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Apolipoprotein E ELISA reagent box and method of producing the same

An apolipoprotein and kit technology, which can be used in biological testing, material testing products, measuring devices, etc., and can solve problems such as difficult clinical diagnosis and promotion.

Inactive Publication Date: 2008-05-14
杭州浙大生科生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The method for determining ApoE4 genotype and gene dosage in human tissue fluid has isoform focusing electrophoresis (KaneJW, Gowland E, 1986, A method for the identification of apolipoprotein E isoform semploying chemical precipitation and flat bed isoelectric focusing in agarose, AnnClin Biochem, 23: 509-513), DNA sequencing (Parker S et al, 1993, Application of denaturing gradient gel electrophoresis to detect DNA sequence difference encoding apolipoprotein E isoforms, Genomics, 16: 245-247), RT-PCR (Bemard PS et al, 1999, Color multiplexing hybridization probes using the apolipoprotein E locus asa model system for genotyping, Anal Biochem, 273: 221-228), etc. These scientific research methods require strong technology and are difficult to promote in clinical diagnosis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Apolipoprotein E4 ELISA kit of the present invention, its composition comprises anti-human ApoE4 monoclonal antibody-coated ELISA plate, enzyme-labeled antibody, ApoE4 standard freeze-dried powder, sample diluent, TMB substrate chromogenic solution, concentrated Washing solution and termination reaction solution; wherein:

[0028] Enzyme-labeled antibody: horseradish peroxidase-labeled anti-human ApoE polyclonal antibody;

[0029] TMB substrate chromogenic solution: 2.08mmol / L 3,3',5,5'-tetramethylbenzidine, 12mmol / L hydrogen peroxide, 10mmol / L β-cyclodextrin, 0.1mol / L acetic acid at pH5.0 buffer;

[0030] Sample diluent: 10mmol / L pH7.4-7.6 phosphate buffer containing 1% BSA, 0.05% Tween-20 and 1mg / L gentamicin;

[0031] Concentrated washing solution: 0.1mol / L pH7.4-7.6 phosphate buffer containing 1% fetal bovine serum, 0.5% Tween-20 and 20mg / L gentamicin;

[0032]Termination reaction solution: 1.0mol / L H 2 SO 4 .

[0033] The above enzyme plate is 96 wells (12 st...

Embodiment 2

[0129] Example 2 The analytical precision (CV%) of the actual serum sample detection kit and the determination of ApoE4 genotype and gene dosage

[0130] Measure the ApoE4 concentration in the serum samples prepared by randomly drawing three blood samples for physical examination with the method of using the aforementioned apolipoprotein E4 ELISA kit and calculate the analytical precision (Table 1) of the method and the serum samples of 92 routine physical examination healthy persons ApoE4 genotype was determined (Table 2).

[0131] Table 1. Intra-analytical precision of the ELISA method of the kit

[0132] serum sample

[0133] Table 2. ELISA method to determine the genotype of ApoE4

[0134]

[0135] The detection results show that the detection precision of the kit of the present invention meets the standard requirements of the ELISA quality index, the analysis sensitivity is less than 8.0ng / ml, the genotype of ApoE4 can be determined, and it is suitabl...

Embodiment 3

[0136] Embodiment 3 Application kit of the present invention detects 451 cases of age 60-90 years old physical examination elderly (wherein 95 cases of normal controls (42 cases of men and 53 cases of women), 125 cases of non-fatty liver (nonfatty liver, NFL) group ( 59 males, 66 females), 126 nonalcoholic fatty liver (NAFL) group (60 males, 66 females), 105 NAFL+MS (metabolicsyndrome, MS) group (Male 52 examples, female 53 examples)) ApoE4 level in serum, the result is as follows:

[0137] group

normal control group

NFL group

NAFL group

MS+NAFL group

Number of cases

95

125

126

105

ApoE4 concentration

(ng / ml)

35.0±22.7

34.4±27.0

48.4±35.3 *

49.8±21.4 *

[0138] Note: * means significant difference (ρ<0.01)

[0139] The results suggest that the concentration of ApoE4 in the blood also tends to increase in the elderly patients with lipid metabolism disorders, and th...

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Abstract

The invention discloses an apolipoprotein E4 ELISA kit and a preparation method thereof, the invention applies the purified human apolipoprotein E4 to prepare an anti-human ApoE4 antibody, and an ELISA plate is coated after the compatible purification. The kit composition includes the ELISA plate coated by the anti-human ApoE4 monoclonal antibody, an enzyme-labeled antibody, ApoE4 standard frozen powder, sample dilution solution, TMB substrate color developing solution, concentration washing liquid and reaction termination liquid. The ApoE4 in the antibody capture standard solution or the sample solution which is fixed on the microporous surface of the ELISA plate is identified by and combined with the enzyme-labeled antibody, so as to form the antibody-ApoE4-antibody-enzyme compound, the absorbance is measured after the reaction and color development of the enzyme and the substrate and the concentration of the ApoE4 in the sample can be calculated by the standard curve. The kit can detect the consistency of the apolipoprotein E4 in human serum, plasma or cerebrospinal fluid, the invention has the advantages of sensitivity, rapidness, simpleness and accuracy, so the invention provides the effective means for in vitro diagnosis and determination of ApoE4 gene type and the gene dosage.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent assay (ELISA) kit for quantitatively detecting apolipoprotein E4 in human serum, blood or cerebrospinal fluid and a preparation method thereof. Background technique [0002] Apolipoprotein (ApoE) is a single-chain protein composed of 299 amino acids of known sequence, with a molecular weight of 34.2kDa. It is the main lipoprotein in human plasma proteins such as chylomicrons, very low-density lipoprotein and high-density lipoprotein. Partially synthesized by liver, brain and other organ tissues. ApoE is involved in the transport of triglyceride-cholesterol between cells and metabolism in vivo, and plays an important role in lipoprotein metabolism. ApoE is also involved in the regulation of the activity of several enzymes and enzymes in the process of lipid metabolism in vivo (Mahley RW, Rall SCJr, 2000, Apolipoprotein E: far more than a lipid transport protein, Annu RevGenomics Human Genet, 1: 50...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/543
Inventor 吴善东
Owner 杭州浙大生科生物技术有限公司
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