Cow Meat identification kit

Cat No. AID 051

Intended use
Cow meat identification kit is intended to be used for detection of fresh or frozen cow meat and to distinguish
it from other meats such as buffalo, goat, sheep, pig and chicken. The kit should not be used to test cooked
meat. This kit is helpful in preventing adulteration of cow meat with other meat for economic, religious or health
reasons. The kit does not distinguish between cow and bullock meat.
The total incubation time of the assay is 40 minutes.

Principle of the test

Antibodies specific to proteins present in cow meat are immobilized on the microwell plates. When the
extracts of meat samples are added to the microtiter wells, cow specific meat proteins bind to the antibodies
immobilized in the well. This binding is subsequently detected by addition of enzyme conjugate. After a washing
step, substrate is added. The reaction is terminated by addition of stopping solution. Absorbance is then measured
on a plate reader. The colour of substrate changes from blue to yellow on addition of stop solution.

Cross-reactivity
The Cow meat identification kit detects specifically cow meat only and it does not cross react with meats such
as buffalo, pig, chicken, goat or sheep.

Precautions
The Cow meat identification ELISA test kit is intended for in vitro use only. The reagents contain Thimerosal
as preservative. Prevent direct skin and eye contact with kit components. Seek medical attention in case of
accidental ingestion of kit components.

Storage of the kit

The kit should be stored at 2 - 8 C. The unopened kit is stable till the expiry date printed on the kit label. The
expiry date of each unopened component is printed on the label of the component.
Contents of the kit
Reagents are sufficient for 96 wells.
1. One plate of 96 wells coated with Antibodies, packed in a laminate bag
with silica gel
2. Enzyme conjugate: 6 ml, One vial, Ready to use
3. Meat extraction buffer: (20X Stock): 50 ml, One vial
4. Wash solution: (10X Stock): 50 ml, One vial
5. TMB Substrate, 12 ml, One vial, Ready to use
6. Stop solution: 12 ml, One vial, Ready to use
7. Positive control: 1 ml, One vial, Ready to use
8. Negative control: 1 ml, One vial, Ready to use
Storage of the kit
The kit should be stored at 2 - 8 C. The unopened kit is stable till the expiry date printed on the kit label. The
expiry date of each unopened component is printed on the label of the component.

Reagent preparation:
For 96 wells kit

Preparation of Working Meat extraction buffer:

To 950 ml of distilled/deionized water, empty entire contents of the extraction buffer vial. Mix well before use.
Store unused portion at 2 - 8 C.

Preparation of Working wash buffer:

To 450 ml of distilled/deionized water, empty entire contents of the wash buffer vial. Mix well before use. Store
unused portion at 2 - 8 C.

Material and equipment required but not provided

v Coffee grinder
v Pipette with disposable plastic tips
v Orbital plate shaker
v Deionized or distilled water
v Graduated cylinders with one liter capacity
v Reagent troughs
v Plate ELISA washer or wash bottle
v Plate ELISA reader with 450/620 nm filter
v Table top centrifuge
v 15 ml centrifuge tubes, 5 ml plastic tubes or 2 ml microfuge tubes
v Marking pen, Parafilm*, Pipette tips, Timer and Paper towels.

Meat Sample preparation

Weigh 10 gram fresh or frozen meat sample and transfer it to a coffee grinder jar. Add 10 ml of meat
extraction buffer to the jar and blend it for two minutes. Transfer the mixture to 15 ml tube and centrifuge it
for 10 minutes at 3000 rpm. Carefully collect the clear supernatant in a plastic tube and use it for analysis.

Assay Procedure
Test protocol. mEstimated procedure time is 40 minutes
1. Allow all reagents to reach room temperature before use.
2. Add 50 l enzyme conjugate to each well.
Add 50 l of positive control in one well and 50 l of negative control in another well.
Add 50 l of meat sample extracts to wells. Mix contents of the plate carefully to avoid cross contamination.
3. Place the plate on orbital shaker for 30 minutes at room temperature.
4. Remove content of the wells by decanting into a sink or a waste container. Add 300 l/well wash solution
to all wells and then empty wells by inverting the plate. Repeat washing three more times. Alternately,
perform four washes by using microtiter plate washer. After last wash, tap the inverted microtiter plate on
paper towel to remove as much liquid as possible.
5. Add 100 l substrate per well.
6. Cover the plate and incubate for 10 minutes at room temperature.
7. Stop the reaction by adding 100 l stop solution.
8. Measure the absorbance at 450 nm (primary filter) and 620/630 nm (secondary filter). Read the plate
within 10 minutes after addition of stop solution.
Notes on technique:
1. Protect the plates from draught, strong light or direct sunlight during the test procedure.
2. Careful aspiration of the washing solution is essential for good assay precision.
3. Since the timing of the incubation steps is important for performance of the assay, use multi channel
pipettes to dispense samples/reagents.
4. Plate readers measure absorbance vertically. Do not touch bottom of the wells.
5. Please follow the procedure accurately to get good results. First incubation has to be on a shaker for 30
minutes.

Interpretation of results:
General test criteria:
* Positive control should have absorbance of at least 1.0
* Negative control should have absorbance below 0.5
If above criteria are not met, the test is invalid and should be repeated.

Positive or Negative Sample

Individual meat samples are either positive or negative.
Negative sample: Sample values with absorbance below 1.0 are considered negative for Cow meat
Positive sample: Sample values with absorbance above 1.0 are considered positive for Cow meat
Low level results normally indicate improper washing, incubation timing, cross contamination or technique. In
such cases, retesting is recommended.

Performance characteristics
Fifty samples (25 cow and 25 buffalo) were tested to check for performance of the kit.
Mean absorbance value of Cow meat sample was 2.6 and the range of absorbance was from 1.93 to 2.85
Mean absorbance value of buffalo meat sample was 0.209 and the range of absorbance was from 0.115 to
0.652
All meat samples which were tested from Goat, Sheep, Pig and Chicken gave absorbances below 0.5
WARRANTY
Amar Immunodiagnostics Pvt. Ltd. warrants that the products sold hereunder (the Product) are defect-free
in material and workmanship, provided they are used in accordance with the prescribed instructions before
the expiry of the products as printed on the product label.

The customer should notify Amar Immunodiagnostics in writing of Warranty defects during the warranty
period, including an offer by the customer to return the Products to Amar Immunodiagnostics for evaluation.
Amar Immunodiagnostics will repair or replace, at its sole option, any product or part thereof that proves
defective in materials or workmanship within the warranty period.
This warranty also does not apply to Products to which changes or modifications have been made or attempted
by persons other than pursuant to written authorization by Amar Immunodiagnostics

THIS WARRANTY IS EXCLUSIVE

The sole and exclusive obligation of Amar Immunodiagnostics shall be to repair or replace the defective
Products in the manner and for the period provided above. Amar Immunodiagnostics shall not have any other
obligation or liability, whatsoever it may be, with respect to the Products or any part thereof.

Under no circumstances, whatsoever the circumstances may be, Amar Immunodiagnostics shall be liable
for incidental, special, or consequential damages.
If any part of this Warranty is determined to be void or illegal, the remainder shall remain in full force and
effect.

*Parafilm is a registered trademark of American Can Corporation ( now Pechinney Plastic Packaging).

For any further information, please contact:

AMAR IMMUNODIAGNOSTICS PVT. LTD.
242/1, Road No. 18, Jubilee Hills, Hyderabad - 500033, India.
Tel : +91 40 2355 2953, 2354 7202 Fax : +91 40 2355 2954
E-mail : info@amarimmunodiagnostics.com
Website : www.amarimmunodiagnostics.com