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KRISHI: Technology Collections Developed by ICAR Institutes

General Part-1



Technology Code:- : 201540287510319
Organization Details...
Subject Matter Division : {{smdOb.smdName}}
Organization Name : {{orgOb.orgName}} ,{{orgOb.City}}
Details of Inventors..
Principal Inventor : Dr. K. Rajukumar
Principal Inventor Designation: : Principal Scientist
Principal Inventor Email : K.Rajukumar@icar.gov.in
Principal Inventor Address : ICAR-National Institute of High Security Animal Diseases, Hathai Kheda Road, Anand Nagar, Bhopal - 462022 Madhya Pradesh
Co-Inventor Name : Dr. Senthil Kumar, D., Dr. Kulkarni, D.D.
Co-Inventor Email : Senthil.D@icar.gov.in,ddkulkar@gmail.com
Technology Name : NIHSAD_Porcine Reproductive and Respiratory Syndrome Antibody ELISA kit
Technology Details..
Major resource : All Not Applicable {{majorOb.majreName}}
Minor Subject Classification : All Not Applicable {{minorOb.minorName}}
Minor Subject Sub Classification : All Not Applicable {{minorOb.minorsubName}}
Technology Group : All Not Applicable {{techgroup.tecg_group_name}}
Technology Related To : All Not Applicable {{techrela.tr_name}}
Complete Details of Technology: :
Porcine Reproductive and Respiratory Syndrome (PRRS) is an economically important viral disease of pigs, characterized by reproductive problems in breeding animals and respiratory disease in pigs of any age. In India, PRRS outbreaks have been occurring in the Northeast India, especially in Mizoram since 2013 and has resulted in death of thousands of pigs, causing huge economic loss to the farmers. Hence, serosurveillance is essential to monitor the disease situation in India. Although commercial kits are available for serodiagnosis of PRRS, their high cost has hindered the surveillance of PRRS. We have developed a cost effective ELISA kit employing antigens suitable for Indian conditions and capable of detecting a wide range of PRRS strains for large scale sero-surveillance. The NIHSAD-PRRS antibody ELISA is a qualitative, recombinant antigen based indirect ELISA for detection of antibodies against PRRS virus in porcine serum samples. The kit contains 8-well strips of ELISA microplate coated with an improvised recombinant antigen mix prepared from PRRSV genotype 1, classical genotype 2 and the Indian PRRSV which is a highly pathogenic variant of genotype 2 (HPPRRS). Due to the presence of mixture of three different antigens, the assay is expected to have a wider antibody coverage against most of the existing PRRS viruses. When incubated with test sera, PRRSV specific antibodies, if present, react with the coated antigen. The antigen-antibody complex formed is detected through a colorimetric reaction. The intensity of colour developed is proportional to the level of anti-PRRSV antibodies present in the serum. The results can be interpreted based on the absorbance (optical density) read at 450 nm (A450) in an ELISA plate reader. Thus, the purpose of NIHSAD-PRRS antibody ELISA is to detect antibodies against PRRS virus in serum samples from domestic pigs. Recombinant N proteins were expressed from plasmid clones containing nucleocapsid gene of PRRSV after induction with IPTG. The proteins from cell pellets were purified using BugBuster protein extraction reagent (Novagen) and HisBind purification kit (MerckMillipore). Expression of the proteins was confirmed by SDS-PAGE and Western blot analysis using anti-PRRSV serum. A mixture of the three purified proteins was used in the ELISA for detection of PRRSV antibodies in porcine sera. The test was optimized using reference serum samples obtained from Poland and evaluated using 360 field serum samples. TG-ROC analysis for recombinant nucleocapsid protein based indirect ELISA developed at NIHSAD was carried out to determine the cut-off SP value. The assay had a sensitivity of 97.5% and a specificity is 93.5% at a cut-off of 0.33. Analytical sensitivity of the assay was found to be 1:4800 using rN hyper immune serum and earliest detection in serum of experimentally infected pigs was on 6th dpi. Analytical specificity was tested and no cross reactivity was observed with CSFV, PRCV, TGEV, PEDV, PRV antibodies. Inter- and Intra-lab performance validation Intra-institutional validation was carried out in three different laboratories of ICAR-NIHSAD, Bhopal, namely, the Immunology Lab, Diagnostic-1 laboratory and Diagnostic-2 laboratory. The agreement ratio was found to be 96.2%. Further, the inter-institutional validation of the kit was carried out at three different laboratories in the NE region, viz, ICAR research complex for NEH region, Barapani, Meghalaya, ICAR- National Research Centre on Pigs, Guwahati and NE-RDDL, Guwahati on a panel of 26 sera (known PRRSV antibody positive and negative sera) and agreement ratio/concordance was 100% in each of the labs. Altogether, the kit composition provides a diagnostic solution based on qualitative detection of antibodies against nucleocapsid protein of PRRS virus in domestic pig serum samples. The ELISA test can be performed by any laboratory personnel with ease and does not require intense training and costly instruments. This ELISA kit will help in cost effective surveillance and monitoring of PRRS in the country, and will aid in the PRRS control programme.
Brief Description of Technology Including Salient Features:
The NIHSAD-PRRS antibody ELISA kit is an indirect Enzyme Linked Immunosorbent Assay for the qualitative detection of antibody against Porcine Reproductive and Respiratory Syndrome (PRRS) virus antibodies in domestic pig serum samples. The kit contains 8-well strips of ELISA microplate coated with an improvised recombinant antigen mix prepared from PRRSV genotype 1, classical genotype 2 and the Indian PRRSV which is a highly pathogenic variant of genotype 2 (HPPRRS). When incubated with test sera at appropriate dilution, PRRS virus specific antibodies, if present, will react with the coated antigen. The antigen-antibody complex formed is detected through immunodetection by anti-pig IgG HRPO enzyme and a colorimetric reaction using the substrate solution. The intensity of colour developed is directly proportional to the level of anti-PRRSV antibodies present in the serum. The reaction can be stopped by adding the stop solution provided in the kit and the results can be interpreted based on the absorbance (optical density) read at 450 nm (A450) in an ELISA plate reader. The salient features of the technology include ? Due to the presence of mixture of three different antigens, the assay is expected to have a wider antibody coverage against most of the existing PRRS viruses. ? As the kit employs recombinant antigen mix, potential hazards associated with handling infectious agents is eliminated. ? The test is easy to perform and require minimal training. ? No cross- reactivity with related swine viruses like CSFV, PRCV, TGEV, PEDV, PRV, etc.
Benefits/Utility :
This indigenously developed PRRS antibody ELISA kit is intended to detect antibodies against PRRS virus in serum samples from domestic pigs with high degree of accuracy. As the cost is expected to be less, this kit can act as an import substitute for expensive commercial ELISA kits used for PRRS diagnosis. The assay can be easily carried out in peripheral diagnostic laboratories for PRRS antibody detection in outbreak investigation and post-outbreak surveillance and monitoring of PRRS. Thus it will aid in the PRRS control programme in India.
Precaution With The Technology : The kit should be stored at 40C in the refrigerator. The manual should be read thoroughly prior to use and the given instructions should be followed properly during the assay.
How To Use :
User manual is provided along with the kit. Preparation of reagents 1. Wash Buffer: Prepare required volume of 1x Wash Buffer. Dilute 1 part of 10x Wash Buffer provided in 9 parts of double distilled water (Add 270 ml of double distilled water to 30 ml of 10x Wash Buffer for a full ELISA plate of 96 wells). Scale down if lesser number of strips are used. 2. Conjugate: The provided conjugate is 100x. Dilute 1 part of provided stock in 99 parts of dilution buffer. (Add 50 Ál of 100x conjugate to 4950 Ál of dilution buffer for a full ELISA plate of 96 wells). Scale down if lesser number of strips are used. 3. Control and Test serum samples It is preferable to clarify all the serum samples by centrifugation at 3000 rpm for 5 minutes. Use all test serum samples as well as the kit controls at a dilution of 1:100 in the 1x dilution buffer provided (by diluting 2 Ál with 198Ál of dilution buffer). A 96-well V-bottom microtitre plate is provided for dilution purpose. Use separate micropipette tip for each serum. Diluted samples must be thoroughly mixed prior to dispensing into the coated ELISA strips. Test Procedure (All reagents should be used after bringing to room temperature) ? Take out the required number of antigen coated ELISA strips from the zip-lock bag and bring to room temperature. (The remaining strips must be placed back in the zip-lock bag and stored at 4░C for future use). ? Dispense 300 Ál of the 1x Wash Buffer to each well. Leave for 3 minutes. ? Discard the liquid by inverting. Drain the remaining liquid by inverting and tapping over paper towel. ? Add 50Ál of diluted test and control serum samples (Refer instructions above) to the corresponding wells in duplicates. Ensure proper spreading of sera in the wells. ? Incubate at 37░C for 1 hour. ? Empty the wells and wash. ? Washing: Dispense 300 Ál of 1x Wash Buffer in to all the wells. Leave for 2 min with intermittent shaking. Discard the liquid by inverting. Drain the remaining liquid by inverting and tapping over paper towel. Repeat the washing two more times. ? Add 50 Ál of diluted (1x) conjugate solution. ? Incubate at room temperature (20 to 27 oC) for 75 minutes (▒ 5 minutes). ? Empty the wells. ? Wash the wells as described above using 1x Wash Buffer four times. ? Dispense 50Ál of Substrate Solution in to each well and incubate in dark for 5 to 7 min at room temperature. ? Stop the colour development by adding 50Ál of Stop Solution to each well. ? Measure absorbance at 450 nm (A450) in an ELISA plate reader. Calculation of S/P value Calculate the S/P value of each test serum as given below S/P value of test sample = (Mean A450 of test serum ? Mean A450 of negative control) (Mean A450 of positive control - A450 of negative serum) Validity criteria ? The mean A450 of the kit positive control should be at least twice that of kit negative control Interpretation of result S/P value more than 0.33  Test serum is positive S/P value less than 0.33  Test serum is negative
TargetUsers/Stake holders : ? Regional and Peripheral veterinary disease diagnosis laboratories ? Research and academic institutions ? Field/farm veterinarians
Technology Contact..
Name : Director
Email : director.nihsad@icar.gov.in
Phone Number : 0755-2759204
Fax Number : 0755-2758842
Address : ICAR-National Institute of High Security Animal Diseases,Anand Nagar, Bhopal-462021.,Bhopal-462021
Alternate Contact..
Name : Dr. K. Rajukumar
Email : K.Rajukumar@icar.gov.in
Phone No : 07552754675
Keyword for Technology : PRRS, PRRS virus, antibody detection, ELISA, pigs


Technology Development Details Part-2

Project Details
(Through which technology was developed)
: 1. Development of enzyme immunoassay and nucleic acid based diagnostic tests for the detection of porcine reproductive and respiratory syndrome in pigs (Institute funded project) 2. Deployment of nucleic acid and ELISA based diagnostic tests to determine incidence of porcine reproductive and respiratory syndrome (PRRS) in the North East and evaluation of the prospects of a potential candidate vaccine against the disease (DBT twinning project in collaboration with ICAR-NEH region, Barapani)
Technology Validated by : OutSide ICAR
Technology Validation Details..
Subject Matter Division : {{smdOb.smdName}}
Organization Name(if within ICAR) : ICAR-National Institute of High Security Animal Diseases,Bhopal
Organization Name(if outside ICAR,Please enter) : North Eastern Regional Disease Diagnostic Laboratory, Guwahati, Assam.
Through Technology Transfer : YES


Applies To(Regional Differentiation)Inform Part-3

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