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Latex microsphere-protein-CMPF conjugate, preparation method thereof and kit for determining content of CMPF in serum

A -CMPF, protein coupling technology, used in biological testing, measuring devices, instruments, etc., can solve the problems of poor reagent stability, inaccurate test results, loss of carrier enzyme activity, etc., to achieve enhanced stability and improved reagent stability. , to avoid the effect of loss of vitality

Inactive Publication Date: 2019-07-12
HANGZHOU BOPU MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to overcome the poor stability of the reagents in the homogeneous enzyme immunoassay CMPF detection kit in the prior art, and at the same time, the activity of the carrier enzyme will be lost during the reaction process, resulting in inaccurate detection results, and provides a A latex microsphere-protein-CMPF conjugate with good stability and unaffected reactivity and a preparation method thereof, which are simultaneously applied to a kit for measuring CMPF content in serum, improving the test stability of the kit sex and accuracy

Method used

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  • Latex microsphere-protein-CMPF conjugate, preparation method thereof and kit for determining content of CMPF in serum
  • Latex microsphere-protein-CMPF conjugate, preparation method thereof and kit for determining content of CMPF in serum
  • Latex microsphere-protein-CMPF conjugate, preparation method thereof and kit for determining content of CMPF in serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A kind of latex microsphere-protein-CMPF conjugate, described conjugate is the coupling product that CMPF and bovine serum albumin (BSA) and latex microsphere are dehydrated successively under the effect of coupling agent, and its preparation The method is as follows.

[0031] (S.1) CMPF and protein coupling

[0032] Weigh CMPF (100mg), dissolve it with 10mL DMSO, weigh 40mg bovine serum albumin (BSA) (Germany Roche Company) and dissolve it in pH7.6, 100mM phosphate buffer, stir at room temperature for 15 minutes, then add 125mg water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) powder and 75mg water-soluble N-hydroxysuccinimide (NHS), gently stir at room temperature in the dark After 180 minutes, after the reaction is over, put it into a dialysis bag (free permeable molecular size <14KD), dialyze at 4°C, change the medium every 6 hours, and continue for 6-8 times to obtain the desired protein conjugate CMPF-BSA.

[0033] (S.2) Activation...

Embodiment 2

[0038] A latex microsphere-protein-CMPF conjugate, said conjugate is a coupling product obtained by successive dehydration of CMPF, hemocyanin (KLH) and latex microspheres under the action of a coupling agent, and its preparation method As follows:

[0039] (S.1) CMPF and protein coupling

[0040] Weigh CMPF (100mg), dissolve it with 10mL DMSO, weigh 30mg hemocyanin (KLH) (Germany Roche Company) and dissolve it in pH7.8, 100mM phosphate buffer, stir at room temperature for 15 minutes, then add 100mg Water-soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) powder and 80mg water-soluble N-hydroxysuccinimide (NHS), gently stir at room temperature for 180 Minutes, after the reaction is over, put it into a dialysis bag (free permeable molecular size <14KD), dialyze at 4°C, change the solution every 6 hours, 6-8 times in a row, and you can get the desired protein conjugate CMPF -KLH.

[0041] (S.2) Activation of CMPF-KLH

[0042] Dilute the CMPF-KLH conjug...

Embodiment 3

[0046] A kind of latex microsphere-protein-CMPF conjugate, described conjugate is the coupling product that CMPF and ovalbumin (OVA) and latex microsphere dehydrate successively under the effect of coupling agent, and its preparation method is as follows Shown:

[0047] (S.1) CMPF and protein coupling

[0048] Weigh CMPF (100mg), dissolve it with 10mL dimethylsulfoxide, weigh 50mg ovalbumin (OVA) (Germany Roche Company) and dissolve it in pH7.6, 100mM phosphate buffer, stir at room temperature for 15 minutes, then add 120mg water-soluble 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) powder and 100 mg water-soluble N-hydroxysuccinimide (NHS), stirred gently at room temperature for 180 minutes in the dark After the reaction, put it into a dialysis bag (free permeable molecule size <14KD), dialyze at 4°C, change the liquid once every 6 hours, and continue for 6-8 times to obtain the desired protein conjugate CMPF- OVA.

[0049] (S.2) Activation of CMPF-prot...

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Abstract

The invention relates to the technical field of biological detection, in particular to a latex microsphere-protein-CMPF conjugate and a preparation method thereof, the conjugate is a coupling productobtained by sequentially dehydrating CMPF, protein and latex microspheres under the action of a coupling agent, and the conjugate can be used in a kit for determining the content of CMPF in human serum by a latex enhanced immunoturbidimetry. The invention overcomes the defects that a reagent in a homogeneous enzyme immunoassay CMPF detection kit in the prior art is poorer in stability; meanwhile,the defect that the yield of reaction products is low due to the fact that the activity of carrier enzyme is lost in the reaction process is overcome, unstable enzyme conjugates in the prior art are replaced by latex microsphere conjugates with good stability, the stability of a reagent is improved, the activity cannot be lost in the reaction process, and interference to a test result is prevented; the kit provided by the invention can also realize rapid high-throughput detection of a sample, and meets clinical detection requirements.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a latex microsphere-protein-CMPF conjugate used for latex-enhanced immune turbidimetry to determine the content of CMPF in human serum, a preparation method thereof, and a kit for determining the content of CMPF in serum . Background technique [0002] The small molecule CMPF involved in the present invention is an endogenous furan fatty acid metabolite, is one of the main uremic toxins, and belongs to protein-binding toxoids. Some recent clinical research results show that the level of CMPF in serum is significantly higher in patients with gestational diabetes mellitus (GDM), impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). Shan Zhang et al. (Shan Zhang et al. Circulating 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) levels are associated with hyperglycemia and β cell dysfunction in a Chinese population. Scientific Reports 7: 3114.) ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/6893G01N33/531
Inventor 朱永良杨晓东陈佳明缪刘
Owner HANGZHOU BOPU MEDICAL TECH