Sheep Prolactin
(
PRL
ELISA
Kit
Experimental Operation Guide
This kit is for research use only. It is not intended for diagnostic or therapeutic purposes.
Experimental Principle
Sheep Prolactin (PRL) ELISA Kit is based on the double antibody sandwich method. The microwell plate is pre-coated with a specific antibody against sheep PRL. After adding the sample, PRL binds to the immobilized antibody. Then, a HRP-conjugated secondary antibody is added, forming an antibody-antigen-enzyme complex. Following washing steps, TMB substrate is added and develops color under the action of HRP. The color intensity is proportional to the PRL concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the PRL level is calculated from a standard curve.
Kit Composition
1. 130x 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 Reagent A – 6ml × 1 bottle
8. TMB Color Reagent 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 not used immediately, store at -20°C. Avoid repeated freeze-thaw cycles.
2. Samples containing NaN3 cannot be tested, as it may inhibit HRP activity.
Kit Steps
1. Standard Dilution: Start with the original standard (400μg/ml). Prepare serial dilutions as follows:
No.5: 150μl original + 150μl diluent
No.4: 150μl No.5 + 150μl diluent
No.3: 150μl No.4 + 150μl diluent
No.2: 150μl No.3 + 150μl diluent
No.1: 150μl No.2 + 150μl diluent
2. Loading: Add 50μl of each standard and 40μl of diluent plus 10μl of sample (final 5x dilution) into respective wells.
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted washing solution (1:30).
5. Add 50μl of enzyme reagent to all wells except blank.
6. Incubate again at 37°C for 30 minutes.
7. Wash again 5 times.
8. Add 50μl of TMB A and 50μl of TMB B, incubate at 37°C for 15 minutes.
9. Stop reaction by adding 50μl stop solution.
10. Measure OD at 450nm within 15 minutes.
Calculation
Plot a standard curve using standard concentrations vs. OD values. Determine the unknown concentration by interpolation or linear regression. Multiply by the dilution factor (×5) to get the actual sample concentration.
Precautions
1. Allow kit to reach room temperature before use. Store unopened plates in sealed bags.
2. If washing solution crystallizes, dissolve in warm water. It will not affect results.
3. Use accurate pipettes. Keep loading time under 5 minutes. For large batches, use a multichannel pipette.
4. Always make a standard curve and duplicate wells. If OD exceeds the first standard, dilute samples accordingly.
5. Use a new sealing film per experiment to prevent cross-contamination.
6. Protect TMB from light.
7. Follow instructions strictly. Results must be confirmed by microplate reader readings.
8. Treat all waste as biohazardous material.
9. Do not mix reagents from different batches.
10. In case of discrepancy, the English manual takes precedence.
Storage Conditions & Expiration
1. Store at 2–8°C.
2. Shelf life: 6 months from date of manufacture.
Dongguan Pinji Electronic Technology Limited , https://www.iquaxusb4cable.com