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NIHSAD_AVIAN INFLUENZA ANTIBODY DETECTION ELISA KIT
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).
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).
Benifits/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.
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.
Performance:
Advantage Over Existing Process/Product
The proposed kit is cheaper and technically more sensitive as per the limited data available from laboratory validation.
Advantage Over Existing Process/Product
The proposed kit is cheaper and technically more sensitive as per the limited data available from laboratory validation.
Impact
Cost-Benefit Ratio
Yet to be determined
Yet to be determined
Name and Address of Firm/entrepreneur to whom the Technology has been Transferred
Special facilities Required:
Precaution with 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 µl 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 µl of diluent cum blocker. Add 200 µl 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 µl of diluent cum blocker for each sample/ control in the wells separately and add 1.5 µl 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 50µl of each of the diluted positive, negative control solutions and the test samples in duplicate (2 wells). For blank, add the diluent cum blocker (50µl/well) in duplicate (2 wells). Incubate the strips covered with plate sealer at 37o C for 60 minutes. 6. Wash the wells with 300µl of washing solution (1X) 4 times with holding time of 1 min at each wash. 7. Add 50 µl of anti-chicken antibody-HRP (1X) to each wells and incubate at 37o C for 60 minutes. 8. Wash each of the wells with 300µl of washing solution (1X) 3 times with holding time of 1 min at each wash. 9. Add 50 µl of substrate (Ready to use) to each well and incubate the wells for 10 minutes at room temp. 10. Add 50 µl 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 ≥ 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.
Precaution with 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 µl 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 µl of diluent cum blocker. Add 200 µl 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 µl of diluent cum blocker for each sample/ control in the wells separately and add 1.5 µl 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 50µl of each of the diluted positive, negative control solutions and the test samples in duplicate (2 wells). For blank, add the diluent cum blocker (50µl/well) in duplicate (2 wells). Incubate the strips covered with plate sealer at 37o C for 60 minutes. 6. Wash the wells with 300µl of washing solution (1X) 4 times with holding time of 1 min at each wash. 7. Add 50 µl of anti-chicken antibody-HRP (1X) to each wells and incubate at 37o C for 60 minutes. 8. Wash each of the wells with 300µl of washing solution (1X) 3 times with holding time of 1 min at each wash. 9. Add 50 µl of substrate (Ready to use) to each well and incubate the wells for 10 minutes at room temp. 10. Add 50 µl 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 ≥ 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.
Contact Details
Director,
ICAR-National Institute of High Security Animal Diseases,Anand Nagar, Bhopal-462021.,Bhopal-462021
Phone No.:0755-2759204, Fax No.:0755-2758842, E-mail:director.nihsad@icar.gov.in
Director,
ICAR-National Institute of High Security Animal Diseases,Anand Nagar, Bhopal-462021.,Bhopal-462021
Phone No.:0755-2759204, Fax No.:0755-2758842, E-mail:director.nihsad@icar.gov.in
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Indian Council of Agricultural Research
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Disclaimer
© 2018 All Rights Reserved •
Indian Council of Agricultural Research
Krishi Bhavan, Dr. Rajendra Prasad Road, New Delhi-110 001. INDIA