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

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Human Glutaminase (GluAP) ELISA Kit

Experimental Procedure

This kit is intended for research use only. It utilizes the sandwich ELISA method to detect human glutaminase (GluAP) levels in samples. The microplate is pre-coated with a specific antibody against GluAP. After adding the sample and HRP-labeled secondary antibody, an immune complex is formed. Following washing steps, TMB substrate is added, leading to a color change that correlates with the amount of GluAP present. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm using a microplate reader. The concentration of GluAP in the sample is then calculated based on a standard curve.

Kit Components

• 130x Washing 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
• Instructions – 1 copy
• Sealing Film – 2 sheets
• Seal Bag – 1
• Standard Dilutions – 1.5ml × 1 bottle

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles.

2. Samples containing NaN3 cannot be used, as it may inhibit horseradish peroxidase activity.

Procedure Steps

1. Dilution of Standards: Prepare dilutions from the original standard according to the provided chart.
2. Loading: Set up blank, standard, and sample wells. Add 50μl standard, 40μl sample diluent, and 10μl sample to each well. Mix gently.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash 5 times with diluted washing solution, ensuring thorough removal of unbound reagents.
5. Add Enzyme: Add 50μl enzyme-labeled reagent to all wells except blanks.
6. Incubation: Repeat the incubation step at 37°C for 30 minutes.
7. Color Development: Add 50μl of TMB substrate A and B, mix gently, and incubate at 37°C for 10 minutes.
8. Stop Reaction: Add 50μl stop solution to each well to halt the reaction.
9. Measurement: Measure OD values at 450nm within 15 minutes of stopping the reaction.

Calculation

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

Precautions

• Allow the kit to reach room temperature before use. Store unused enzyme-labeled plates in a sealed bag.
• Concentrated washing solution may crystallize; dissolve by heating if necessary.
• Use accurate pipettes and avoid cross-contamination. Keep loading time under 5 minutes.
• Always run a standard curve and consider diluting high-concentration samples.
• Use a new sealing film for each experiment to prevent contamination.
• Keep TMB away from light and handle all waste as biohazardous material.
• Do not mix components from different batches.
• Follow all instructions carefully and ensure results are confirmed by a microplate reader.

Storage and Shelf Life

• Store the kit at 2–8°C.
• Shelf life: 6 months from the date of manufacture.