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Improved solid phase fluorescent immunizing detecting method

An immunoassay method and solid-phase fluorescence technology, which is applied in the field of improved solid-phase fluorescence immunoassay, can solve the problems of affecting the repeatability of measurement results, affecting the sensitivity of measurement results, and greatly affecting the dryness of carriers, so as to avoid instability and shorten Effects of detection time, improved sensitivity and accuracy

Active Publication Date: 2011-09-07
上海北加生化试剂有限公司
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AI Technical Summary

Problems solved by technology

However, this method uses carrier dry measurement, which requires strong drying equipment. The humidity of the measurement environment has a great influence on the dryness of the carrier, which affects the repeatability of the measurement results.
Moreover, the fluorescence excited by the laser is only limited to a certain point at the bottom of the micropores of the carrier, so that the fluorescent markers bound to the surface of the micropores of the carrier cannot be used, resulting in a low measured fluorescence value, which affects the sensitivity of the measurement results and limits the promotion.

Method used

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  • Improved solid phase fluorescent immunizing detecting method
  • Improved solid phase fluorescent immunizing detecting method
  • Improved solid phase fluorescent immunizing detecting method

Examples

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Embodiment 1

[0044] Europium chelate (BCPDA-Eu 3+ )-labeled antibody, using the double-antibody sandwich method to measure alpha-fetoprotein (AFP) in serum as an example, the specific steps are as follows:

[0045] A.Eu 3+ - Labeling of BCPDA-goat anti-human AFP-IgG antibody:

[0046] 1) BCPDA-labeled goat anti-human AFP-IgG antibody:

[0047] Take 100 μl of goat anti-human AFP-IgG antibody (1.0 mg / ml Medix Biochemical) and add it to 900 μl of carbonate buffer solution with pH 9.1 to prepare 0.1 mg / ml goat anti-human AFP-IgG antibody carbonate solution. At the same time, 1 mg of BCPDA was dissolved in 50 μl of dimethylformamide (DMF) to obtain a BCPDA solution, and the BCPDA solution was equally divided into 4 parts. Add 1 part of BCPDA solution to the 0.1 mg / ml goat anti-human AFP-IgG antibody carbonate solution with stirring every 2 minutes, and complete the addition within 8 minutes. Stir the reaction for 30 minutes, take G-50 glucose as a gel and a 1cm×15cm layer suction column, an...

Embodiment 2

[0057] Using europium-containing chelate-avidin-biotin-labeled antibody to measure serum prostate-specific antigen (PSA) by the double-antibody sandwich method as an example, the specific detection steps are:

[0058] A.Eu 3+ -Preparation of BCPDA: Tris-HCl buffer (containing Eu 3+ 10 -6 mol L -1 ) to dilute the hydrolyzed BCPDA solution, place the above solution in a constant temperature water bath at 37°C for a heating reaction for 2 hours, and obtain Eu 3+ - BCPDA.

[0059] B.Eu 3+ -Preparation of BCPDA chelate-labeled streptavidin (SA):

[0060] Take 1mg SA and dissolve it in 0.5ml 0.05mol / L pH9.6 carbonate buffer, add Eu which is equivalent to 12 times the molar amount of SA 3+ - BCPDA chelate, react overnight at 4° C., the product is subjected to SephadexG-50 column chromatography, and 0.05 mol / L Tris-HCl solution (containing 0.9% NaCl) of pH 7.75 is rinsed. Purified Eu 3+ - BCPDA-SA, marker ratio Eu 3+ / SA is 10.

[0061] C. Preparation of biotin-labeled goat ...

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Abstract

The invention relates to an improved test method for solid phase fluorescence immunity. The invention is characterized in that the supplied eluent can be utilized to mark the immune complex and form a dissociation labeled substances, and the testing of the dissociation labeled substances can be carried on. The solute of the eluent is a negative ion surface active agent SDS, and the menstruum of the eluent is the water or Tris aqueous solution; and according to a chemical cross-linking or physical adsorption method, a dissociation labeled substances is formed, the sensitivity of the test is improved. The invention solves the pollution problem of lanthanon ion in the solid phase time-resolved fluorescence immunity, the drying craft in the prior art is spurned, and the repetitiveness and stability of the test are improved.

Description

technical field [0001] The invention relates to an improved solid-phase fluorescence immunoassay method, specifically, the invention is an improved solid-phase fluorescence immunoassay method belonging to the field of immunoassay. It belongs to the field of fluorescence immunoassay. Background technique [0002] Time-resolved fluorescence analysis (TRIFA) is a new non-radioactive microanalysis technique. The theory of "time-resolved fluorescent immunoassay" was proposed by Finns Soini and Hemmia in 1979. Its characteristic is that the use of trivalent rare earth ions and their chelates is a tracer with a long fluorescence emission time, and the method of avoiding the natural fluorescence of the sample during the measurement period is used to replace fluorescent substances, isotopes and enzymes to label biologically active substances. . Due to its high sensitivity (up to 10pmol / L), easy operation, stable tracer, wide range of standard curve, no interference from natural fl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/58G01N33/543G01N21/00
Inventor 张瑞镐忻鼎广
Owner 上海北加生化试剂有限公司
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