Human nerve injury-inducible protein 1 (NINJ1) ELISA kit experimental operation skills - Database & Sql Blog Articles

Human Nerve Injury-Inducible Protein 1 (NINJ1)

ELISA Kit

Experimental Skill

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

Experimental Principle

The Human NINJ1 ELISA Kit uses a sandwich immunoassay to detect the presence of NINJ1 in biological samples. The process involves coating a microplate with a specific antibody against NINJ1, followed by incubation with the sample. A secondary HRP-labeled antibody then binds to the captured NINJ1, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, which changes color in the presence of HRP. The intensity of the color is directly proportional to the concentration of NINJ1 in the sample. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm using a microplate reader. A standard curve is used to calculate the exact concentration of NINJ1 in the sample.

Kit Components

1. 130x Washing Solution – 20ml × 1 bottle
2. Stop Solution – 6ml × 1 bottle
3. Enzyme Standard Reagent – 6ml × 1 bottle
4. Standard (960pg/ml) – 0.5ml × 1 bottle
5. Enzyme-Labeled Coating Plate – 12 wells × 8
6. Sample Diluent – 6ml × 1 bottle
7. TMB Substrate A – 6ml × 1 bottle
8. TMB Substrate 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 tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.
2. Avoid using samples containing NaN3, as it may inhibit HRP activity.

Procedure

1. Prepare standards by serial dilution according to the provided instructions.
2. Add 50μl of standard and 50μl of diluted sample to the plate, ensuring proper mixing.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing solution.
5. Add 50μl of enzyme-labeled reagent to each well except blank wells.
6. Incubate again for 30 minutes.
7. Wash the plate again.
8. Add 50μl of TMB A and B, incubate at 37°C for 10 minutes.
9. Stop the reaction with 50μl of stop solution.
10. Measure OD values at 450nm within 15 minutes.

Calculation

Plot the standard curve using OD values vs. concentrations. Use linear regression to determine the sample concentration from its OD value. Multiply by the dilution factor to obtain the actual concentration.

Precautions

1. Allow all reagents to reach room temperature before use.
2. Handle concentrated wash solution carefully; heat if crystallized.
3. Use accurate pipetting and ensure consistent timing during loading.
4. Always run standards in duplicate. If sample OD exceeds the highest standard, dilute and retest.
5. Do not reuse sealing films to prevent cross-contamination.
6. Protect TMB from light.
7. Follow the manual strictly and rely on instrument readings.
8. Treat all waste as biohazardous materials.
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.