Sheep prolactin (PRL) ELISA kit experimental operation guide - Database & Sql Blog Articles

Sheep Prolactin

(

PRL

ELISA

Kit

Experimental Operation Guide

This kit is for research use only and not intended for human or animal consumption.

Experimental Principle

The Sheep Prolactin (PRL) ELISA Kit is designed to measure the levels of PRL in various biological samples using a double-antibody sandwich immunoassay. The microtiter plate is pre-coated with a specific antibody against sheep PRL. After adding the sample, PRL binds to the immobilized antibody. A second HRP-conjugated antibody is then added, forming an antibody-antigen-enzyme complex. Following washing steps, the TMB substrate is introduced, which changes color in the presence of HRP. The intensity of the color is directly proportional to the amount of PRL in the sample. The optical density (OD) is measured at 450 nm, and the concentration of PRL is determined by comparing the OD values to a standard curve.

Kit Composition

1. 130× Washing Solution – 20ml × 1 bottle

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

4. Standard (800pg/ml) – 0.5ml × 1 bottle

5. Enzyme-labeled Coating Plate – 12 wells × 8

6. Sample Diluent – 6ml × 1 bottle

7. TMB Color Developer A – 6ml × 1 bottle

8. TMB Color Developer B – 6ml × 1 bottle

9. Standard Dilutions – 1.5ml × 1 bottle

10. Instructions – 1 copy

11. Sealing Film – 2 sheets

12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If testing cannot be performed immediately, store samples at -20°C. Avoid repeated freeze-thaw cycles.

2. Do not use samples containing NaN3, as it may inhibit the activity of horseradish peroxidase (HRP).

Procedure

1. Standard Dilution: Prepare standards from the original stock according to the provided dilution series.

2. Loading: Add 50μl of standard and 50μl of sample diluent to each well, followed by 10μl of the sample. Mix gently.

3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.

4. Washing: Rinse the plate with diluted washing solution (1:30) five times.

5. Enzyme Addition: Add 50μl of enzyme-labeled reagent to each well except the blank.

6. Incubation: Repeat the incubation step at 37°C for 30 minutes.

7. Washing: Repeat the washing procedure.

8. Color Development: Add 50μl of TMB developer A and B, incubate at 37°C for 15 minutes.

9. Stop Reaction: Add 50μl of stop solution to each well to terminate the reaction.

10. Measurement: Read the absorbance at 450 nm within 15 minutes after stopping the reaction.

Calculation

Plot the standard curve using standard concentrations and corresponding OD values. Determine the unknown sample concentration based on the standard curve. Multiply by the dilution factor to obtain the actual concentration.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme reagents in a sealed bag.

2. If the washing solution crystallizes, warm it 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 consider diluting high-concentration samples if needed.

5. Use the sealing film only once to prevent cross-contamination.

6. Protect the substrate from light.

7. Follow the manual strictly and rely on the microplate reader for results.

8. Treat all waste materials as biohazardous.

9. Do not mix components from different batches.

10. In case of discrepancies, the English manual takes precedence.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.