Homotype cysteine detecting method

A homocysteine ​​and amino acid technology, applied in the field of small molecules to be detected, can solve problems such as poor preparation, infeasibility of antibody preparation, and invalid expression of antibodies

Inactive Publication Date: 2009-02-04
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polymorphisms in the gene for tetrahydrofolate reductase lead to folic acid deficiency which affects the sensitivity of homocysteine ​​levels (Boers, et al., J. Inher. Metab. Dis., 20:301-306 (1997) )
First, for many methods, specific binders are not easy to prepare and lack specificity, which affects the specificity of detection
Although this defect can be made up by preparing high-molecular-weight antibodies, especially purified monoclonal antibodies, this process takes a lot of time and costs a lot
In addition, for some small-molecular-weight analytes, the option of preparing antibodies becomes infeasible, because small-molecular ...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0235] Preparation of mutated SAH hydrolase-encoding nucleic acid

[0236] Human SAH hydrolase gene encoding (SEQ ID No. 1) EcoR I recombined expression plasmid PKK223-3 (Pharmacia Biotech, Piscataway, N.J.). Plasmid PKK223-3 includes a strong TAC promoter upstream of the multiple cloning site and a strong rrnB ribosomal terminator downstream controlling protein expression. An expression plasmid containing the SAH hydrolase gene was transcribed into E. coli single-chain JM109 (Invitrogen, Carlsbad, Calif.). The direct mutation of the SAH hydrolase site is through the following two forms: 1) single-stranded DNA mutation by M13 method 2) double-stranded DNA mutation by PCP method.

[0237] ssDNA variation

[0238] Single-stranded DNA mutation is a method developed by Taylor et al, Nucleic Acids Res., 13:8765-8785 (1985), which uses inactive Ncil to unwind sulfur-containing DNA strands. The Sculptor.TM. in vitro mutation system RPN1526 (Amersham Life science, UK) was used. The ...

example 2

[0285] Large-scale expression and purification of wild-type and mutant SAH hydrolases

[0286] purification

[0287] IPTG mutagenesis E.Coli JM109 medium (PKK-223-3 carrier combined with SAH hydrolase gene) cell extract was mixed with DEAE-cellulose (Sigma, St.Louis, Mo.), mixed with 0.1M sodium phosphate, pH 7.2, the buffer solution containing 1mM EDTA equilibrated the mixture of cell extract and DEAE-cellulose to vacuum filter, washed with 3 volumes of Buffer A. The filtrate was precipitated with amino acid salts. The filtered protein was collected by centrifugation at 13,000 rpm and redissolved in 50 mM phosphate buffer pH 7.2 containing 1 mM EDTA. This protein was dyed through a DEAE-sephrose ion exchange column (2.5times, 100cm) (Pharmacial Biotech, Piscataway, N.J.), and then washed with Nacl DEAE-Sepharose ion exchange column (2.5.times.30cm) of different gradient concentrations, obtained from DEAE -The main protein peak eluted by Sepharose ion exchange is detected b...

example 3

[0295] Reagent preparation

[0296] Preparation of fluorescently labeled adenine and SAH analogs

[0297] method 1

[0298]ADO-5′-carboxylic acid (Sigma, St.Louis, Mo.) derived from 9-hydroxymethylanthracene (HMA) (Fluka, Buchs, Switzerland), added 50mg HOBT, dry chemical staining under nitrogen, added 300mgN -ethyl-N'(3-dimethylaminopropyl)carbodiimide hydrochloride in 300ml of chloroform, add 5ml of triethylamine. The above liquid was left at 0°C for 30 minutes, 200mg of HMA and 100ml of chloroform were added, the mixture was left at room temperature for 10 minutes, dried in a nitrogen stream, and the residue was dissolved in 10ml of HPLC mobile phase (methanol:water=90:10). 1ml of the above liquid was injected into a semi-prepative column (Econosphere, C18, 7.times.300mm, Alltech, Dearfield, Ill.), using the equilibrium filtration method, the flow rate was 2m / min, 260nm was the detection peak, and the fluorescence analysis was at 415nm and 365nm, the substance at 260nm a...

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PUM

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Abstract

The present invention relates to a method of producing homocysteine diagnostic kit, which is based on the small molecule capture technology. The method adopts the combination of gene mutation enzyme and small molecule substance to be detected; the gene mutation enzyme is named as small molecule capture enzyme; and the small molecule capture enzyme after mutation and modification maintain and even enhance the affinity to the substrate or the small molecule substance to be detected, but the catalytic capability and the decomposition capacity of the small molecule capture enzyme after mutation and modification to the substrate or the small molecule substance to be detected is weakened. The invention provides a method of researching and producing the homocysteine (HCY) diagnostic kit, which is based on the small molecule capture technology, and also provides mutated S-adenosine homocysteine hydrolase; and the specific S-adenosine homocysteine (SAH) capture enzyme can fully maintain and even enhance the affinity to the S-adenosine homocysteine (SAH), but the catalytic capability to the HCY or the S-adenosyl homocysteine (SAH) is weakened. The method is in particular suitable for small molecule substances, such as inorganic ions, amino acids, polypeptides, nucleotides, oligonucleotide, vitamins, monosaccharides, oligosaccharides, lipids and organic acids.

Description

technical field [0001] The invention relates to components and a detection method, and is mainly aimed at small molecules to be detected. The detection method for homocysteine ​​is developed and produced based on the small molecule capture technology. This method uses a gene mutant enzyme to combine with the small molecule substance to be tested. This gene mutant enzyme is called a small molecule capture enzyme. These mutated and modified small molecules The capture enzyme retains or even enhances its affinity to the substrate or the small molecule substance to be tested, but weakens its ability to catalyze and decompose the affinity to the substrate or the small molecule substance to be tested. The method of developing and producing homocysteine ​​(HCY) diagnostic kit based on small molecule capture technology provides this, and also provides mutant S-adenosyl homocysteine ​​hydrolase, specific S-adenosyl homocysteine Acid (SAH) trapping enzymes can fully retain or even enha...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/00C12Q1/68
Inventor 刘希
Owner BEIJING STRONG BIOTECH INC
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