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Preparation method for rheumatoid factor detection reagent

A technology for rheumatoid factor and detection reagent, applied in the field of preparation of rheumatoid factor detection reagent, can solve the problems of limited sample volume, high cost, low sensitivity, etc., and achieves convenient large-scale industrial production, low production cost and simple process. Effect

Active Publication Date: 2010-02-10
BEIJING LEADMAN BIOCHEM
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Although the rheumatoid factor detection reagent obtained by the former method has high reaction specificity, it has the problem of low sensitivity
For example, Coleman L. et al. use gradient electrophoresis to separate Fab and Fc fragments (see Non-Patent Document 1), but this method requires the preparation of electrophoresis equipment, which requires high pH and ionic strength of the separation system, and the processing sample volume Limited, there is a problem that the operation process is time-consuming, and the cost is high, and it is difficult to be used in large-scale industrial production

Method used

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  • Preparation method for rheumatoid factor detection reagent
  • Preparation method for rheumatoid factor detection reagent
  • Preparation method for rheumatoid factor detection reagent

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preparation example Construction

[0027] The preparation method of the rheumatoid factor detection reagent of the present invention comprises the following steps: coupling IgG molecules with latex particles to obtain IgG molecule sensitized latex; digesting and decomposing the obtained IgG molecule sensitized latex with enzymes to obtain Fab fragments and Fc fragments A mixture of sensitized latex; separating the Fab fragment and Fc fragment sensitized latex to obtain the Fc fragment sensitized latex; making the Fc fragment sensitized latex thermal fusion to obtain denatured Fc fragment sensitized latex, and making it into a rheumatoid factor detection reagent .

[0028] The above-mentioned denaturation can be carried out by conventional methods, not limited to thermal fusion methods.

[0029] In addition, after separating the Fab fragment and the Fc fragment sensitized latex, further purification may be performed.

[0030] In the preparation method of the rheumatoid factor detection reagent of the present in...

Embodiment

[0041] The present invention is further described by the following examples, but it should not be construed as a limitation of the present invention.

[0042] [Experimental Materials]

[0043] Latex carrier (manufactured by JSR Corporation);

[0044] Papain (purchased from MERCK company, CB5125);

[0045] Human IgG (purchased from Chinese Academy of Military Medical Sciences);

[0046] Control 1 (Human IgG Fc Fragment (Human IgG Fc Fragment Plasma (trade name), manufactured by Calbiochem);

[0047] Reference substance 2 (RF detection reagent obtained by coupling and denaturing the Fc fragment of reference substance 1 to latex particles);

[0048] Reference substance 3 (RF detection kit (RHEUMATOID FACTOR Reagent kit (trade name)), manufactured by Randox Company, the RF detection kit of immunoturbidimetric method, the detection principle is to detect with anti-human rheumatism factor antibody and RF generation immune reaction );

[0049] Centrifuge (Beckman Allegra 64R hig...

preparation example 1

[0051] (A) Coupling of IgG to latex particles

[0052] 1) Weigh 10.6mg of human IgG and dissolve it in 5ml of 100mM PBS buffer to make solution 1;

[0053] 2) Take 0.1 g of 10% (latex particle content) polymethyl methacrylate latex with a particle diameter of 135 nm, and use 50 mM PBS buffer solution to dissolve it to 5 ml to make solution 2;

[0054] 3) Slowly add solution 2 to solution 1, the temperature of the water bath is 40°C, and stir slowly (rotating speed 100rpm) for 2 hours;

[0055] 4) After centrifugation at 10,000g for 20 minutes, wash with 10ml of 50mM PBS buffer, and resuspend with pH 8.2200mM glycine buffer (under the condition of 200w-5min) to make latex 1.

[0056] (B) Digestion of IgG-sensitized latex

[0057] 1) Add 2KU / L papain to latex emulsion 1, and act at 42°C for 2 hours;

[0058] 2) Add 2KU / L of papain and continue to act for 2 hours to make digestive juice 1.

[0059] (C) Separation of Fc fragment sensitized latex and Fab fragment

[0060] 1) C...

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Abstract

The invention provides a preparation method for rheumatoid factor detection reagent. The method comprises the following steps of: coupling IgG molecules and latex particles to obtain IgG sensitizing latex; digesting and decomposing obtained IgG sensitizing latex by enzyme to obtain mixture of Fab fragment and Fc fragment sensitizing latex; separating and purifying the Fab fragment and Fc fragmentsensitizing latex to obtain the Fc fragment sensitizing latex; causing the Fc fragment on the sensitizing latex to be denatured, and obtaining the denatured Fc fragment sensitizing latex which is prepared into rheumatoid factor detection reagent. The method not only can produce rheumatoid factor detection reagent with high specificity, but also has low production cost, simple technique and is convenient to large-scale industrialization production.

Description

technical field [0001] The invention relates to a preparation method of a rheumatoid factor (RF) detection reagent, which is used for determining the content of the rheumatoid factor in serum. Background technique [0002] Rheumatoid factor (RF) is a specific antibody against the antigenic determinant on the Fc fragment of human IgG molecule. It is an autoantibody against IgG. The increase in serum is common in rheumatoid arthritis. Rheumatoid factor detection kit is clinically used to determine the content of rheumatoid factor in serum. At present, the detection methods of rheumatoid factor kits that can be used in biochemical analyzers are roughly divided into two types according to the measurement principles: one is to use anti-RF antibodies to react with them to generate turbidity for detection, that is, immunoturbidimetric method, for example, currently The commercially available products that utilize this method to detect include the rheumatoid factor test kit of Brit...

Claims

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Application Information

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IPC IPC(8): G01N33/50G01N33/564
Inventor (请求不公开姓名)
Owner BEIJING LEADMAN BIOCHEM
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