Human glutamine (Gln) ELISA kit operating instructions - Database & Sql Blog Articles

Human Glutamine (Gln) ELISA Kit

Instructions

This kit is intended for research use only. It should not be used for diagnostic or therapeutic purposes.

Experimental Principle

The Human Glutamine (Gln) ELISA Kit utilizes a double-antibody sandwich method to quantify Gln levels in the sample. The microtiter plate is pre-coated with a specific antibody against human glutamine. After adding the sample, the target antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following a thorough wash, the substrate TMB is introduced, producing a blue color that turns yellow in the presence of an acidic stop solution. The intensity of the color correlates directly with the concentration of Gln in the sample. The absorbance at 450 nm is measured using a microplate reader, and the Gln concentration is determined by comparing the sample's OD value to a standard curve.

Human Glutamine (Gln)

ELISA

Kit

Composition

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

2. Stop Solution – 6ml × 1 bottle

3. Enzyme Standard Reagent – 6ml × 1 bottle

4. Standard (64pg/ml) – 0.5ml × 1 bottle

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

6. Sample Diluent – 6ml × 1 bottle

7. TMB Color Development Agent A – 6ml × 1 bottle

8. TMB Color Development Agent 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, avoiding repeated freeze-thaw cycles.

2. Samples containing NaN3 are not suitable for this assay, as sodium azide may inhibit HRP activity.

Human Glutamine (Gln)

ELISA

Kit

Procedure

1. Standard Dilution: This kit includes one original standard. Prepare serial dilutions according to the provided instructions.

2. Loading: Set up blank, standard, and sample wells. Add 50μl of standard, 40μl of sample diluent, and 10μl of sample to each well. Ensure proper mixing without touching the well walls.

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

4. Washing: Use 30x diluted washing solution. Wash 5 times, ensuring complete removal of unbound reagents.

5. Enzyme Addition: Add 50μl of enzyme-labeled reagent to all wells except blank controls.

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

7. Washing: Perform the same washing steps as above.

8. Color Development: Add 50μl of TMB A and B solutions to each well. Incubate at 37°C for 15 minutes.

9. Stop Reaction: Add 50μl of stop solution to each well to terminate the reaction. The color will turn from blue to yellow.

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

Calculation

Plot a standard curve using the OD values of the standards versus their concentrations. Determine the unknown sample concentration based on the curve. Multiply the result by the dilution factor if applicable. Alternatively, calculate the linear regression equation and use it to determine the sample concentration.

Human Glutamine (Gln)

ELISA

Kit

Precautions

1. Allow the kit to reach room temperature (15–30 minutes) before use. Store any unused enzyme-labeled reagents in a sealed bag after opening.

2. If the washing solution crystallizes, warm it gently in a water bath before use. This will not affect the results.

3. Use a pipette for accurate measurements. For large numbers of samples, consider using an automated pipetting system to improve efficiency and accuracy.

4. Always prepare a standard curve during each experiment. If the sample OD exceeds the highest standard, perform a preliminary dilution before measurement.

5. Use a new sealing film for each experiment to prevent cross-contamination.

6. Keep the TMB substrate away from light to maintain its stability.

7. Follow the operating instructions carefully. Results must be confirmed using a microplate reader.

8. All samples, washes, and waste should be treated as biohazardous materials.

9. Do not mix reagents from different batches.

10. In case of discrepancy between the Chinese and English manuals, the English version takes precedence.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

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