Antibody conjugate and method thereof

An antibody conjugation and antibody technology, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of low coupling efficiency, low antibody coupling efficiency, and difficulty in finding coupling conditions, and achieve mild reaction conditions and improved Conjugation conditions, the effect of improving sensitivity

Active Publication Date: 2014-11-19
GENRUI BIOTECH INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The current antibody coupling technology is relatively immature, the coupling efficiency of the antibody is relatively low during the coupling process, and the size of the coupled latex particles is not suitable
There are mainly the following disadvantages: 1. The description of coupling conditions is not clear enough, it is difficult to find suitable coupling conditions; 2. There are many coupling methods, and it is difficult to find suitable coupling methods; 3. The activation of latex particles The requirements of the group are relatively broad, which makes it difficult to find a suitable group for coupling; 4. The description of various buffers in the coupling process is not clear enough, which makes it difficult to grasp the concentration of various ions in the coupling; 5. The evaluation methods for the effect of latex after coupling are not uniform, resulting in a lack of convincing judgment on the coupling effect
[0003] In the existing technology, there are also many shortcomings: 1. The coupling efficiency is low, resulting in a large waste of latex and antibodies, which makes the cost of coupling remain high; 2. The coupling efficiency is low, resulting in the concentration range of the detected items Cannot meet the requirements (such as CRP, IgG, IgA, etc.); 3. The size of the coupled latex particles is not suitable, resulting in the detection sensitivity not meeting the requirements; 4. The overall coupling effect is poor, and the stability of the coupled product cannot be guaranteed 5. It is not convenient and fast enough to evaluate the effect of latex after coupling

Method used

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  • Antibody conjugate and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Clean the latex, wash twice with 0.05M, pH5.5 MES buffer; the size of the selected latex is 80nm;

[0049] 2. Add NHS and EDC 10 times the molar number of latex-COOH to the washed latex, and react for 30 minutes at room temperature under vigorous stirring;

[0050] 3. After centrifugation at low temperature (4°C), discard the supernatant and redissolve with activation buffer; repeat this step twice, discard the supernatant after the last centrifugation, do not redissolve, and set aside;

[0051] 4. Dilute the antibody to 1.2mg / ml with 0.04M, pH7.2 phosphate buffer, namely the reaction buffer;

[0052] 5. Reconstitute the activated latex with deionized water, the concentration is 10mg / ml, and quickly add the reconstituted latex to the antibody diluted in equal volume;

[0053] 6. React for 4 hours under room temperature and vigorous stirring;

[0054] 7. After centrifugation at low temperature (4°C), suck out the supernatant, and put the supernatant into a centrifug...

Embodiment 2

[0058] 1) Choose the size of latex as 120nm. Dilute the latex to 10 mg / ml with the activation buffer, remove the supernatant after centrifugation; redissolve the precipitate with the activation buffer to 10 mg / ml;

[0059] 2) Centrifuge the solution obtained in step 1 at 4°C, remove the supernatant, and redissolve the precipitate with the activation buffer to 10 mg / ml;

[0060]3) To the latex obtained in step 2, add NHS and EDC 5-15 times the number of moles of carboxyl groups in the latex, and stir vigorously at room temperature for 15-30min;

[0061] 4) Centrifuge the solution obtained in step 3 at 4°C, remove the supernatant, and redissolve the obtained precipitate with the activation buffer;

[0062] 5) After repeating step 4 twice, centrifuge at 4°C to remove the supernatant and set aside;

[0063] 6) Dilute the antibody to 1-6 mg / ml with the reaction buffer;

[0064] 7) Reconstitute the latex obtained in step 5 with deionized water to a concentration of 10 mg / ml; quic...

Embodiment 3

[0076] Verification of coupling efficiency: ①Use the absorbance at 260nm and 280nm of the UV spectrophotometer to determine a standard curve of antibody concentration; ②Use the UV spectrophotometer at 260nm and 280nm to measure the absorbance of the antibody before and after coupling respectively; ③Use the standard of antibody concentration Curve to calculate the antibody concentration before and after coupling; ④Divide the antibody concentration after coupling by the antibody concentration before coupling to calculate the percentage, and then subtract the calculated percentage from 1 to obtain the coupling efficiency; ⑤This type of coupling The antibody coupling efficiency of the coupling method can reach about 90%.

[0077] Table 1: Coupling efficiency

[0078]

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Abstract

The invention provides an antibody conjugate and a method thereof, and the antibody conjugate comprises: (1) an activation buffer which is a 0.05 M MES buffer with a pH of 5.0-6.0; (2) a reaction buffer which is a 0.04 M phosphate buffer with a pH of 7.0-8.5; (3) a stopping solution which comprises a 0.04 M phosphate buffer with a pH of 7.0-8.5, and 0.04 M Gly; (4) a blocking solution which comprises a 0.04 M phosphate buffer with a pH of 7.0-8.5, and a BSA solution with a mass fraction of 0.01%-1%; (5) a preservation solution which is a 0.02 M Tris buffer with a pH of 7.0-7.8; (6) a reaction buffer which comprises a 0.02 M phosphate buffer with a pH of 7.0-7.5, 0.15 M sodium chloride, and sodium dodecyl benzene sulfonate with a mass fraction of 0.005%-0.5%; (7) latex which is latex particles with carboxyls.

Description

technical field [0001] The present invention relates to an antibody conjugate and its method, in particular to an antibody conjugate with high conjugation efficiency and its method. Background technique [0002] The current antibody coupling technology is relatively immature, the antibody coupling efficiency is relatively low during the coupling process, and the size of the coupled latex particles is not suitable. There are mainly the following disadvantages: 1. The description of coupling conditions is not clear enough, it is difficult to find suitable coupling conditions; 2. There are many coupling methods, and it is difficult to find suitable coupling methods; 3. The activation of latex particles The requirements of the group are relatively broad, which makes it difficult to find a suitable group for coupling; 4. The description of various buffers in the coupling process is not clear enough, which makes it difficult to grasp the concentration of various ions in the coupli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/531G01N33/544
Inventor 郑焕巍李华涛陈亚球
Owner GENRUI BIOTECH INC
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