Pig progesterone / progesterone (PROG) elisa kit experimental description - Database & Sql Blog Articles

Kaixin Micro Test

Test - Lowercase JPG

Test Probe PH-5H

Porcine Progesterone (PROG) ELISA Reagent Kit

This kit is for research use only. Not for human diagnostic use.

Experimental Principle

The Porcine Progesterone (PROG) levels in samples are determined using a double-antibody sandwich ELISA method. A microtiter plate is pre-coated with a specific anti-PROG antibody. After adding the sample and a labeled secondary antibody, an immune complex is formed. The enzyme-labeled antibody binds to the antigen, and after washing, TMB substrate is added. The reaction is terminated with an acidic solution, resulting in a color change that correlates with PROG concentration. The absorbance at 450 nm is measured, and the concentration is calculated based on a standard curve.

Kit Components

- 130x Washing Solution: 20ml × 1 bottle
- Stop Solution: 6ml × 1 bottle
- Enzyme Standard Reagent: 6ml × 1 bottle
- Standard (4800 pmol/L): 0.5ml × 1 bottle
- Enzyme-Labeled Coating Plate: 12 wells × 8 plates
- Sample Diluent: 6ml × 1 bottle
- TMB Substrate A: 6ml × 1 bottle
- TMB Substrate B: 6ml × 1 bottle
- Instruction Manual: 1 copy
- Sealing Film: 2 sheets
- Sealed Bag: 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles.
2. Avoid using samples containing NaN3, as it may inhibit HRP activity.

Procedure

1. Prepare standard dilutions according to the provided table.
2. Add 50 μl of standard or sample diluent to each well, then add 10 μl of sample (final 5× dilution).
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted washing solution.
5. Add 50 μl of enzyme-labeled reagent to each well (except blank wells).
6. Incubate again for 30 minutes.
7. Wash again.
8. Add 50 μl of TMB A and 50 μl of TMB B, incubate at 37°C for 10 minutes.
9. Stop the reaction by adding 50 μl of stop solution.
10. Measure OD450 within 15 minutes using a microplate reader.

Calculation

Plot a standard curve using standard concentrations vs. OD values. Calculate sample concentration from the curve, then multiply by the dilution factor.

Precautions

1. Let the kit equilibrate to room temperature before use. Store unopened enzyme reagents in a sealed bag.
2. If washing solution crystallizes, warm it gently in a water bath before use.
3. Use a pipette for accurate measurements. Keep loading time under 5 minutes.
4. Always prepare a standard curve and perform duplicate tests. For high-concentration samples, dilute before testing.
5. Use a new sealing film for each experiment to avoid contamination.
6. Protect TMB from light.
7. Follow the manual strictly and rely on instrument readings.
8. Treat all waste as biohazardous material.
9. Do not mix components from different batches.

Storage and Shelf Life

1. Store the kit at 2–8°C.
2. Shelf life: 6 months from the date of manufacture.