Instructions for Daidzein ELISA Detection Kit for Rats

BS-8567 Rat Daidzein ELISA Kit

Product Overview

This kit uses competitive enzyme-linked immunosorbent assay (ELISA) and is specifically designed for quantitative detection of daidzein content in biological samples such as rat plasma, serum, or tissue homogenates. Daidzein is a natural flavonoid compound with biological activities such as antioxidant, anti-inflammatory, and immune regulation. It is widely present in leguminous plants, and its content detection is crucial for pharmacological research and nutritional evaluation. This reagent kit is based on the principle of antigen antibody specific binding and achieves high-sensitivity detection through enzyme-linked immunosorbent assay signal amplification. It is suitable for routine laboratory analysis.

Composition of reagent kit

Pre coated microporous plate: 96 holes (12 × 8 holes), detachable Flat noodles, coated with daidzein specific antibody.

Standard: 6 concentration gradients (0.13-61.02 ng/mL), freeze-dried powder, redissolved with standard diluent before use.

Enzyme marker: Competitive antigen labeled with horseradish peroxidase (HRP), stored in brown and dark.

Chromogenic substrate: TMB solution, colorless and transparent liquid.

Termination solution: 2M sulfuric acid solution, used to terminate the reaction.

Washing solution: 20 x concentrated buffer solution, diluted with deionized water before use.

Sample diluent: used to adjust the sample concentration to the detection range.

Sealing film: 2 sheets, used for sealing microporous plates.

Instruction manual: 1 copy.

Storage conditions

Unopened reagent kit: stored at 2-8 ° C in the dark, with a shelf life of 12 months.

After opening: Standard samples, enzyme markers, and chromogenic substrates need to be packaged and frozen (-20 ° C) to avoid repeated freezing and thawing; Microplate should be dried and sealed for storage.

Do not freeze concentrated detergent to prevent crystallization.

sample processing

Plasma/serum samples: Immediately centrifuge (3000 rpm, 10 minutes) after collection, collect the supernatant, and store at -80 ° C. Avoid hemolysis.

Tissue homogenate: Weigh the tissue, add physiological saline (1:9 w/v) homogenate, centrifuge to obtain the supernatant.

Attention: Daidzein in the sample may exist in a bound state. It is recommended to use β - glucuronidase hydrolysis to improve detection sensitivity. When the sample is turbid, it needs to be centrifuged (5000 rpm, 10 minutes) or filtered (0.22 μ m membrane).

Experimental Procedure

1. Preparation of reagents

Washing solution: Dilute 20 x concentrated solution with deionized water to 1 x (e.g. 10 mL concentrated solution+190 mL water).

Standard gradient: Dissolve the freeze-dried standard in a standard dilution solution and dilute the gradient to 0.13, 0.39, 1.17, 3.51, 10.53, and 61.02 ng/mL.

Sample dilution: Dilute the test sample to the detection range with sample diluent (it is recommended to determine the dilution factor through preliminary experiments).

2. Sample addition and incubation

Add 100 μ L of standard or sample to each well (assuming blank wells only contain sample diluent).

Add 50 μ L of enzyme marker to each well, shake gently and mix well.

Incubate at 37 ° C in the dark for 60 minutes.

3. Washing

Discard the liquid in the well, add 350 μ L of washing solution to each well, let it stand for 30 seconds, shake dry, repeat 5 times, and pat dry.

4. Color rendering and termination

Add 100 μ L of chromogenic substrate to each well and incubate at 37 ° C in the dark for 15 minutes.

Add 100 μ L of termination solution to each well, shake gently and mix well.

5. Measurement

Immediately measure the absorbance (OD value) using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm, with a reference wavelength of 630 nm.

Result Calculation

Standard curve drawing: Fit a four parameter logic curve with standard concentration (ng/mL) as the horizontal axis and OD value as the vertical axis (professional software is recommended).

Sample concentration calculation: Substitute the sample OD value into the standard curve equation and multiply it by the dilution factor to obtain the actual concentration.

performance parameters

Sensitivity: Detection limit of 0.13 ng/mL.

Precision: The coefficient of variation (CV) within the board is less than 10%, and the CV between boards is less than 15%.

Recovery rate: 80-120% (validation method: spiked recovery experiment).

Linear range: 0.13-61.02 ng/mL, correlation coefficient r ² ≥ 0.98.

Specificity: Cross reactivity with compounds with similar structures (such as lignin) is less than 5%.

Precautions

Operating standards: Avoid contact between enzyme markers and chromogenic substrates with metal ions; Measure within 30 minutes after color development to prevent signal attenuation.

Quality control: Each batch of experiments should include standard curves and quality control samples (recommended high, medium, and low concentrations).

Safety protection: If the termination liquid contains strong acid, gloves and goggles should be worn during operation.

Waste disposal: Enzyme containing waste needs to be inactivated before disposal.

frequently asked questions

Low signal: Check if the incubation time or temperature is insufficient; Confirm if the washing steps are sufficient.

High background value: It may be due to improper washing or interference from the sample matrix. It is recommended to increase the washing frequency or optimize sample pretreatment.

Abnormal standard curve: When dissolving the standard, it is necessary to mix thoroughly to avoid bubbles affecting the accuracy of concentration.

Note: This product is for scientific research purposes only and is not intended as experimental evidence. Please refer to the actual reagent kit instructions for specific operations.

Tianjin Bensheng has always regarded quality control as the lifeblood of the enterprise and pursued continuous improvement of its competitiveness, using the Daidzein ELISA detection kit for rats. The company always adheres to the corporate spirit of abiding by laws and regulations, being strict with oneself, being tolerant and willing to take on responsibilities as the standard in its operations. It maintains and expands the market with excellent quality and service, and maximizes the satisfaction of customer needs. Our development goal is to achieve mutual benefit with our customers. This student! A partner you trust. We are willing to cooperate sincerely with you and create a better future together. Bensheng reagent kit