Human glutaminase (GluAP) enzyme-linked immunoassay kit experimental operation - Database & Sql Blog Articles

Human Glutaminase (GluAP) ELISA Kit

Experimental Procedure

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

Experimental Principle

The Human Glutaminase (GluAP) ELISA Kit utilizes the sandwich immunoassay technique to quantify the levels of human glutaminase in biological samples. The microplate is pre-coated with a specific monoclonal antibody against GluAP. After adding the sample, the target antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following a washing step, the substrate TMB is introduced, which changes color under the catalytic action of HRP. The reaction is stopped with a stop solution, and the final color intensity is measured at 450 nm using a microplate reader. The absorbance is directly proportional to the concentration of GluAP in the sample, allowing for quantification via a standard curve.

Kit Components

- 130x Wash Solution: 20ml × 1 bottle - Stop Solution: 6ml × 1 bottle - Enzyme Standard Reagent: 6ml × 1 bottle - Standard (960pg/ml): 0.5ml × 1 bottle - Enzyme-Labeled Coating Plate: 12 wells × 8 - Sample Diluent: 6ml × 1 bottle - TMB Substrate A: 6ml × 1 bottle - TMB Substrate B: 6ml × 1 bottle - Standard Dilutions: 1.5ml × 1 bottle - Instruction Manual: 1 copy - Sealing Film: 2 sheets - Ziplock Bag: 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not used immediately, store at -20°C, avoiding repeated freeze-thaw cycles. 2. Avoid using samples containing NaN3, as it may inhibit HRP activity and interfere with the assay results.

Procedure Steps

1. Standard Dilution: Prepare a serial dilution of the standard from 960pg/ml down to 30pg/ml using the provided diluent. 2. Loading: Add 50μl of each standard and 50μl of sample (after 5-fold dilution) into designated wells. Ensure proper mixing without touching the well walls. 3. Incubation: Seal the plate and incubate at 37°C for 30 minutes. 4. Washing: Rinse the plate five times with diluted wash solution, 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: Repeat the washing procedure. 8. Color Development: Add 50μl of TMB A and 50μl of TMB B to each well, incubate at 37°C for 10 minutes. 9. Termination: Add 50μl of stop solution to each well to halt the reaction. 10. Measurement: Read the OD values at 450nm within 15 minutes of stopping the reaction.

Calculation

Plot the OD values of the standards against their concentrations to create a standard curve. Use this curve to determine the concentration of GluAP in the unknown samples. Multiply by the dilution factor to obtain the actual sample concentration. Alternatively, calculate the linear regression equation and apply it to the sample OD value for accurate quantification.

Precautions

1. Allow the kit to reach room temperature (15–30 minutes) before use. Store any unused enzyme reagent in a sealed bag. 2. The concentrated wash solution may crystallize; heat gently if needed, but this will not affect the results. 3. Use a pipette for accuracy and ensure consistent timing during loading. For large batches, consider using a multichannel pipette. 4. Always prepare a standard curve with duplicates. If the sample OD exceeds that of the highest standard, perform a preliminary dilution before testing. 5. Use the sealing film only once to prevent cross-contamination. 6. Protect the substrate from light exposure. 7. Strictly follow the protocol to ensure reliable results. 8. All waste materials must be handled as biohazardous. 9. Do not mix reagents from different batches.

Storage and Shelf Life

- Storage Conditions: 2–8°C - Shelf Life: 6 months from the date of manufacture