Preparation method of rheumatoid factor antigen, detection kit and preparation method of detection kit

A technology for rheumatoid factor and detection kits, applied in measurement devices, instruments, scientific instruments, etc., can solve the problem of kit linear range, sensitivity, specificity, stability is not ideal, can not meet the requirements of clinical detection, stability and batch. It can improve the performance of reagents, detect serum samples quickly and have good stability.

Active Publication Date: 2018-12-25
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the domestic RF latex immunoturbidimetric kits in the market generally have poor reaction specificity
The main raw material denatured IgG (rheumatoid factor antigen) is usually prepared by heat denaturation, which cannot guarantee to maintain the exposed state of the binding site, and the stability and batch-to-batch difference are poor; which further leads to the linear range and sensitivity of the kit. , specificity, stability and other indicators are not ideal, these technical issues need further in-depth research
[0005] Chinese patent CN105911285A discloses a kit for measuring rheumatoid factor, aiming to solve the problem of complex operation and low measurement accuracy in the detection process of rheumatoid factor in the prior art. The kit used is composed of reagent R1 and reagent R2. Grouped into kits, w...

Method used

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  • Preparation method of rheumatoid factor antigen, detection kit and preparation method of detection kit
  • Preparation method of rheumatoid factor antigen, detection kit and preparation method of detection kit
  • Preparation method of rheumatoid factor antigen, detection kit and preparation method of detection kit

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preparation example Construction

[0055] A preparation method of rheumatoid factor antigen, adopting the following steps:

[0056] Mix the IgG solution with the denaturing buffer and equilibrate overnight at 4°C;

[0057] After the IgG molecules have fully reacted with the denaturant, thermally polymerize in a constant temperature water bath at 55°C to 65°C for 30 to 60 minutes to obtain polymeric denatured IgG;

[0058] Select appropriate dialysis equipment, for example, you can use dialysis bag, hollow fiber column or ultrafiltration cup, or other dialysis equipment, and use the dialysis buffer matched with the dialysis equipment to filter out impurities;

[0059] Dilute it with storage buffer to a concentration of 5-30mg / mL and store it. It can be stored at 4°C for short-term storage or -20°C for long-term storage. It can be used in the detection kit for detecting rheumatoid factor.

[0060] In the preparation method of rheumatoid factor antigen, the denaturing buffer used is composed of the following comp...

Embodiment 1

[0088] Preparation of rheumatoid factor antigen (polymeric IgG)

[0089] (1) Preparation of denaturing buffer: take 995mL of water, add 2.42g of tris, 303g of urea, 1mL of β-mercaptoethanol (14.4mol / L), and 1g of sodium azide in sequence, stirring until Completely dissolve, adjust the pH value to 8.0; filter with a 0.22μm filter membrane to make 1L denaturation buffer.

[0090] (2) Preparation of dialysis buffer: Take 995mL of water, add 0.59g of sodium dihydrogen phosphate dihydrate, 5.80g of sodium dihydrogen phosphate dodecahydrate, and 1g of sodium azide in sequence. Stir each addition until completely dissolved, and adjust pH to 7.4.

[0091] (3) Preparation of storage buffer: take 995mL water, add 0.59g sodium dihydrogen phosphate dihydrate, 5.80g sodium dihydrogen phosphate dodecahydrate, 17.42g L-arginine, 30g trehalose, 1g sodium azide in sequence , Stir until the material is completely dissolved each time, adjust the pH value to 7.4; filter with a 0.22 μm filter me...

Embodiment 2

[0097] Preparation of rheumatoid factor latex immunoturbidimetric kit

[0098] The main raw materials involved in 1, embodiment 2 kits are as follows:

[0099] Rheumatoid factor antigen (polymeric IgG: prepared in Example 1)

[0100] Latex microspheres: polystyrene latex particles with a diameter of 160nm and carboxyl groups (Jesus Ya, Japan)

[0101] II, the main reagent preparation method of the present embodiment is as follows:

[0102] Preparation of reagent R1: Take 995g of water, add 4.1g of sodium dihydrogen phosphate, 5.64g of disodium hydrogen phosphate dodecahydrate, 2g of disodium edetate, 2g of bovine serum albumin, 15g of sodium chloride, 20g of polyethylene glycol Glycol 20000, 5g Tween-20, 5g ethylenediamine polyoxypropylene polyoxyethylene ether, 1g sodium azide, stir until completely dissolved each time, adjust the pH value to 6.5; filter with a 0.22μm filter membrane , to make 1 L of reagent R1. The R1 reagent adds an anti-interference surfactant, which h...

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Abstract

The invention relates to a preparation method of a rheumatoid factor antigen, a detection kit and a preparation method of the detection kit. The preparation method of rheumatoid factor antigen comprises the following steps: uniformly mixing an IgG solution with a denaturing buffer at 4 DEG C; after IgG molecules and the denaturant are fully reacted, carrying out heat polymerization reaction in a constant-temperature water bath at 55-65 DEG C for 30-60 min to obtain multimeric modified IgG; filtering to remove impurities by using a dialysis buffer; diluting to the proper concentration with a stock buffer solution and preserving, and applying the prepared rheumatoid factor antigen to the detection kit. Compared with the prior art, the preparation method of rheumatoid factor antigen, detection kit and preparation method of detection kit can greatly improve the reagent performance of such products, and has a simple preparation method, the difference between the preparation batches is controllable, and large-batch industrial production can be performed.

Description

technical field [0001] The present invention relates to biological reagents, in particular to detection kits, in particular to the preparation of a highly specific polymeric IgG (rheumatoid factor antigen) and a rheumatoid factor latex with wide linear range, high sensitivity and low preparation cost Immunoturbidimetric detection kit and preparation method thereof. Background technique [0002] Rheumatoid factor (Rheumatoid Factor, RF) is an autoimmune antibody targeting denatured IgG, which was first discovered in the serum of patients with rheumatoid arthritis. Its essence is a kind of immunoglobulin, mainly 19S IgM rheumatoid factor, and 7S IgG and IgA, while IgE and IgD rheumatoid factor are relatively rare. Rheumatoid factor can recognize the CH2 and CH3 parts of denatured IgG Fc fragments, and has no species specificity. When the two form immune complexes, they can activate complement and cause tissue damage. RF mainly exists in the serum and joint fluid of patients ...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/544
CPCG01N33/531G01N33/544
Inventor 郭雅莉景晟孙卫兵
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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