, as devoid of handling infectious material, the live virus. Possess high sensitivity so detecting rotavirus infection at even early stages of establishment. Applicable in multiple species (Bovine, Sheep, Goat, Porcine, and Poultry), thus a single kit will work for all species. Highly specific as no cross-reactivity with other enteric viruses. Time and cost-effective making applicability for its use as screening test and eliminates need of importing ELISA kits which are costly.
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Recombinant NS1 protein based indirect IgG ELISA for sero-diagnosis of Japanese Encephalitis in swine and kit there of.
Japanese encephalitis (JE) is a mosquito-borne viral zoonotic disease due to which hundreds of children die every year in India. Swine act as sentinel animal for control of disease in humans.
The present invention refers to development of an ELISA for diagnosis of Japanese encephalitis in swine using a recombinant non-structural protein 1 (NS1). The described invention is unique and novel as other ELISA available in the market either employ whole JE virus (JEV) antigen or Envelope antigen, both of which suffer from specific limitations. The preparation of whole JEV antigen involves risk of exposure to live virus. On the other hand, recombinant envelope protein possesses cross reacting epitopes which results in decreased specificity of the assay. Technology Details An indigenously manufacture kit for sero-monitoring of JE in swine is made available with this invention. Handling live JEV is not required. Crossreaction with other closely related Flavivirus is eliminated. No need of importing ELISA kits which are costly. This indigenously developed ELISA kit can be readily in-cooperated into surveillance program which in turn will save the life of several children. Patent has been applied in Indian Patent office (Application No.201611024016 A). Date of filing: 13/7/2016.
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Japanese Encephalitis - IgM ELISA kit for swine
Japanese encephalitis virus (JEV) is one of the major causes of encephalitis in children and it is estimated that annually JEV causes 50,000-67,900 cases leading to 10,000-20,000 deaths globally. Increasing global population, novel agricultural practices and increasing global temperature have facilitated the perpetuation of JE vector, Culex spp., thereby, increasing the human JE cases.
Swine, being an amplifier host of JEV, plays an important role in its epidemiology. Therefore, early detection of either JEV or antibo dies against JEV in swine is a feasible alternative for initiating necessary measures to prevent the spread of infection to humans. However, since the viraemia lasts for a brief period of 2-3 days, the detection of JEV in swine is not always successful, and hence, the detection of antibodies against JEV using ELISA is the best alternative. Technology Details: Earlier, IgG ELISA kit for sero-surveillance of JE in swine was developed by ICAR-IVRI and has been extensively used to screen the swine serum samples from various parts of our country. Though the IgG ELISA is good for surveillance but by using this kit we cannot estimate that JE virus infection is active or not in the swine population of the area under surveillance as IgG antibodies survive for several months in swine serum unlike IgM antibodies which survive for less than one month. Moreover, none of the JE IgM ELISA kits for swine are manufactured indigenously and the high cost of the imported kits is a huge economical burden, especially for developing countries. The kit can be readily incorporated in the JE control program of the Govt. of India for surveillance of active JE virus infection in swine population to forecast JE disease in humans. The kit developed by ICAR-IVRI is recombinant protein-based and does not involve the handling of live JE virus Our kit is cost-effective as compared to existing commercial kits which are being imported.
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Synthetic peptide antigen for diagnosis of Peste Des Petits Ruminants (PPR)
The invention relates to synthetic peptide mixotope antigen comprising peptides from different immunodominant regions identified in proteins of peste des petits ruminants (PPR) virus, which specifically react immunochemically with antibodies to PPR virus.
The mixotope antigen can react with different antibodies present in the polyclonal sera to generate strong signals in immunoassays. This antigen can successfully be used for improving diagnostic methods by reducing the variability and high backgrounds associated with methods that rely on use of whole virus or recombinant antigens for sero-diagnosis of PPR. The synthetic peptide antigen is safe, non-infectious and non-hazardous and very stable at room temperature; and as coating antigen for visual immuno dot assay on polystyrene comb. The results could be obtained in less than 30 minutes without the need of any instrument for interpretation of results. Therefore, the test can provide rapid sero-diagnosis of PPR at the door-step of livestock owners. In a limited study on 840 goat serum samples, the immuno dot assay could detect PPR virus specific antibodies in test sera samples with an accuracy of 97%, at the same time, it was found to be 98% sensitive and 96.5% specific in comparison to c-ELISA for detection of serum antibodies against PPR virus.
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Multiple antigenic peptide assay for detection of Peste Des Petits Ruminants (PPR) virus specific antibodies.
The invention relates to synthetic multiple antigenic peptides (MAPs) mixotope reagent for detecting antibodies to peste des petits ruminants (PPR) virus in the test sera. The invention also relates to a method for detecting and quantitating antibodies to PPR virus in an indirect-ELISA, wherein, the MAP mixotope reagent is used as coating antigen. The assay using MAP antigen specifically detects antibodies to PPR virus. The novel diagnostic method using MAP antigen has its application in sero-monitoring of PPR in target population. The MAP assay can suitably be used for quantitative assessment of humoral immune response in the vaccinated animals and also for diagnosis of PPR in naturally infected animals, if paired sera samples are available. In a limited experimental analysis comprising 480 goat serum samples, the MAP-ELISA detected antibodies to PPR virus with sensitivity of 98.3% and specificity 97.5% in comparison to c-ELISA. The MAP antigen developed in present invention is safe, homogenous, non-infectious, non-hazardous and stable at room temperature, therefore, it is aptly suited for serological surveys in different geographical and climatic situations.
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Recombinant Antigen Based Sero-diagnosis of Infectious Bronchitis.
This assay is designed to measure the relative levels of antibody to IBV in chicken serum. A micro titration format has been developed in which a recombinant protein derived from IBV is coated onto each well. The antibody specific to IBV forms a complex with the coated virus antigen.
After washing away unbound materials from the wells, anti-chicken horseradish peroxidase (HRP) is added and colour development occurs in presence of substrate. The assay is useful to assess the immune status of vaccinated birds and the management of large flocks. The innovation has the following advantages. Recombinant antigen-based single serum dilution ELISA reduces the reagent costs and increases the number of samples to be screened. Has excellent diagnostic specificity and sensitivity. A total of 45 sera samples in duplicate can be analyzed and their antibody titres quantified with sufficient accuracy in a single microtitre plate, whereas in serial dilution ELISA only 8– 10 samples can be analyzed. The recombinant antigen is non-infectious and free from the risk of bio-hazard. The test is very specific as it avoids cross-reactivity among related proteins. The recombinant antigen is specific for all pathotypes of the virus. The kit is available in the form of recombinant antigen-coated ELISA plates with a shelf life of 6 months under refrigerated conditions.
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IVRI M PPRV Antigen Capture ELISA Kit
Government of India has recently launched a Peste des Petits Ruminants Control Programme (PPR-CP). The PPR control programme involves intensive vaccination of all susceptible goats and sheep and three subsequent generations. Although the currently used sandwich ELISA kit is a successful technology and got well accepted across India, it has two major issues namely a) it employs whole live virus as antigen in the control panel and the production of the whole virus-based control antigen is a cumbersome procedure. Further, the control programme in India is in the mid-way of PPR eradication, use of live virus particles in diagnostic kits will not be allowed after acquiring the PPR free status. b) The kit uses a lower sensitive heterologus capture antibody in the assay which needs to be replaced with a more sensitive homologus capture antibody. To solve the above problems, we have developed the IVRI-M PPRV Antigen capture ELISA kit. The following are the solution we offered Whole virus antigen has been replaced with recombinant N antigen thereby completely eliminating the need to handle live virus at any stage from production till end use. The heterologous capture antibody has been replaced with a more sensitive homologus capture antibody.
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Monoclonal antibody-based blocking ELISA for FMD: A Highly Sensitive Assay for Detection of FMD Non-Structural Protein (NSP) Antibodies in All Species.
In India, non-structural protein (NSP) based serological tests have been developed by both ICAR-DFMD, Mukteswar (3AB indirect ELISA, Mohapatra et al., 2014) and ICAR-IVRI (3ABC multi-species indirect ELISA, Hosamani et al., 2015). Indirect ELISA developed by ICAR-DFMD uses species specific conjugates and need optimization with each species conjugate and also conjugate lots supplied by the vendors. The multi-species 3ABC iELISA developed earlier by ICAR-IVRI employs protein G-HRPO conjugate, as Protein G has affinity for IgG from different species. This test is usable in many susceptible species but not in some species such as pigs and wild animals. In view of these short falls with indirect ELISA formats, there is need to develop an improved NSP assay system that can be used across all the susceptible species including wild animals. Use of the new blocking ELISA developed here is based on the monoclonal antibody that could serve as a single platform system to assay the serum samples from all animals including wild animals that are susceptible to FMD. The assay is inexpensive to produce as it does not depend on commercial conjugated antibodies. NSP antibody detection ELISA currently used in India is based on indirect ELISA that needs species specific conjugate, and needs to be optimized for each animal species. The commercially available conjugates are expensive and their application in ELISA needs to be standardized for each lot of conjugate supplies. Thus, it does not offer a single assay system for all species of animals and not usable for samples from wild animals. To overcome these limitations of indirect ELISA, ICAR-IVRI has developed a monoclonal antibody based blocking ELISA that can be used in all species. The newly developed blocking ELISA uses recombinant antigen produced in insect cells. The diagnostic sensitivity and specificity of the assay was 93.7% and 99.8%, respectively at 50% inhibition value as the cut-off. The assay is validated at different laboratories and at different institutes. The assay was also validated in an international laboratory (Wageningen Bioveterinary Research Lelystad, Netherlands). The assay performance was highly comparable to a similar commercial test (PrioCHECK FMD NS test) based on the testing a panel of sera (n=2434) available in Netherlands laboratory. The test is performed on samples from different species including cattle, buffalo, sheep, goats, Mithun and Yak and wild animal sera. It is validated with sera from animals infected with all the seven serotypes of FMDV.
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Competitive ELISA Kit for Detection Of Antibodies to Bluetongue Virus
Bluetongue (BT) is one of the most important and OIE ‘notifiable’ multiple species viral diseases of global significance due to its economic impact and embargo on animal trade. It is an arthropod transmitted haemorrhagic infectious viral disease of wild and domestic ruminants including the camelid species. The disease is enzootic in India and wide sero-prevalence is reported in sheep, goats, cattle, buffaloes and camels. A large number of sheep get affected and die of disease every year. Control of bluetongue is attempted with vaccination of susceptible animals, vector control and quarantine of infected animals with good management practices. Apart from vaccination, early diagnosis and isolation of the infected animals is the commonly suggested practice for control of the disease. Bluetongue laboratory of IVRI Mukteswar campus has developed the bluetongue competitive ELISA (cELISA) kit for detection of antibodies to bluetongue virus. Indirect ELISA (iELISA) kits are available for detection of antibodies to BTV in sheep and goats. However, requirement of the species-specific secondary antibody conjugate in an iELISA is the major hurdle for sero-diagnosis of a multiple species disease like BT. The cELISA kit has been developed by using recombinant antigen and polyclonal antibodies to the virus core antigen. The cELISA is suitable for sero-diagnosis and sero-epidemiological studies of BT in species independent manner. The relative diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the cELISA kit is 97.7% and 97.2%, respectively. The kit has been validated at different laboratories within the institute and at different other institutions. The kit is routinely being used in the laboratory and more than 2600 samples from sheep, goat, cattle, buffalo and camel were screened using the cELISA kit. The kit do not contains any live pathogen and is a suitable alternate of the commercially available kits.
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Competitive-ELISA kit for PPR antibody detection
Peste des petits ruminants (PPR) also known as goat plague is an OIE list A disease and characterized by fever, oculo-nasal discharge, and necrotic ulceration in the oral mucosa, pneumonia, diarrhea, dehydration and death. A monoclonal antibody (Hybridoma clone 4B11) based competitive-ELISA kit for PPR antibody detection (sero-surveillance and sero-monitoring) has been developed.
The kit includes critical reagents such as antigen, different level of antibody controls (negative, weak positive, strong positive), monoclonal antibody specific to PPR virus. Several of these components are byproducts of PPR vaccine production/ manufacturing units. The kit has profound export potential especially to Asian countries, Middle East and African Continent, as 0.1% animals need to be screened during sero-surveillance and sero-monitoring in view of FAO/ OIE Global PPR Eradication Programme by the year 2030. This kit is many folds cheaper than equivalent kit produced abroad. Such kit will be in high demand for the next 15-20 years for Global PPR Eradication Programme in line with Global Rinderpest Eradication.
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Recombinant antigen (VP7 protein) based indirect ELISA Blue Tongue antibody detection kit.
Bluetongue, caused by bluetongue virus (BTV), is a disease of sheep and characterized by high fever, excessive salivation, swelling of the face and tongue and cyanosis of the tongue.
The virus can infect all ruminants. The virus has 27 serotypes and is transmited by the vectors of Culicoides sp. The disease is diagnosed by detection of antibodies against the group-specific antigen VP7. In indirect or competitive ELISA, the whole BTV is used as antigen to which the group-specific antibodies bind. In India, BTV sero-surveillance or antibody detection is mainly done by imported kits, which are costly with limited shelf life.