Ovucheck Plasma (Canada)

Measurement Of Progesterone In Plasma Or Serum

Insert / Product #c005

2007-10-25

The OVUCHECK^® PLASMA ELISA kit is an immunoenzymatic test which provides a simple, reliable and precise measurement of progesterone in plasma or serum from a number of species (for example cows, horses, dogs, sheep, pigs, goats, and rabbits). Each kit contains sufficient reagents for up to 92 tests plus four standards.

OVUCHECK^® PLASMA is used for oestrus detection and assessment of pregnancy status/luteal function.

Principle Of The Test

The OVUCHECK^® PLASMA test is based on the competitive binding of unlabelled progesterone present in the standard or sample, and a fixed quantity of progesterone labelled with the enzyme alkaline phosphatase (AP), to binding sites on a limited amount of specific progesterone antibodies.

The wells are pre-coated with antibodies, providing a solid phase for the convenient separation of the bound progesterone from the free progesterone in the sample. After incubation, all components other than those bound to the plate wells are washed away.

The amount of bound AP-labelled progesterone remaining on the wells is inversely proportional to the concentration of the unlabelled progesterone present in the sample. The bound labelled progesterone is then measured by reacting the AP with its substrate during a second incubation.

The colour produced is measured spectrophotometrically and the concentration of progesterone in the sample is determined from a standard curve.

Execution

A. Substrate Preparation

Add 3 substrate tablets to the substrate buffer and shake it to dissolve.

If all the substrate is not required immediately, it can be dispensed into clean plastic containers and stored at 4 ± 2°C for 1 week or frozen for 3 months at -20 ± 4°C (-4°F).

B. Sample Collection

Plasma

- Blood samples should be collected into heparinised collection vessels.

- Centrifuge the blood sample within 30 minutes of collection and draw off the plasma into a clean container. The plasma sample will remain usable for up to 48 hours if kept cool or will remain usable indefinitely if kept frozen.

- If centrifugation is not immediately available, keep the blood sample as cool as possible and centrifuge within 24 hours of collection. Progesterone levels fall by as much as 30% under these conditions.

- Centrifugation is generally not required for equine samples. The blood will separate naturally into plasma and red cells if left standing for 10 to 20 minutes.

Serum

- Blood samples should be collected into a non-heparinised tube. Once the clot has formed, centrifuge the tube within 30 minutes and draw off the serum into a clean container.

- If a centrifuge is not available, the serum should be drawn off as soon as the clot has settled. If kept cool, serum samples will remain usable for up to 24 hours.

C. Test Procedures

1. Bring all components to room temperature before use (approximately 30 minutes).

2. Shake samples and standards just before use.

3. Leave the microtitration plate from the protected packaging. Take the quantity of microwells needed for the test (4 wells for the controls and 1 well per sample to be tested). Wells can be separated from one another. Return unused microwells strips to storage at 4 ± 2°C.

4. To prevent any mistake, it is recommended to identify the wells used by making a schematic representation.

5. Add 10 µL of each four standards to be used to the appropriate wells.

6. Add 10 µL of each sample to be tested to the appropriate wells.

7. Add 200 µL of conjugate to every exposed well.

8. Cover all wells with a sheet of clean paper and let stand for 30 minutes at room temperature (23 ± 2°C).

9. Empty wells and wash by filling with warm tap water and emptying. Repeat twice more. Tap dry on absorbent paper.

10. Add 200 µL of substrate (see section A) to all wells.

11. Cover all wells with a sheet of clean paper and let stand for 30 minutes at room temperature (23 ± 2°C).

12. Add 100 µL stop solution to all wells.

13. Read the results at 405 nm - see « RESULTS » section.

14. At the end of testing, return components to storage at 4 ± 2°C. Used wells should be emptied, but should not be used again. It is recommended to identify, with a waterproof marker, the wells that have been used.

Material

Materials Required But Not Provided

- Purified water

- Adjustable single- and multi-channel micropipettes

- Single-use micropipette tips

- ELISA microplate washer

- Test tubes for sample collection

- ELISA 96-well microplate reader equipped with 405 nm filter

Reagents Provided With The Kit

Precautions

- Store the kit at 4 ± 2°C. DO NOT FREEZE.

- The standards and the conjugate contain a preservative. When emptying the well contents into a sink, thoroughly flush away with an excess volume of tap water.

- For in vitro veterinary diagnostic use only. The components or their residues must not be allowed to come into contact with livestock.

- Do not use the kit after the expiry date indicated on the package.

- Do not mix the reagents from different lot numbers.

- Keep out of reach of children.

- Do not take by mouth. If eyes or skin are splashed, wash thoroughly with tap water.

Interpretation

Results

Set spectrophotometer or plate reader to read absorbance at 405 nm. Zero instrument on air. Draw a standard curve by plotting standard absorbance values on the graph paper provided.

The progesterone concentration of the samples can then be read from the curve.

Progesterone Levels

Specificity

The interference (cross-reactivity) from steroids other than progesterone is insignificant (less than 1%) except for:

Biovet Inc. 4375, Beaudry ave, St. Hyacinthe (Qc) J2S 8W2 Canada

Tél:1-888-8BIOVET Fax: (450) 771-2996 Technical services: support@biovet-inc.com www.biovet.ca

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