Human serum albumin (HSA) quantitative detection kit based on time-resolved fluorescence immunoassay technology

A technology of time-resolved fluorescence and human serum albumin, applied in the fields of nanobiology and bioanalytical chemistry, can solve the problems of no literature reports, etc., and achieve the effect of wide range of standard curve, good repeatability, and no radioactive pollution

Inactive Publication Date: 2013-06-19
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, TRFIA technology has been widely used in various clinical detectio

Method used

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  • Human serum albumin (HSA) quantitative detection kit based on time-resolved fluorescence immunoassay technology
  • Human serum albumin (HSA) quantitative detection kit based on time-resolved fluorescence immunoassay technology
  • Human serum albumin (HSA) quantitative detection kit based on time-resolved fluorescence immunoassay technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Preparation of the kit of the present invention

[0027] (1) Preparation steps of anti-HSA microwell reaction plate:

[0028] Coating: Dilute the anti-HSA antibody to 3ug / mL with coating buffer, coat 100uL / well onto a 96-well microplate, vortex and mix, and place overnight at 4°C. Take it out and wash the plate 4 times the next day.

[0029] Blocking: Add 150ml of blocking solution to the washed microwell plate, shake and mix well, and place at 4°C overnight.

[0030] Storage: The next day, take the plate and pat it dry. After vacuuming, store at 2-8°C.

[0031] The coating buffer used above is: Tris-HCl (50 mmol / L), pH7.2 buffer.

[0032] The blocking buffer used above is: Tris-HCl (50mmol / L), containing 0.5% hydrolyzed casein, 10% sucrose, pH7.2 buffer.

[0033] (2) The preparation steps of europium-labeled anti-HSA antibody are:

[0034] Antibody purification and concentration: Add 1 mg of anti-HSA monoclonal antibody to 0.5 ml of labeling buffer (5...

Embodiment 2

[0043] Embodiment 2 The usage method of the kit of the present invention

[0044] (1) Sample collection

[0045] Take 100uL of finished or semi-finished vaccines or other biological preparations containing HSA to be tested, and store them at 2-8°C for one week. For longer storage, store at -20°C.

[0046] (2) Preparation of reagents

[0047] 1) Washing liquid: Mix 50mL concentrated washing liquid and 1200mL deionized water as working washing liquid.

[0048] 2) Marker working solution: Dissolve each bottle of marker with 1 mL of deionized water one hour before use, and dilute 1:50 times with analysis buffer as the europium-labeled antibody working solution.

[0049] 3) 96-well micro-well reaction plate: take it out and equilibrate at room temperature for 15 minutes before use.

[0050] (3) Operation steps

[0051] 1) Take 100uL of each well of the calibrator and the sample to be tested, and incubate with shaking at room temperature for 60min. After the reaction, the plat...

Embodiment 3

[0055] Embodiment 3 The methodological test of the kit of the present invention

[0056] The test kit prepared in Example 1 is tested according to the routine manufacturing and testing procedures in the art, and the results are as follows:

[0057] (1) Accuracy

[0058] After analyzing and measuring the national standard substance of the calibrator and the corresponding concentration simultaneously, with double logarithmic mathematical model (log-log) fitting, two dose-response curves are basically parallel ( figure 1 : the standard substance dose-response curve prepared for pure HSA product of SIGMA company (product number: A8763, agarose gel electrophoresis detection purity≥99%); figure 2 : is the dose-response curve of the calibrator; Cps is the fluorescence count per second), the slopes of the two curves are 0.98 and 0.97 respectively (t test P>0.05). With the HSA national standard as the reference substance, the ratio of the measured potency to the marked potency of th...

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Abstract

The invention discloses a human serum albumin (HSA) quantitative detection kit based on a time-resolved fluorescence immunoassay (TRFIA) technology. The kit comprises: a 96-pore microporous reaction plate coated with anti-HSA antibody, a calibrator, europium-labeled anti-HSA antibody, an assay buffer, a washing solution, and an enhancing solution. With a double-antibody sandwich immune reaction, a monoclonal antibody composition with coated anti-HSA antibody-HSA molecule-europium-labeled anti-HAS is formed; the enhancing solution is added, and value measuring is carried out by using a time resolution instrument. The method provided by the invention has the technical advantages of TRFIA, and can satisfy the requirement of quantitative detection of high-concentration (microgram-grade) HSA in biological product production. Therefore, the dilution of a product requiring detection during application is avoided, such that working time is greatly reduced, and working efficiency is greatly improved.

Description

technical field [0001] The invention belongs to the field of bioanalytical chemistry and nanobiotechnology, in particular to a HSA quantitative detection kit based on time-resolved fluorescent immunoassay technology, which can be used to determine the concentration of human serum albumin contained in vaccine-related biological products , to assist the control of product quality in the production process. Background technique [0002] Human serum albumin (HSA) is a protein in human plasma, and its non-glycosylated single-chain polypeptide contains 585 amino acids with a molecular weight of 66 kD. Its concentration in plasma is 42g / L, accounting for about 60% of total plasma protein. Human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules in body fluids, while maintaining normal blood osmotic pressure. [0003] In the production of biological products, especially vaccines, HSA, as a commonly used ...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 万勇吴英松陈殷陈少琅黄黉姚小艳
Owner DAAN GENE CO LTD
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