AffiVET® Bovine Brucella Antigen Rapid Test Kit

AffiVET® Bovine Brucella Antigen Rapid Test Kit

Species: Bovine
Disease or Pathogen: Brucellosis
Target: Antigen
Test Type: Rapid Tests

1. What is Bovine Brucella?

Bovine brucellosis is a contagious disease of livestock typically caused by Brucella abortus which may also infect people. The disease is also known as “contagious abortion” or “Bang's disease” in livestock and as “undulant fever” in people because of the recurring fevers accompanying the infection.

2. Materials Provided : 

3. Materials Needed, but Not Provided :

4. Precautions :

In order to obtain reproducible results, the following rules must be observed :

1. For in vitro diagnostic use only.
2. Store the components at 2-8 ℃ right after use. Do not reuse microwells or pour reagents back into their original bottles once dispensed.
3. Do not intermix components from kits with different batch numbers.
4. Do not use reagents after the expiry date
5. Do not reuse containers and residues to avoid contamination in reagents due to samples or other reagents
6. Handle all reagents and samples as biohazardous materials
7. Use fresh samples. Hemolyzed or contaminated samples may give erroneous results.
8. Remove the red blood cell in samples clearly. It may give non-specific reaction.
9. Wear the gloves when you handle the potentially infectious materials. After handling, wash hands with sanitizers.
10. Keep all reagents away from skin and eyes. If exposure should occur, immediately rinse with fresh cold water
11. Dispose of containers and residues safely in accordance with national and local regulations
12. TMB Substrate and Stop Solution can cause irritation or burns to the skin and eyes. In case of an accident, rinse immediately with fresh cold water
13. If the color reaction starts to appear from one part of the well, it is not considered negative.

5. Collection and Storage of Sample :

[Serum or Plasma]

1. Fresh serum or plasma samples should be used for this assay.
2. Hemolyzed or contaminated samples may give erroneous results
3. If samples are not immediately tested, they should be refrigerated at 2~8 ℃. For longer storage, freeze the samples at -20 ℃ or below. Avoid repeated freezing and thawing.
4. For serum samples, the heat-inactivation (56℃, 30 minutes) does not affect the judgment result.

 [Milk]

1. Milk samples must be centrifuged for 15minutes at 2000 Xg to remove the lipid layer
2. If samples are not immediately tested, they should be refrigerated at 2~8 ℃.
3. A pooled milk sample can be used for testing.

6. Preparation of Reagent 1) Unused :

1. Unused microplate wells must be sealed with silica gel in enclosed sealing bag and stored at 2~8 ℃.

2. 101X Enzyme Conjugate: Dilute 101X Enzyme Conjugate 1:100 with Conjugate Diluent before use.
   (e.g. add 10 ㎕ of 101X Enzyme Conjugate to 1 ml of Conjugate Diluent and mix well.)

3. 5X Sample Diluents: Dilute 5X Sample Diluent 1:4 with distilled/deionized water before use. (e.g. add 10 ㎖ of 5X Sample Diluent to 40 ㎖ of distilled water and mix well.)                
4. 10X Washing Solution: Dilute 10X Washing Solution 1:9 with distilled/deionized Water before use. (e.g. add 50 ㎖ of 10X Washing Solution to 450 ㎖ of distilled water and mix well. If undissolved crystals are present, re-suspend the solution by warming the bottle at 37 ℃ for few minutes.)                                                                                                               

5. The storage and stability conditions of the prepared test reagents are as follows:

7. Procedure of the Test :

1. Allow all reagents and samples to equilibrate at room temperature (18~25℃) for 30 minutes and shake them gently before use.
2. Take out only the number of wells required for the test. Then seal the remaining wells back using the silver foil with silica gel and store at 2~8 ℃.
3. Dilute serum or plasma samples 1:49 with diluted sample diluent (e.g., by diluting 10 ㎕ of sample with 490 ㎕ of diluted sample diluent). (*NOTE: Do Not Dilute Negative Control (NC), Strong Positive Control (SPC), Weak Positive Control (WPC) serum.)
4. Add 100 ㎕ of prepared sample (serum or plasma; diluted, milk; not diluted) into each sample well.
5. Add 100 ㎕ of controls(not diluted) into appropriate wells.
6.           -   NC (serum); 2 wells, WPC (serum); 2 wells, SPC (serum); 3 wells,
7.           -   NC (milk); 2 wells, PC (milk); 3 wells
8.  Cover the microplate with adhesive plate sealer and mix well on vibrating mixer. Incubate the wells for 60 minutes at 37 ℃ ± 1 ℃.
9. Prepare the 10X Washing Solution in accordance to ‘Preparation of Reagent’
10. Aspirate all liquid from the wells and rinse the wells 5 times with 350 ㎕ of diluted washing solution. Remove any remaining washing solution by inverting the plate and blotting it against a clean paper towel.
11. Prepare the 101X Enzyme Conjugate in accordance to ‘Preparation of Reagent’.
12. Add 100 ㎕ of diluted Enzyme Conjugate to each well.
13. Cover the wells with plate sealer and incubate for 30 minutes at 37±1 ℃.
14. Wash the wells as described above in Step (8).
15. Add 100 ㎕ of TMB Substrate to each well.
16. Cover the wells with plate sealer and incubate for 15 minutes at room temperature (18~25 ℃) in the dark.
17. Add 100 ㎕ of Stop Solution to each well. Gently mix well until the blue color changes completely to yellow.
18. Read the absorbance values of the wells at 450 nm in a dual wavelength reader (with reference wavelength at 620 nm) right after from the end of assay, within 30 minutes

8. Interpretation of the Result:

1) Test validation :

[Serum & Plasma]

1. The OD value of NC (serum) (average of two wells) should be ranged from -0.005~0.2000
2. The OD value of WPC should be above 0.500
3. The OD value of SPC (serum) (average of three wells) should be above 1.000

[Milk]

1. The OD value of NC milk should be ranged from 0.000~0.2000.
2. The OD value of PC milk should be above 1.000

2) Calculation of Percent Positivity (%P) value

[Serum & Plasma] 

%P = OD of samples x 100 / Average OD of SPC serum 

[Milk]

%P = OD of samples x 100 /  Average OD of PC milk

3) Interpretation of Result :

1. The result should be determined based on the following %P criteria

2. 	Serum / Plasma	Milk
   Positive	≥ 25	≥ 15
   Negative	< 25	< 15

3. Negative: Specimens with a %P value less than the criterion value are considered negative
4. Positive: Specimens with a %P value greater than or equal to the criterion value are considered positive
5. If the result is positive, retest the sample in duplicate. If one or more wells show a positive response, then the result is considered positive
6. Professional veterinarian should make a final diagnosis based on the results of this product, other test results and clinical findings.

4) Example : 

OD of sample = 1.589, average OD value of PC milk = 2.056

%P value= (1.589 / 2.056) x 100 = 77

→ This sample is regarded as positive to bovine brucellosis

9. Limitations and Interferences :

1. 1) The test procedure, precautions and interpretation of results sections for this test kit must be followed closely when testing.
2. 2) This test kit detects antibodies against brucella specific antigen in serum and plasma samples and thus is useful as a screening procedure.
3. 3) Failure to add sample in the procedure could lead to a false negative result. Repeat testing should be considered where there is clinical suspicion of infection

10. Stability and Storage :

1. 1) All reagents should be stored at 2~8 ℃. Do not freeze the test kit
2.  2) Shelf life is 12 months. Use all reagents before the expiry date on the kit.

11. Packaging Unit :