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Method for detecting immune body affinity

A technology for detecting antibodies and affinity, applied in the field of medicine and biology, can solve problems such as long time required, and achieve the effects of good repeatability, stable and reliable detection results, and high sensitivity

Inactive Publication Date: 2011-09-14
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the antigen coating needs to be overnight at 4°C, and there are multiple washing steps, which take a long time

Method used

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  • Method for detecting immune body affinity
  • Method for detecting immune body affinity
  • Method for detecting immune body affinity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Take the affinity detection of three different antibodies (cloning numbers M94139.7, 220-5011, and 220-5013) to β-HCG (β-subunit human chorionic gonadotropin) as an example.

[0066] The steps of the antibody affinity detection method are as follows:

[0067] Antigen-coated microspheres:

[0068] -Use a vortex oscillator or ultrasonic to suspend the microspheres (Luminex, USA), about 20s;

[0069] - Take 50μL of microspheres in a 1.5ml centrifuge tube and centrifuge at a speed of ≥8,000g for 1-2min;

[0070] -Remove the supernatant, resuspend the microspheres in 100 μL double distilled water, suspend the microspheres with a vortex shaker or ultrasonic wave for about 20 seconds, and centrifuge at a speed of ≥8000g for 1-2 minutes;

[0071] - Discard the supernatant, add 80 μL of phosphate buffer (pH 6.2), vortex for about 20 seconds, and ultrasonically suspend the microspheres for about 20 seconds;

[0072] -Add 10μL 50mg / mL Sulfo-NHS and mix gently with a vortex shak...

Embodiment 2

[0119] Affinity detection of three antibodies (cloning numbers: M701042, PS5, BM215) of PSA (Prostate Specific Antigen).

[0120] The method used is the same as that of Example 1. The result is as follows,

[0121] Table 2

[0122] μg / ml mol / L Negative logarithm of concentration M701042(MFI value) PS5(MFI value) BM215(MFI value)

[0123] 100 6.25E-07 6.20412 6323.5 5369 10054.5

[0124] 10 6.25E-08 7.20412 6479 4481.5 7657

[0125] 1 6.25E-09 8.20412 2031.5 1910.5 3447.5

[0126] 0.1 6.25E-10 9.20412 640 183.5 418

[0127] 0.01 6.25E-11 10.20412 336.5 97 125

[0128] 0.001 6.25E-12 11.20412 438.5 52.5 57

[0129] The result is calculated as follows:

[0130] Calculation of the affinity constant of the M701042 antibody:

[0131] y=(6323.5-438.5) / 2=2942.5, substitute into the formula y=-4447.5x+38174, and get x=7.92164

[0132] Affinity constant K=1 / (10 -7.92164 )=8.35E+07

[0133] Similarly, the affinity constants of PS5 and BM215 antibodies can be calculated, which...

Embodiment 3

[0135] Affinity detection of three antibodies to TNF-α (Tumor necrosis factor-alpha, tumor necrosis factor α) (cloning numbers are respectively TNF-αF6, D2, 28401.111).

[0136] The method used is the same as that of Example 1. The result is as follows,

[0137] table 3

[0138] Negative logarithm of μg / ml mol / L concentration TNF-αF6 (MFI value) D2 (MFI value) 28401.111 (MFI value)

[0139] 100 0.000625 3.204119983 10233.5 20352 16397

[0140] 10 6.25E-05 4.204119983 5137.5 15397.5 12433

[0141] 1 6.25E-06 5.204119983 270.5 6122 5077

[0142] 0.1 6.25E-07 6.204119983 0 779 722.5

[0143] 0.01 6.25E-08 7.204119983 0 70.5 73

[0144] 0.001 6.25E-09 8.204119983 2 15.5 16

[0145] 0.0001 6.25E-10 9.204119983 16

[0146] The result is calculated as follows:

[0147] Calculation of TNF-αF6 antibody affinity constant:

[0148] y=(10233.5-2) / 2=5116.75, substitute into the formula y=-4981.5x+26157, and get x=4.2237

[0149] Affinity constant K=1 / (10 -4.2237 )=1.67E+04

[...

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Abstract

The invention discloses a method for testing the affinity of antibody, which mainly comprises the following steps: firstly, coating the antibody with microspheres; secondly, testing the antibody affinity; thirdly, processing data. The invention has the advantages of simple and fast test, high sensitivity, good repeatability, and stabler and more reliable testing result.

Description

technical field [0001] The invention belongs to the category of medical biology, and in particular relates to a method for detecting antibody affinity. technical background [0002] Antibody affinity is one of the important parameters to determine the properties of antibodies, and at the same time, it is also an important indicator of the stability of monoclonal antibodies (McAb) secreted by hybridomas. It indicates how tightly the antibody binds to the antigen. Antibodies with high affinity are useful for quantitative detection and oriented diagnosis and treatment, while antibodies with low affinity are better for immunopurification. Therefore, in order to select an antibody with a certain affinity according to the purpose of the application, it is necessary to test the affinity of the antibody first. [0003] Antibody affinity refers to the firmness of antibody-antigen binding, which is often expressed by the affinity constant Ka. Ka is the ratio of the forward / reverse ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/531
Inventor 彭臻菲杨惠夷许嘉森
Owner SUREXAM BIO TECH
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