Chicken Infectious Bronchitis Virus Antibodies ELISA Kit

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Test Principle

This kit is comprised by HRP conjugate, other reagents and ELISA Microtiter plate pre-coated with recombinant chicken infectious bronchitis Virus (IBV) N proteins. Apply the principle of enzyme-linked immunoassay (ELISA) to detect IBV-Ab in serum, plasma of chicken. During the experiment, add control and samples into the ELISA Microtiter plate, IBV-Ab will be bound with the antigen on the ELISA Microtiter plate. Then wash the plate to remove unbound components, horseradish peroxidase (HRP) conjugate is added to each ELISA Microtiter plate well. The unbound HRP Conjugate will be removed by washing and substrate reagent is added for color development. At last, end the reaction by adding Stop Solution to produce a yellow product. There is a positive correlation between the OD value of samples and the concentration of IBV-Ab. Measure the absorbance value of each well by using a microplate reader with 450 nm (630 nm) wavelength, then we can judge whether IBV antibody exist in the sample.

Kit Components

Item Specifications

ELISA Microtiter plate

96 wells

Dilution plate

86 wells

HRP Conjugate

11 mL

Sample Diluent

50 mL

20×Concentrated Wash Buffer

40 mL

Substrate Reagent A

6 mL

Substrate Reagent B

6 mL

Stop Solution

6 mL

Positive Control

1 mL

Negative Control

1 mL

Plate Sealer

3 pieces

Sealed Bag

1 piece


1 copy


All reagent bottle caps must be tightened to prevent evaporation and microbial pollution

Experimental Instrument

  • Microplate Reader with 450 nm wavelength filter or dual-wavelength (450/630 nm)
  • High-precision transferpettor, EP tubes and disposable pipette tips
  • 37℃ incubator or water bath
  • Deionized or distilled water
  • Absorbent paper

Sample Preparation

1. Use the conventional method to prepare animal serum/plasma, the serum/plasma must be clear, no hemolysis and no pollution. Samples can be conserved in 2~8℃ in 1 week, and it should be stored at - 20℃ for a long term storage.
2. Diluted serum/plasma: Dilute the sample serum with the Sample Diluent at 1:9 (10 μL of sample serum or plasma and 90 μL of sample diluent, mix fully).The positive/negative control do not need to be diluted.
3. Wash Buffer: The 20×Concentrated Wash Buffer should be adjusted to room temperature to make the sediment dissolved fully before use, then dilute it with deionized water at 1:19.

Assay Procedure

Restore all reagents and samples to room temperature (25℃) before use. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. The unused ELISA Microtiter plate should be sealed as soon as possible and stored at 2~8℃.
1. Number: number the sample and control in order (multiple well), and keep a record of control wells and sample wells. Set 1 well for blank control and 2 wells for negative/positive control respectively. Samples need test in duplicate.
2. Add sample: add 100 µL of Sample Diluent to the blank control well, add 100 μL of positive/negative control to positive/negative control well. Add 10 µL of diluted serum/plasma and 90 µL of Sample Diluent to the sample wells.
3. Incubate: cover the plate sealer and mix thoroughly, incubate at 37℃ for 30 min in shading light.
4. Wash: remove the liquid in each well. Immediately add 300 μL of Wash Buffer to each well and wash. Repeat wash procedure for 5 times, 30 s intervals/time. Invert the plate and pat it against thick clean absorbent paper (If bubbles exist in the wells, clean tips can be used to prick them).
5. HRP Conjugation: add 100 µL of HRP Conjugate into each well (except the blank control well), over the plate sealer and incubate at 37℃ for 30 min in shading light.
6. Wash: repeat step 4 for washing.
7. Color Development: add 50 µL of Substrate Reagent A and 50 µL of Substrate Reagent B into each well. Cover the plate sealer and mix thoroughly, incubate at 37℃ for 10 min in shading light.
8. Stop reaction: add 50 µL of Stop Solution into each well, mix thoroughly.
9. OD Measurement: adjusted zero with the blank control, measure the absorbance value (A-value) of each well by using a Microplate Reader with 450 nm (it is recommended to set the dual wavelength at 450 nm/630 nm) wavelength. Blank well is not needed when using dual wavelength 450 nm/630 nm for detection.