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

Human Glutamine (Gln) ELISA Kit

Instructions

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

Experimental Principle

The Human Glutamine (Gln) ELISA Kit utilizes a sandwich immunoassay technique to quantify the concentration of Gln in biological samples. The microtiter plate is pre-coated with a specific anti-Gln antibody. After incubation with the sample, a HRP-conjugated secondary antibody binds to the captured Gln, forming an immune complex. Following washing steps, the substrate TMB is added, which turns blue under HRP catalysis and then changes to yellow when acid is introduced. The intensity of the color is directly proportional to the amount of Gln present in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the Gln concentration is determined by comparing the sample OD to a standard curve.

Human Glutamine (Gln)

ELISA

Kit

Composition

1. 130× 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 Developer A – 6ml × 1 bottle 8. TMB Developer B – 6ml × 1 bottle 9. Standard Dilutions – 1.5ml × 1 bottle 10. Instructions – 1 copy 11. Sealing Film – 2 sheets 12. Seal Bag – 1

Sample Requirements

1. Samples should be processed and tested as soon as possible after collection. If not used immediately, store at -20°C, avoiding repeated freeze-thaw cycles. 2. Samples containing NaN3 are not suitable for testing, as it may inhibit HRP activity.

Human Glutamine (Gln)

ELISA

Kit

Procedure

1. Standard Dilution: The original standard can be diluted according to the provided dilution chart. For example, add 150μl of standard diluent to 150μl of the original standard (32pg/ml), creating a 16pg/ml solution for No.4 standard, and so on. 2. Loading: Set up blank, standard, and sample wells. Add 50μl of standard, 40μl of sample diluent, and 10μl of sample into each well. Ensure gentle mixing without touching the well walls. 3. Incubation: Seal the plate and incubate at 37°C for 30 minutes. 4. Washing: Use a 30× diluted washing solution and wash the plate 5 times. 5. Enzyme Addition: Add 50μl of enzyme-labeled reagent to all wells except the blank. 6. Incubation: Repeat the 37°C incubation for 30 minutes. 7. Washing: Repeat the washing procedure. 8. Color Development: Add 50μl of TMB A and 50μl of TMB 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 450nm within 15 minutes of stopping the reaction.

Calculation

Plot a standard curve using standard concentrations and their corresponding OD values. Determine the sample concentration from the curve, then multiply by the dilution factor. 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 min) before use. Store unopened enzyme reagents in a sealed bag. 2. If the washing solution crystallizes, warm it gently in a water bath before use. 3. Use a pipette for accuracy. Keep loading time under 5 minutes. 4. Always run a standard curve and consider diluting high-concentration samples if needed. 5. Use the sealing film only once to prevent contamination. 6. Protect the substrate from light. 7. Follow the manual strictly. Results must be confirmed by a microplate reader. 8. Treat all waste as biohazardous material. 9. Do not mix components from different batches. 10. In case of discrepancy, 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.