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ELISA detection kit based on molecular chaperone adding technology and method thereof

A molecular chaperone and kit technology, applied in the biological field, can solve the problems of protein denaturation and inactivation, and achieve the effect of low cost and simple technology

Inactive Publication Date: 2010-02-17
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Simulate the in vivo environment. In the actual production of the ELISA kit, the components of the kit, such as antibodies, standards, and enzyme markers, are all protein components. During transportation and packaging, due to the relatively low protein concentration, the reaction conditions External factors such as temperature and environment are unstable, and these proteins are easily affected and denatured and inactivated

Method used

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  • ELISA detection kit based on molecular chaperone adding technology and method thereof
  • ELISA detection kit based on molecular chaperone adding technology and method thereof
  • ELISA detection kit based on molecular chaperone adding technology and method thereof

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Experimental program
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Embodiment 1

[0031] The preparation of embodiment 1HSP60

[0032] HSP60 protein exists widely in various organisms, and its encoding gene can generally be obtained from organism cDNA by RT-PCR. In the present invention, the plasmid pDNA3.1-HSP60 is used as a template to amplify.

[0033] 1. Clone PET28a-HSP60:

[0034] The expression vector PET28a was selected as the cloning vector (see figure 1 ), according to its multi-cloning site and HSP60 sequence characteristics, design primers at both ends to PCR amplify the gene fragment expressing HSP60, with NdeI and XhoI restriction sites at both ends, and insert NdeI and XhoI digested Pet28a after restriction digestion , to obtain plasmid PET28a-HSP60;

[0035] 2. Expression and purification of PET28a-HSP60:

[0036] The plasmid HIS-HSP60 was expressed in E. coli Rossetta. First, through the identification of small amount of expression, the induced strain has an obvious specific band at about 60KD compared with the uninduced strain (see ...

Embodiment 2

[0042] Example 2 Performance comparison of NGAL ELISA kits with and without HSP60 added

[0043] 1. Sensitivity comparison: prepare NGAL standard solutions with concentrations of 0.0002, 0.002, 0.04, 0.10, 0.20, 0.50, 2.00, and 5.00 μg / ml. Using the supplemented HSP60 and unadded NGAL ELSIA kits that were placed at 4°C for two months, respectively, were operated according to the indirect competitive ELISA method. Inhibitory medium concentration (IC50) and minimum detection limit (IC10).

[0044] Inhibition rate (%) = (OD max -OD min )-(OD x -OD min ) / (OD max -OD min ) × 100 (see Table 5 for the results)

[0045] Where: OD max Absorbance value without NGAL, OD x is the absorbance value when NGAL is added, OD min is the absorbance value of the blank control well.

[0046] The standard inhibition curve of NGAL was established by IC-ELISA method. In the concentration range of 0.002~0.5μg / ml, the logarithm of the concentration in this range and the inhibition rate were ...

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Abstract

The invention relates to an ELISA detection kit based on a molecular chaperone adding technology and a method thereof. The ELISA detection kit is prepared by adding molecular chaperone protein purified by in vitro expression to various components in ELISA of NGAL. The molecular chaperone protein provided by the invention has extremely good solubility and extremely high activity; and compared withkits without protein addition, the ELISA detection kit has the advantages of good stability, long storage life and high accuracy. Motif of the molecular chaperone is derived from human protein and purification host is Escherichia coli, thus cross reaction does not exist in application; and probability of events affecting activity of antibodies, standard substances and other substances by certain extreme condition is greatly lowered due to characteristics of the molecular chaperone of the protein.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method for improving the quality of an ELISA detection kit by using a molecular chaperone as an additive. Background technique [0002] During protein expression, many proteins do not fold in the correct way. Misfolded proteins often expose carbon-rich amino acids on the surface, rather than wrapping them up. These carbon-rich groups bind tightly with similar groups in other proteins to form macromolecular polymers. These polymers themselves lose their biological activity and can aggregate and precipitate surrounding proteins, which are often lethal when present in cells. [0003] A chaperone is a protein that guides the correct folding of proteins. When proteins fold, they protect protein molecules from interference by other proteins. Many molecular chaperones are heat shock proteins, which are synthesized in large quantities when cells are heated. Heat shock reduces the stability of most...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/53
Inventor 华权高
Owner CUSABIO TECH LLC
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