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Stereotactic fixing method of IgG antibody on surface of polystyrene carrier

A carrier surface, polystyrene technology, applied in the field of immunoassay, can solve the problems of losing binding antigen, inability to effectively expose antibody binding sites, unfavorable antibody-antigen binding, etc., and achieve the effect of improving specificity

Inactive Publication Date: 2013-03-27
WEIFANG MEDICAL UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0008]The antibody molecules immobilized directly on the surface of the carrier through physical adsorption, when the functional domains of the solid phase surface tend to go down or to the side, it is not conducive to the binding of antibody-antigen , if the Fab end of the antibody is adsorbed on the surface of the solid phase, the antigen-binding domain of the antibody will be covered and lose the ability to bind the antigen; and the PS microwell plate needs to be washed several times during the ELISA operation, so that the physically adsorbed antibody is easy to fall off and become active Reduced, resulting in significantly reduced sensitivity, repeatability and stability
[0009]The immobilization behavior of the chemical method is composed of active groups such as -NH2, -COOH, -OH or -SH of protein molecules and The active groups on the surface of the solid-phase carrier are covalently bonded. Due to the uncertainty and non-uniqueness of the position of the above-mentioned active groups in the protein molecule, there is no fixed binding mode for this covalent bond, and the chemical connection is often accompanied by varying degrees. When the covalent bond acts on the active site of the antibody or nearby groups, it affects the activity of the antibody; the modification of solid-phase materials such as the amination of the PS carrier and the activation of succinimide lipid can only increase the amount of protein adsorption. , can not efficiently guarantee antibody directional immobilization and high immunological activity
[0010]The indirect coating method can expose the antigen binding site of the antibody, which is beneficial to capture the antigen in the system, and improves the specificity and sensitivity of the immunoassay to a certain extent. However, the first antibody or receptor protein (such as SpA) is still chemically covalently coupled (including polymer chemical coupling) by physical adsorption (including molecular coating membranes), and more importantly, the above methods are still not effective. Directional immobilization of the primary antibody or receptor protein, that is, the protein coated on the carrier is limited by the coating method, and the antibody binding site cannot be effectively exposed and loses the ability to bind the antibody

Method used

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  • Stereotactic fixing method of IgG antibody on surface of polystyrene carrier
  • Stereotactic fixing method of IgG antibody on surface of polystyrene carrier
  • Stereotactic fixing method of IgG antibody on surface of polystyrene carrier

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1. Introduction of IDA-Ni on the surface of a polystyrene microwell plate 2+ active functional group

[0026] In order to bind the active Ni of His-tag on the surface of polystyrene microwell plate through metal affinity 2+ , first to activate the surface of polystyrene and introduce Ni that can chelate 2+ chemical groups such as figure 1 shown.

[0027] 1. Add 100 μL of 10% HNO to the microwells of the PS microplate 3 The glacial acetic acid solution was incubated at 60°C for 3 h, the reaction solution was removed, and the microplate was rinsed with deionized water.

[0028] 2. Add 100 μL containing 50% HNO to the microwell of PS microplate 3 Concentrated H 2 SO 4 The solution was kept at 60°C for 15 minutes, the reaction solution was removed, and the microplate was rinsed with deionized water.

[0029] 3. Add 100 μL of 1% Na 2 S 2 o 4 NaOH (0.1M) solution, kept at 60°C for 3h, removed the reaction solution, and rinsed the microplate with ...

Embodiment 2

[0039] Example 2, Preparation and Purification of ZZ-His Affinity Peptide by Genetic Engineering Technology

[0040] 1. Amplification of ZZ affinity peptide gene sequence, construction of vector and genetically engineered bacteria. Design primers: Primer 1: 5'-CCGGAATTCCATGGCGCAACACGATGAAG-3' and

[0041] Primer 2: 5'-CCCAAGCT TTTTATGATGATGATGATGATGATGATTCGCGTC-3'; using pEZZ18 as a template, the above primers PCR amplified the ZZ affinity peptide gene with His tag, cloned into pUC 18 vector to construct pUC-ZZ-His, and transformed E. coli DH5α, construct pUC-ZZ-His / E. coli DH5α engineering bacteria.

[0042] 2. Expression and purification of ZZ-His affinity peptide. pUC-ZZ-His / E. coli The DH5α engineered bacteria were activated overnight, transferred to fresh LB medium (ampicillin concentration 100 μg / mL) according to 5% inoculum, cultured with shaking at 37°C for 17-24 hours, and centrifuged to collect the bacteria. Resuspend the bacteria in PBS solution containi...

Embodiment 3

[0043] Embodiment three, PS-IDA-Ni 2+ Analysis of the directional immobilization ability of His tag protein

[0044] 1. Add 100 μL / well of ZZ-His (50 ng / mL, the solvent is PBS solution containing 20 mM imidazole, pH8.0) to PS–IDA–Ni 2+ Shake the microplate at room temperature for 30 minutes, remove the solution in the well, wash with PBST (containing 20nm imidazole) for 3 times, each time for 5 minutes, and dry it for later use.

[0045] 2. In order to determine whether the protein immobilized on the microwell plate is directional immobilized, that is, the binding ability of IgG antibody Fc segment, 100 μL / well rabbit anti-HRP antibody (200ng / mL) was added to ZZ-His / PS–IDA–Ni 2+ Microplate, incubated at 37°C for 1 hour, washed 3 times with PBST, added 100 μL / well of HRP (200ng / mL), incubated at 37°C for 1 hour, washed 3 times with PBST, HRP substrate 3,3' ,5,5'-Tetramethylbenzidine (TMB) for color development. The control experiment was the same concentration of ZZ-Hi...

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Abstract

The invention discloses a stereotactic fixing technology of an IgG antibody, wherein the IgG antibody fixed on the surface of a polystyrene carrier is uniform, the Fab segment is exposed adequately and the high antigen binding activity of the IgG antibody is maintained, so that the sensitivity, specificity and stability of solid phase immunoassay are effectively improved. The stereotactic fixing method of IgG antibody provided by the invention specifically comprises the following steps: grafting a 'metal ion arm' on the surface of polystyrene by a chemical modification method; constructing a ZZ affinity peptide with Histag by a genetic engineering technology; mediating the ZZ affinity peptide based on Histag and metal ion affinity characteristics to be stereotactically fixed on the surface of polystyrene, so as to exert the immunological properties of the ZZ affinity peptide; specifically combining the Fc section of the IgG antibody to obtain a metal ion-(ZZ-His)-Fc assembled body in a biological affinity mode, so as to stereotactically fix the Fab segment of the IgG antibody.

Description

technical field [0001] The invention belongs to the field of immunoassay, and in particular relates to the stereospecific immobilization of antibodies on the surface of carriers to keep their antigen-binding sites fully exposed to space and improve the sensitivity, specificity and stability of solid-phase immunoassays. Background technique [0002] Solid-phase immunoassay technology is an important part of clinical immunology testing, such as the classic enzyme-linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) and new technologies such as immunosensors and immunodiagnostic chips. Immunoassays are coated on a solid-phase carrier, and generally coated antibodies are more commonly used. The spatial configuration, quantity and immunological activity of the antibody immobilized on the carrier surface are the key factors affecting the stability, sensitivity and selectivity of solid-phase immunoassay technology. The ideal antibody immobilization technology is to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/545
Inventor 杨洪鸣唐金宝梁淑娟陈永
Owner WEIFANG MEDICAL UNIV
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