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KRISHI: Technology Collections Developed by ICAR Institutes

General Part-1

Technology Code:- : 201538127945396
Organization Details...
Subject Matter Division : {{smdOb.smdName}}
Organization Name : {{orgOb.orgName}} ,{{orgOb.City}}
AICRP name if any (AICRP) : All Not Applicable {{aicrpOb.aicrpName}}
Details of Inventors..
Principal Inventor : Dr. Sandeep Bhatia
Principal Inventor Designation: : Principal Scientist
Principal Inventor Email :
Principal Inventor Address : Sandeep Bhatia Principal Scientist & I/c PME Cell ICAR-National Institute of High Security Animal Diseases (NIHSAD) Anand Nagar, Bhopal-462021. Phone Off: 0755-2754673; 2754674-76 ext. 340(lab), 304(office) Fax: 0755-2758842 Res: 0755-2529195, Mob: +91-9893893659
Co-Inventor Name : Dr, Atul Kumar Pateriya, Dr. Richa Sood, Dr. Naveen Kumar
Co-Inventor Email :,,
Technology Name : NIHSAD_Avian Influenza Antibody Detection 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: :
Avian influenza (AI), commonly known as Bird Flu has been responsible for huge economic losses to the poultry farmers in India through several outbreaks and culling of chickens. In this scenario, surveillance is one the best tool for risk assessment in the poultry population. Costly commercial kits are available but their high cost coupled with huge poultry population have largely hindered the surveillance of AI. Therefore, we have developed cost effective kit suitable in Indian conditions and could be employed for AI surveillance at large scale. The kit is effective in the detection of avian influenza A virus infection in chicken serum samples. The highly conserved nucleoprotein (NP) enables this kit to detect AIV antibodies irrespective of HA subtypes (H1-H16), with a high degree of accuracy. Basically, pre-coated recombinant NP on to the solid support (microplate) is made to react with test sample in this test. AIV specific antibodies, if present in the test serum sample bind to the NP and this antigen-antibody complex is detected by anti-Chicken HRP conjugate and visualized by the addition of substrate. The color development is directly proportional to the anti-AIV antibodies in the test serum sample. The validation of the assay has been carried out as per the OIE Manual for Antibody Detection Assays and various performance characteristics designate this kit as an ideal surveillance tool for detection of AIV infection in chicken at large scale implementation. Besides, this kit has several applications viz. (1) establish freedom from infection in a defined population of chickens, (2) assessment of risk analysis in non-vaccinated chickens, (3) determine the immune status in chickens post-vaccination, (4) could serve as DIVA test (Differentiation between Infected and Vaccinated Animals) especially in chickens immunized with HA, NA or M antigen based vaccines, and (5) monitor the influenza A virus infection in specific pathogen free (SPF) chickens. Production and characterization of recombinant NP The full length of NP gene (GenBank Acc. No. EF010524) was amplified from the Indian isolate of H5N1 (A/chicken/Jalgaon/India/8824/2006) virus and cloned in pET 28b+ expression vector. The expression of NP gene with polyhistidine-tag in E. coli BL21(DE3)pLysS cells was optimized in such a way that over-expression was achieved in soluble fraction of induced cell lysate, thus avoided the inclusion bodies formation which would otherwise have required complete denaturation of protein (chances of losing immune-dominant epitopes while refolding of denatured protein). The 6X His-tagged NP fusion protein in soluble fraction was purified by nickel affinity chromatography. Development and validation indirect ELISA Checkerboard titration was performed by varying the concentration of purified NP fusion protein and standard AIV positive and negative sera to determine the optimal concentration to be used in indirect ELISA. Analytical Specificity (ASp) and Sensitivity (Asn) For analytical specificity, the AIV Ab detection ELISA was tested for inclusivity and exclusivity criteria as per OIE norms. For inclusivity, a panel of OIE reference sera, received from National Veterinary Services Laboratory (NVSL), Ames, Iowa, USA (OIE Reference Laboratory for Avian Influenza) representing all AI haemagglutinin serotypes (H1 ? H16) were tested. The assay could detect antibodies against all the sub-types of avian influenza (H1-H16). For exclusivity, NDV specific serum was tested which the assay clearly differentiated from other avian influenza A viruse HA subtypes, suggesting that this assay is specific to avian influenza viruses and does not cross-react with related viruses such as NDV. In the case of Analytical Sensitivity (ASe), AIV Ab detection ELISA kit was compared with a reference test (AGID) by testing sera collected from experimentally infected SPF chickens with LPAI H9N2 virus starting from day 0 to 60 days post-infection (dpi). ELISA could detect the infection as early as 5th dpi, as compared to 6th dpi in AGID, clearly indicating the comparatively high sensitivity of the ELISA. Additionally, in-house ELISA was compared with commercially available type A Influenza Ab detection ELISA kit (Bionote) by testing the two-fold dilutions of an AIV positive sample spiked with negative serum. The in-house ELISA was equally sensitive to commercially available ELISA in its ability to detect the AIV specific antibodies up to 1:128 dilution of spiked positive serum. Diagnostic Sensitivity (DSe) and Specificity (DSp) A total of 715 sera samples collected from field, experimentally infected/vaccinated and SPF chickens, formed the basis for calculation of DSe and DSp. The status of each serum was cross-checked with reference tests (HI & AGID). A cut off value of the assay was determined by Receiver Operating Characteristic (ROC) curve analysis so that DSn and DSp were maximized while the sums of false negative and false positive results were minimized. Based on ROC, at 20% PPV cut off, DSp and Dse of the ELISA kit were 99% and 99.01%, respectively. Evaluation of assay Repeatability The AIV Ab detection kit was tested for repeatability using three replicates of eight samples which included four negative sera, two low positive sera, two strong positive sera. The intra- and inter-plate coefficient of variations (CV %) was in the range of 0.71? 8.12 and 6.14? 20.15, respectively, all of which were in the acceptable range. Inter- and Intra- lab performance validation The kit has been validated by a number of labs across the country (including NABL Accredited Lab of ICMR-NIV, Pune; ICAR-IVRI, Izatnagar; ICAR-NRCE, Hisar; NERDDL, Assam; SRDDL, Bangalore; WRDDL, Pune) on a panel of 25 sera (positive and negative AIV sera whose status was known) and agreement ratio/concordance was 100% at each time. Altogether, the kit composition provides a diagnostic solution based on qualitative detection of antibodies against nucleoprotein of avian influenza virus in chicken serum samples. Given that the kit detects antibodies to nucleoprotein which is highly conserved among all HA subtypes (H1-H16) of type A influenza viruses, the kit can be used for surveillance of type A influenza in chickens. The ELISA test can be performed by any laboratory personnel with ease and does not require intense training and costly instruments. The invention finds an important role in overall control of AIVs in the country since it can be applied for surveillance of avian influenza.
Brief Description of Technology Including Salient Features:
The Avian Influenza Antibody ELISA kit is an indirect Enzyme Linked Immunosorbent Assay for the qualitative detection of antibody against H1-H16 avian influenza virus (AIV) subtypes in chicken serum samples. The kit contains uncoated ELISA microplate in 8x12 strip format and the 100X recombinant nucleoprotein (NP) antigen which is to be coated on the wells before testing. All the unoccupied sites on the well are blocked by blocking buffer. For testing of field sera, ELISA plates coated with the Nucleoprotein (NP) antigen are incubated with test sera (1:100) for 60 minutes at 370C. During this incubation, AIV antibodies, if present in the test sample bind to the antigens in the well. Following this incubation, all unbounded antibodies are removed by washing. Anti-Chicken HRP conjugate detects these NP-bound antibodies and this binding is visualized by addition of the substrate solution. The color development in each well is directly proportional to the anti-AIV antibodies in the samples. The reaction is stopped by adding a stop solution and colorimetric reading is performed by using a spectrophotometer at 450nm. The highly conserved NP protein enables this test to detect AIV antibodies of all subtypes, with a high degree of accuracy. The unique features of this kit include (1) Devoid of infectious contents, thus eliminate the potential hazards associated with viruses (2) High diagnostic sensitivity and specificity, (3) No cross- reactivity with related viruses like NDV, (4) easy to perform and does not require intense training and costly instruments; (5) Time and cost effective (applicability as potential surveillance tool for implementation at a larger scale).
Benefits/Utility :
This indigenous kit is useful for the detection of avian influenza infection in chickens with high degree of accuracy. The test can be performed by any laboratory personnel with ease and does not require intense training and costly instruments. This kit provide a solution to expensive commercial ELISA kits, and thus could be used for large scale surveillance of avian influenza virus infection in chicken. Largely, this kit would serve as an important tool for cost effective monitoring and control of the avian flu disease in chickens.
Precaution With The Technology : The kit should be stored in the refrigerator (4oC).
How To Use :
Reagents Preparation: 1. Antigen (100X concentrated): Antigen should be diluted 1:99 in coating buffer. Mix well the 100 X antigen before use. 2. All the serum samples as well as the positive and negative controls should be diluted 1:100 in diluent cum blocker separately in the microtiter plate provided before addition to antigen coated wells. 3. Washing solution (10X concentrated): The washing solution must be diluted 1:9 with distilled/deionized water before use. i.e. add 50 ml of Washing solution to 450 ml of distilled water and mix well. 4. Conjugate solution (100X concentrated): The conjugate solution must be diluted 1:99 with diluent just before use every time. Procedure of the test: 1. Take out ELISA strips (8 well) as per the number of samples to be tested and fit in the frame provided. 2. Add 50 Ál of the antigen solution (1X) made in coating buffer and keep the strips covered with plate sealer at 37o C for 60 minutes. 3. Discard the contents of the wells and wash the wells once with 200 Ál of diluent cum blocker. Add 200 Ál diluents cum blocker in each well and keep the strips covered with plate sealer at 37o C for 60 min. 4. During the incubation time at step 3 above, dilute the positive control, negative control and the field/test samples (1:100) in diluents cum blocker in the microtiter plate provided. (Procedure for making these dilutions in the 96-well microtiter plate provided separately: Dispense 150 Ál of diluent cum blocker for each sample/ control in the wells separately and add 1.5 Ál of the sample/ control accordingly. Keep the plate at room temperature and wait till the incubation step at 3 is over.) 5. Discard the contents of the ELISA strips after incubation and add 50Ál of each of the diluted positive, negative control solutions and the test samples in duplicate (2 wells). For blank, add the diluent cum blocker (50Ál/well) in duplicate (2 wells). Incubate the strips covered with plate sealer at 37o C for 60 minutes. 6. Wash the wells with 300Ál of washing solution (1X) 4 times with holding time of 1 min at each wash. 7. Add 50 Ál of anti-chicken antibody-HRP (1X) to each wells and incubate at 37o C for 60 minutes. 8. Wash each of the wells with 300Ál of washing solution (1X) 3 times with holding time of 1 min at each wash. 9. Add 50 Ál of substrate (Ready to use) to each well and incubate the wells for 10 minutes at room temp. 10. Add 50 Ál of stopping solution to each well. 11. Read the absorbance of the wells with a spectrophotometer at 450nm wavelength. Interpretation of the Test: PP value calculation- Calculate the mean ODs of positive and negative controls, test sera and calculate the corrected mean ODs for the samples and the controls by subtracting the mean OD of blank wells. Corrected mean OD of test sample/ control= Mean OD of test sample/ control- Mean OD of blank Calculate the PP (Percent positive) value of each serum, using the formula- PP value= Corrected mean OD of the test sample X 100 Corrected mean OD the of positive control sera Based on the PP value, each test serum can be classified as positive, if PP value is &#8805; 20%. Test validation- The results are valid only if average OD450 value of the positive control is more than 1.00 and PP value of the negative control is less than <20%. In case, these values are out of range, this test should be considered invalid and the samples should be re-tested.
TargetUsers/Stake holders : Regional Disease Diagnostic Laboratories and State Disease Investigation Laboratories
Technology Contact..
Name : Director
Email :
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. Sandeep Bhatia
Email :
Phone No : 07552754673
Keyword for Technology : Avian influenza,indirect ELISA, Diagnostic kit

Technology Development Details Part-2

Project Details
(Through which technology was developed)
: Development of recombinant nucleoprotein based ELISA for Avian Influenza antibody detection under Consortia Research Platform on Vaccines & Diagnostics
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) : National Institute of Virology, Pune
Through Technology Transfer : YES

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