Blood 25-hydroxy vitamin D3 adenocarcinoma assay kit and preparation method thereof

Abstract

The invention provides a blood 25-hydroxy vitamin D3 adenocarcinoma assay kit and a preparation method thereof, and relates to an assay kit. The kit comprises calibrators of different concentrations, an ELISA (enzyme-linked immuno sorbent assay) plate of a 25-hydroxy vitamin D3 coated antibody, horse radish peroxidase marked 25-hydroxy vitamin D3, a quality control product, a substrate chromogenic solution, a reaction stop buffer and a washing fluid. By adopting a series of chemical modification, the 25-hydroxy vitamin D3 is modified into 25-hydroxy vitamin D3 hemisuccinate ester. Hapten is transformed into complete antigen by an improved carbodiimide method and protein vector BSA (bovine serum albumin) protein coupling. Furthermore, the blood25-hydroxy vitamin D3 adenocarcinoma assay kit is developed by combining the ELISA method. The kit can be used in cancer assay in clinical application.

Description

technical field

[0001] The invention relates to a detection kit, in particular to a blood 25-hydroxyvitamin D3 adenocarcinoma detection kit and a preparation method thereof. Background technique

[0002] As early as more than 100 years ago, vitamin D was discovered as a basic component for maintaining bone health, and it was widely used to monitor the occurrence of bone diseases such as rickets and rickets. Now, researchers have found that vitamin D is also associated with many other chronic diseases, especially cancer.

[0003] 1. Vitamin D source and metabolism

[0004] Vitamin D (Vitamin D, VitD) is a fat-soluble steroid derivative. There are five forms, and the ones closely related to health are VitD2 and VitD3. Among them, the former comes from ergosterol in yeast cells, and the latter comes from animal subcutaneous 7-dehydrocholesterol.

[0005] Usually, as long as we receive enough sunlight every day, we can meet 90% of the human body's VitD3 requirement, and some ...

Examples

Embodiment 1

[0086] Synthesis of 25-hydroxyvitamin D3-3-hemisuccinate-BSA

[0087] In this synthesis method, the 3-position of 25-hydroxyvitamin D3 is chemically activated and coupled with BSA protein as a carrier. This synthesis goes through intermediate steps such as 25-hydroxyvitamin D3-hemisuccinate and 25-hydroxy-vitamin D3-3-succinimidyl ester. The structural formula of 25-hydroxyvitamin D3 is as follows:

[0088]

[0089] 1. Preparation of 125-hydroxyvitamin D3-3-hemisuccinate

[0090] Take 10mg (25umol) of 25-hydroxyvitamin D3 dissolved in 1ml of anhydrous pyridine, mix it with 125mg (1.25mmol) of succinic anhydride and stir in dark room at room temperature for 4 days. After diluting the reaction mixture with 10 ml of ethyl acetate, it was washed with 2×10 ml of water, 0.1 M hydrochloric acid, and then washed with water again.

[0091] The organic phase was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo. The remaining solid was dried under...

Embodiment 2

[0101] 2.1 Immunization to produce antibodies against 25-hydroxyvitamin D

[0102] Four 6-week-old female BALB / C mice were immunized with 25-hydroxyvitamin D3-hemisuccinate-BSA, and 25-hydroxyvitamin D3-hemisuccinate-BSA was dissolved in sterile PBS solution for the first immunization and mixed with The same amount of complete adjuvant (FCA) was mixed and fully emulsified, and the immunization dose was 50 μg (0.3 mL) per mouse, and injected subcutaneously in the abdominal cavity of the back at 4 to 6 points; 3 weeks after the first immunization, also dissolved 25- Hydroxyvitamin D3-hemisuccinate-BSA was mixed with an equal amount of incomplete adjuvant (FIA) to fully emulsify and immunize mice. The immunization dose was 30 μg (0.3 mL) per mouse, and the injection site was the same as before. A total of 5 immunizations were performed, with an interval of 2 weeks between each time, and the last immunization was boosted by intravenous injection. On the third day, blood was collec...

Embodiment 3

[0106] Blood 25-hydroxyvitamin D3 adenocarcinoma detection kit (enzyme-linked immunosorbent assay) detection operation steps

[0107] 3.1 Prepare the glass tubes and mark them. Each calibrator CAL, quality control CTRL and sample corresponds to a tube.

[0108] 3.2 Take 10ul each of the calibrator CAL, the quality control substance CTRL and the sample and add them to correspondingly labeled test tubes.

[0109] 3.3 Add 0.4ml of 25-hydroxyvitamin D3 horseradish peroxidase solution VD3+HRP to each test tube. Place on a vortex shaker and mix for 15-30s.

[0110] 3.4 Add 200ul diluted calibrator, quality control or sample to the microwell of the corresponding microtiter plate AbPLAT. Paste plastic wrap and incubate at 18-25c for 2h.

[0111] 3.5 Wash the plate 3 times with WASHBUF, either manually or automatically. NOTE: Before proceeding to the next step, tap the inverted plate vigorously on absorbent paper to remove excess rinse solution.

[0112] 3.6 Add 200ul equal volume...

technical field

[0001] The invention relates to a detection kit, in particular to a blood 25-hydroxyvitamin D3 adenocarcinoma detection kit and a preparation method thereof. Background technique

[0002] As early as more than 100 years ago, vitamin D was discovered as a basic component for maintaining bone health, and it was widely used to monitor the occurrence of bone diseases such as rickets and rickets. Now, researchers have found that vitamin D is also associated with many other chronic diseases, especially cancer.

[0003] 1. Vitamin D source and metabolism

[0004] Vitamin D (Vitamin D, VitD) is a fat-soluble steroid derivative. There are five forms, and the ones closely related to health are VitD2 and VitD3. Among them, the former comes from ergosterol in yeast cells, and the latter comes from animal subcutaneous 7-dehydrocholesterol.

[0005] Usually, as long as we receive enough sunlight every day, we can meet 90% of the human body's VitD3 requirement, and some ...

Examples

Embodiment 1

[0086] Synthesis of 25-hydroxyvitamin D3-3-hemisuccinate-BSA

[0087] In this synthesis method, the 3-position of 25-hydroxyvitamin D3 is chemically activated and coupled with BSA protein as a carrier. This synthesis goes through intermediate steps such as 25-hydroxyvitamin D3-hemisuccinate and 25-hydroxy-vitamin D3-3-succinimidyl ester. The structural formula of 25-hydroxyvitamin D3 is as follows:

[0088]

[0089] 1. Preparation of 125-hydroxyvitamin D3-3-hemisuccinate

[0090] Take 10mg (25umol) of 25-hydroxyvitamin D3 dissolved in 1ml of anhydrous pyridine, mix it with 125mg (1.25mmol) of succinic anhydride and stir in dark room at room temperature for 4 days. After diluting the reaction mixture with 10 ml of ethyl acetate, it was washed with 2×10 ml of water, 0.1 M hydrochloric acid, and then washed with water again.

[0091] The organic phase was dried over anhydrous sodium sulfate, filtered and the solvent was removed in vacuo. The remaining solid was dried under...

Embodiment 2

[0101] 2.1 Immunization to produce antibodies against 25-hydroxyvitamin D

[0102] Four 6-week-old female BALB / C mice were immunized with 25-hydroxyvitamin D3-hemisuccinate-BSA, and 25-hydroxyvitamin D3-hemisuccinate-BSA was dissolved in sterile PBS solution for the first immunization and mixed with The same amount of complete adjuvant (FCA) was mixed and fully emulsified, and the immunization dose was 50 μg (0.3 mL) per mouse, and injected subcutaneously in the abdominal cavity of the back at 4 to 6 points; 3 weeks after the first immunization, also dissolved 25- Hydroxyvitamin D3-hemisuccinate-BSA was mixed with an equal amount of incomplete adjuvant (FIA) to fully emulsify and immunize mice. The immunization dose was 30 μg (0.3 mL) per mouse, and the injection site was the same as before. A total of 5 immunizations were performed, with an interval of 2 weeks between each time, and the last immunization was boosted by intravenous injection. On the third day, blood was collec...

Embodiment 3

[0106] Blood 25-hydroxyvitamin D3 adenocarcinoma detection kit (enzyme-linked immunosorbent assay) detection operation steps

[0107] 3.1 Prepare the glass tubes and mark them. Each calibrator CAL, quality control CTRL and sample corresponds to a tube.

[0108] 3.2 Take 10ul each of the calibrator CAL, the quality control substance CTRL and the sample and add them to correspondingly labeled test tubes.

[0109] 3.3 Add 0.4ml of 25-hydroxyvitamin D3 horseradish peroxidase solution VD3+HRP to each test tube. Place on a vortex shaker and mix for 15-30s.

[0110] 3.4 Add 200ul diluted calibrator, quality control or sample to the microwell of the corresponding microtiter plate AbPLAT. Paste plastic wrap and incubate at 18-25c for 2h.

[0111] 3.5 Wash the plate 3 times with WASHBUF, either manually or automatically. NOTE: Before proceeding to the next step, tap the inverted plate vigorously on absorbent paper to remove excess rinse solution.

[0112] 3.6 Add 200ul equal volume...

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