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Detection kit for enzyme linked immunosorbent assay applied for diethylstilbestrol residue analysis

An enzyme-linked immunosorbent assay and diethylstilbestrol technology, which is applied in analytical materials, biological tests, measuring devices, etc., can solve the problems of difficulty in adapting analytical methods to requirements, and achieve the effects of simple pretreatment process, less time-consuming, and long storage time.

Inactive Publication Date: 2007-05-23
李季
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AI Technical Summary

Problems solved by technology

With the rapid increase in the amount of samples to be tested, especially the samples that require rapid on-site detection, traditional analysis methods are difficult to meet the requirements. Therefore, it is urgent to develop and apply high-efficiency and rapid enzyme-linked immunosorbent assay technology

Method used

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  • Detection kit for enzyme linked immunosorbent assay applied for diethylstilbestrol residue analysis

Examples

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Effect test

Embodiment 1

[0020] Example 1 Kit operation and result calculation

[0021] After the samples to be tested were pretreated, they were made to volume with PBST for later use. Unpack the vacuum packaging bag and take out the microtiter plate, and equilibrate at room temperature for 5 minutes for later use. Prepare 0ng / mL, 1ng / mL, 2.5ng / mL, 5ng / mL, 12.5ng / mL, 25ng / mL, 50ng / mL, 100ng / mL diethylstilbestrol standard solution, add 50μL standard sample or processed sample to each In the well, make 4 replicates of the standard sample and the sample, add 50 μL of diluted antibody, and incubate at 37°C for 30 minutes; pour out the liquid in the well, wash 5 times with diluted PBST, and put the microplate upside down on absorbent paper. Dry; add 100 μL of enzyme-labeled goat anti-mouse secondary antibody diluted at 1:1000, incubate at 37°C for 30 minutes; pour out the liquid in the well, wash the plate 5 times with PBST, and pat dry; take equal volumes of liquid A and liquid B Mix well, add 100 μL t...

Embodiment 2

[0023] Example 2 Synthesis of hapten diethylstilbestrol monoether n-butyrate (DES-CP)

[0024] Preparation of potassium phenate: Add 0.96g (0.0036mol) DES to a 100mL three-neck flask, dissolve in 15mL methanol, add 0.44g (0.0079mol) KOH under nitrogen protection, heat to 40-45°C, stir for 20min, monitor with TLC . After the reaction was completed, the methanol was distilled off under reduced pressure to obtain the potassium salt.

[0025] Preparation of intermediates: Add 10 mL of dimethylformamide (DMF) to potassium salt at room temperature, raise the temperature to 60-80 ° C, add 15 mL of DMF solution of ethyl γ-bromobutyrate dropwise, and drop it in about 10 minutes. The reaction was carried out for 2h, monitored by TLC. After completion of the reaction, cool to room temperature, pour into 50 g of ice water, adjust the pH to 7 with dilute HCl, extract the aqueous phase twice with 50 mL of diethyl ether, combine the organic phase, wash the organic phase twice with 100 mL o...

Embodiment 3

[0027] Example 3 Preparation of Diethylstilbestrol Monoclonal Antibody

[0028] Couple the hapten with bovine serum albumin by the mixed anhydride method, dissolve 80 μg of the immunogen in 80 μL of sterile saline, mix with 80 μL of complete Freund’s adjuvant, fully emulsify, and subcutaneously multi-point on the abdomen of Balb / c female mice Injection, booster immunization once every two weeks after that, use incomplete Freund's adjuvant to mix with the immunogen, and the immunization site is subcutaneous on the back of the neck. From the third immunization, blood is collected from the orbit of the mouse one week after each immunization to detect the serum effect. price. A total of 5 immunizations were performed. One week after the last immunization, the splenocytes of the immunized mice were fused with SP2 / 0 myeloma, and then subcloned 4 times to screen hybridoma cells that could stably secrete diethylstilbestrol monoclonal antibody. The cell culture supernatant was collect...

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Abstract

The invention disclosed a enzyme-linked immunosorbent test reagent kit applied for diethylstilbestrol residue analysis, and it is characterized by ELISA plate covered by diethylstilbestrol antigen, sponge stand, concentrated detergent solution (PBST), diethylstilbestrol standard product, diethylstilbestrol monoclonal antibody, ELISA two-antibody, color solution and reaction termination solution. During detection course, the coating antigen absorbed in ELISA plate hole-wall and the analyte diethylstilbestrol compete and react with the antibody, and the competition result reflects through the color reaction. Detecting known concentration diethylstilbestrol and drawing the standard curve line, it can calculate the analyte diethylstilbestrol concentration. The advantages of the invention are sensitive and accurate to detect diethylstilbestrol residue in the water and meats, and the sample pre-handling process is simple and less time-consuming, and it can simultaneously detect a large number of samples, sample testing cost far lower than traditional equipment detection method, and the reagent of the invention has long time preservation, no radioactive contamination.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent assay (ELISA) kit, in particular to an enzyme-linked immunosorbent assay kit suitable for diethylstilbestrol (DES) residue analysis. Background technique [0002] The routine analysis of artificial hormone residues is mainly carried out in the laboratory by using instruments such as gas chromatography (GC) and high performance liquid chromatography (HPLC). Applying these physical and chemical analysis techniques to analyze trace amounts of diethylstilbestrol residues in environmental, biological, and food samples not only requires a high degree of instrumentation, but also requires complicated pretreatment processes such as separation, extraction, purification, and derivation, which cannot achieve large sample Simultaneous detection requires high professional requirements for operators, and a large amount of organic solvents are used in the pre-treatment process, which causes new environmental pol...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/577G01N33/543G01N33/532
Inventor 王文君李季许艇
Owner 李季
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