Deoxy pyridinoline immunogen, antibody, detection reagent and preparing method

A deoxypyridinol and anti-deoxypyridinol technology, applied in the field of deoxypyridinol detection reagent and its preparation, anti-deoxypyridinol specific antibody, can solve the problem of high-throughput, rapid detection and small-scale detection of DPD content , high cost and other issues, to achieve high-throughput rapid determination, high accuracy, and easy operation

Inactive Publication Date: 2016-07-27
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are time-consuming, costly, and small-scale, and cannot meet the requirements of high-throughput and rapid detection of DPD content in the urine of a large number of clinical patients.
At present, there is a lack of DPD detection reagents with good stability, high sensitivity and strong specificity in the market, especially good quality automatic detection reagents

Method used

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  • Deoxy pyridinoline immunogen, antibody, detection reagent and preparing method
  • Deoxy pyridinoline immunogen, antibody, detection reagent and preparing method
  • Deoxy pyridinoline immunogen, antibody, detection reagent and preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Synthesis of embodiment one deoxypyridinol immunogen

[0048] The deoxypyridinol immunogen is composed of bovine serum albumin (BovineSerumAlbumin, BSA) and deoxypyridinol represented by formula (Ⅱ) The specific steps are as follows:

[0049] 1. Dissolve 200mg of bovine serum albumin in 50ml of 0.2M, pH8.5 phosphate buffer;

[0050] 2. Add the following chemicals into a small beaker and stir to dissolve: 200mg of deoxypyridinol, 3.5ml of dimethylformamide, 3.5ml of ethanol, 7.0ml of 10mM potassium phosphate buffer at pH 5.0, 200mg of 1-ethyl-3- (-3-Dimethylaminopropyl) carbodiimide, 50mg N-hydroxyl sulfosuccinimide, these chemicals were stirred and dissolved at room temperature for 30 minutes;

[0051] 3. Add the dissolved solution dropwise to the BSA solution, and stir overnight at 2-8° C. to obtain the antigen; purify the synthesized antigen through dialysis to obtain the deoxypyridinol immunogen.

Embodiment 2

[0052] Example 2: Preparation of anti-deoxypyridinol specific antibody

[0053] The deoxypyridinol immunogen prepared in Example 1 was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization. The specific steps were as follows:

[0054] 1. Dilute the above synthesized deoxypyridinol immunogen to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with an equal amount of Freund's complete adjuvant, and inject the experimental animal rabbit.

[0055] 2. After 2-3 weeks, inject 1.0ml of the same antigen solution and the same amount of Freund's incomplete adjuvant to the above-mentioned experimental animal rabbit once, and then inject once every four weeks, a total of 4 injections.

[0056] 3. Take blood from the experimental animal rabbit in step 2, separate and purify to obtain anti-deoxypyridinol specific antibody with a titer of 1:30000-1:50000.

Embodiment 3

[0057] Embodiment three: ELISA test of deoxypyridinol

[0058] 1. Establishment of standard curve for ELISA detection of deoxypyridinol

[0059] (1) Preparation of standard products

[0060] Deoxypyridinol powder (purchased from Sigma Company) was dissolved in methanol solution to prepare a 1000 μmol / L stock solution. The stock solution was diluted sequentially with ELISA buffer to 300.00nmol / L, 100.00nmol / L, 30.00nmol / L, 10.00nmol / L, 3.00nmol / L and 0.00nmol / L standard solutions. Among them, the ELISA buffer contains 50.0mM Tris, 145mM NaCl and 0.25% BSA.

[0061] (2) Utilize the ELISA test method of deoxypyridinol to prepare standard curve

[0062] Dilute the anti-deoxypyridinol-specific antibody prepared in Example 2 with PBS to a final concentration solution of 1:5000, coat 100 μL / well on a 96-well enzyme-linked plate, and place it at 4°C for 12-24 hours; After the 96-well enzyme-linked plate coated with anti-deoxypyridinol antibody was washed three times, 200 μL / well o...

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Abstract

The invention discloses deoxy pyridinoline immunogen, an antibody, a detection reagent and a preparing method.The prepared deoxy pyridinoline immunogen is high in immunogenicity and capable of obtaining high-potency specific antibody resisting deoxy pyridinoline through induction and has no cross reaction with 62 common dugs; the deoxy pyridinoline detection reagent prepared from the antibody can accurately and fast determine the content of deoxy pyridinoline in urine and other biological biological samples.Compared with existing detection reagents in the market, the detection reagent has the advantages of being easy to operate, high in sensitivity and specificity, accurate in result, capable of effectively reducing detection cost of deoxy pyridinoline and beneficial for large-scale clinical utilization and popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a deoxypyridinol immunogen, an anti-deoxypyridinol specific antibody, a deoxypyridinol detection reagent and a preparation method thereof. Background technique [0002] Deoxypyridinoline (DPD), its structural formula is shown in formula (II): [0003] [0004] Formula (Ⅱ) [0005] Deoxypyridinol exists only in type Ⅰ collagen fibers of bones, and it is a cross-link between molecules when mature type Ⅰ collagen fiber molecules form collagen fibers, and plays a role in stabilizing the collagen chain. During bone resorption, DPD, as a degradation product of type I collagen fibers, is released into the blood circulation in the form of free or peptide binding, and is directly excreted through the kidneys without being degraded by the liver, and is excreted with urine. DPD is not affected by diet and can appear before changes in bone density. Studies have shown that: 35-40 years old is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/795C07K14/435C07K16/44C07K16/06G01N33/53G01N33/535
CPCC07K19/00C07K14/435C07K14/765C07K14/795C07K16/06C07K16/44G01N33/5308G01N33/535
Inventor 虞留明程黎明陈鸣
Owner 苏州博源医疗科技有限公司
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