Human glucagon like peptide 1 (GLP-1) ELISA kit

　　Human glucagon like peptide 1 (GLP-1) ELISA kitUser Manual

BS-0203-A Human Glucagon like Peptide 1 (GLP-1) ELISA Kit 96T

BS-0203-B Human Glucagon like Peptide 1 (GLP-1) ELISA Kit 48T

1、 Experimental principle

This kit uses a double antibody sandwich method to detect GLP-1 levels in human serum, plasma, or cell culture supernatants. The core steps include:

Solid phase antibody coating: Microplate pre coated purification of anti-GLP-1 monoclonal antibody.

Antigen antibody binding: GLP-1 in the sample binds to solid-phase antibodies and then forms complexes with horseradish peroxidase (HRP) labeled secondary antibodies.

Color reaction: Add substrate TMB for color development, and after acid termination, measure the absorbance at a wavelength of 450nm. The concentration is proportional to the depth of color development.

2、 Composition of reagent kit

Component specification remarks

Pre packaged microplate with 12 holes and 8 strips containing anti-GLP-1 antibodies

Standard 0.5ml/bottle freeze-dried powder, needs to be redissolved

HRP labeled secondary antibody 6ml stored in the dark

Chromogenic substrate (TMB) solution A 6ml+solution B 6ml ready to use and prepare

Termination solution 6ml 2M sulfuric acid

20ml concentrated washing solution needs to be diluted 1:20

6ml of sample Xishi solution containing stabilizer

Note: There may be differences in the components of different brands, please refer to the specific instructions for details.

3、 Sample processing

1. Sample type

Serum/plasma: anticoagulant with EDTA or heparin, centrifuge to obtain supernatant.

Cell supernatant: Centrifuge at 1000 × g for 20 minutes to remove impurities.

Tissue homogenate: After lysis, centrifuge to obtain the supernatant.

2. Storage conditions

Short term: Store at 2-8 ℃ for ≤ 48 hours.

Long term: Freeze at -20 ℃ or -80 ℃ after packaging to avoid repeated freeze-thaw cycles.

3. Dilution suggestion

Conventional sample: It is recommended to dilute 5-20 times (such as taking 20 μ L of sample+180 μ L of diluent).

High concentration samples: Pre experiments are required to determine the dilution factor, with a maximum dilution of 100000 times (three-step dilution method).

4、 Operation steps

Standard product preparation

Dissolve the freeze-dried standard in 1mL of diluent, mix well, and let it stand for 10 minutes.

Perform a series of dilutions (such as 8pmol/L → 4pmol/L →... → 0.5pmol/L).

Add sample

Standard well: 50 μ L/well.

Sample well: First add 40 μ L of diluent, then add 10 μ L of sample (total dilution 5-fold).

Blank hole: No sample or secondary antibody added.

Incubation

Incubate at 37 ℃ for 120 minutes, wash the plate 3 times (soak for 1 minute each time).

Color rendering and termination

Add 100 μ L of TMB chromogenic solution to each well and incubate at 37 ℃ in the dark for 15 minutes.

Add 50 μ L of termination solution to terminate the reaction, and measure the OD value within 30 minutes.

5、 Precautions

Sample avoidance: Hemolysis, high-fat samples, and samples containing NaN3 (inhibiting HRP activity).

Reagent equilibrium: Room temperature equilibrium is required for 20 minutes before use, and the concentrated washing solution needs to be dissolved in a 40 ℃ water bath for crystallization.

Quality control: It is recommended to set up multiple holes, and the standard curve R ² should be ≥ 0.95.

6、 Result analysis

Standard curve: Plot a four parameter equation of OD value versus concentration.

Concentration calculation: Substitute the sample OD value into the curve equation and multiply it by the dilution factor.

Note: The above is for reference only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

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