Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry

A technology of alpha-fetoprotein and immune turbidimetry, which is applied in the field of medical immunological in vitro diagnosis, can solve the problems of small supply, low degree of automation, and increased cost of manufacturers, and achieve easy control of batch differences, easy clinical promotion, uniformity and stability The effect of using

Active Publication Date: 2012-09-12
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the sensitivity and linearity of the chemiluminescence method are relatively high, and the detection range can reach 1-1000 ng/mL, but the reagent cost is relatively high and the supporting large-scale instruments are relatively expensive
Enzyme-linked immunoassay is widely used, but this method is a heterogeneous immunoassay system. Its advantages are: high sensitivity and high level of accuracy can be achieved by using excessive amounts of immobilized antibodies and labeled antibodies; the disadvantages are: the determination process Washing and separation steps are required in the process, which takes a long time and lacks unit test running time; the degree of automation is not high, and the difference between batches and repeatability are relatively large; different steps need to be equipped with a variety of special equipment, which increases the product to a certain extent. cost
However, it is rare to apply it to cl...

Method used

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  • Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry
  • Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry
  • Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Preparation of chicken anti-human alpha-fetoprotein polyclonal antibody (IgY antibody) by affinity purification

[0043] Preparation of chicken anti-human alpha-fetoprotein polyclonal IgY antibody:

[0044] Eight to 10 hens were used in each immunization experiment, and a pair of eggs were collected from each hen before immunization began. IgY purified from these eggs served as control IgY. 0.1 mg of highly purified human alpha-fetoprotein antigen (from Fitzgerald) was dissolved in pH 7.8 phosphate buffer, emulsified with Freund's complete adjuvant, and injected into the breast muscle of hens with repeated injections every 4 weeks. 10 weeks after initiation of injections, eggs were collected. Yolks were separated from the eggs, and the IgY fraction from the yolks was initially isolated by ammonium chloride precipitation.

[0045] Purification of chicken anti-human alpha-fetoprotein polyclonal IgY antibody:

[0046] First, the separation column used for af...

Embodiment 2

[0049] Embodiment 2: Preparation of alpha-fetoprotein R2 reagent

[0050] The process is divided into three steps: antibody cross-linking, latex washing, latex suspension

[0051] Antibody cross-linking: Dilute 0.5 mg of the above-prepared IgY antibody with 5 mL of 0.1 M phosphate buffer (pH7.8), add surface-modified carboxyl groups, polystyrene latex solution with a diameter of 200 nm (from Merck), and then add 5 mg of EDAC , reacted at 37° C. for 6 hours, added 0.5 mL of 0.1 M glycine buffer (pH 8.5) and stirred for 1 hour to terminate the reaction.

[0052] Latex cleaning: Centrifuge to remove the supernatant to remove excess antibodies, cross-linking agents and by-products of cross-linking reactions, etc., and the bottom sediment is the coated latex. Wash 3 times with 20 mL of 50 mM glycine-NaOH buffer.

[0053] Latex suspension: Add 20mL of the same glycine-NaOH buffer solution to the above precipitate, ultrasonicate for 4min, and mix to obtain a uniform milky white lat...

Embodiment 3

[0055] Example 3: Preparation of alpha-fetoprotein R1 reagent

[0056] Add 0.9% sodium chloride, 0.2% Tween 20, 5% PEG-6000, 0.2% BSA, 0.5% EDTA, 0.1% sodium azide to 50mM Tris-HCl buffer solution at pH 7.2, stir evenly to adjust the pH To 8.0, that is the R1 reagent.

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Abstract

The invention relates to a kit for determination of human alpha-fetoprotein content by a latex-enhanced immunoturbidimetry. The kit provided by the invention comprises an alpha-fetoprotein R1 reagent, an alpha-fetoprotein R2 reagent and an alpha-fetoprotein calibrator, wherein the alpha-fetoprotein R1 reagent comprises one or more electrolytes, a coagulation accelerator, one or more stabilizing agents, one or more surfactants, an antiseptic and a buffer solution; the alpha-fetoprotein R2 reagent comprises latex particles coated by polyclonal antibodies capable of resisting a human alpha-fetoprotein, one or more electrolytes, one or more stabilizing agents, one or more surfactants, an antiseptic and a buffer solution; and the alpha-fetoprotein calibrator comprises an antiseptic, one or more electrolytes, one or more stabilizing agents, alpha-fetoprotein antigens and a buffer solution. The kit has strong specificity and high sensitivity, is homogeneous and stable, can be used fast and conveniently and can be used for various biochemical analyzers.

Description

technical field [0001] The invention relates to a kit for measuring alpha-fetoprotein content in human body fluids (including serum and amniotic fluid) by using a latex-enhanced immune turbidimetric method, which belongs to the field of in vitro medical immunodiagnosis. Background technique [0002] Alpha-fetoprotein (α-fetoprotein, or AFP), with a molecular weight of 68kD, is a polypeptide chain glycoprotein consisting of 590 amino acids and containing 4% sugar. The gene of alpha-fetoprotein is located in the 4q11-q22 region of the autosome, the gene is about 20kb long, contains 15 exons, and 14 introns, and belongs to the same family as serum albumin and vitamin D binding protein, and is related to albumin 50% of the amino acids are the same, the isoelectric point is 4.7-4.8, and it is located between albumin and α-globulin during electrophoresis. [0003] During fetal development, the liver is the main site for the synthesis of alpha-fetoprotein, followed by the yolk sac...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 徐丽高爱民刘希
Owner BEIJING STRONG BIOTECH INC
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