Antibody horseradish peroxidase marker and preparation and application thereof

A technology of horseradish peroxidase and marker, applied in the field of enzyme immunity

Pending Publication Date: 2021-06-08
山东莱博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Pretreatment of P6-DG desalting column

[0039] 1) A-pillar treatment:

[0040] Rinse with 1*PBS (0.15mM sodium chloride, pH6.0), flow rate 3.0mL / min, rinse for 2 hours after use (as long as possible); rinse for 1 hour before use (as long as possible).

[0041] 2) B-pillar treatment:

[0042]Rinse with 1*PBS (0.15mM sodium chloride, pH7.2) at a flow rate of 3.0mL / min; rinse with 0.1M PBS (0.15mM sodium chloride, pH7.2) for 30 minutes after use, then rinse with 1*PBS ( 0.15mM sodium chloride, pH7.2) for 2 hours (as long as possible); before use, wash with 1*PBS (0.15mM sodium chloride, pH7.2) at a flow rate of 3.0mL / min for 1 hour (as long as possible) long time).

Embodiment 2

[0043] Embodiment 2: Preparation of antibody horseradish peroxidase marker

[0044] (1) Sulfhydryl modification of HRP:

[0045] 1) Dissolve 100 mg of horseradish peroxidase (HRP) in 10 mL of PBS with pH 7.2 and 0.1 M, and gently invert and mix for 2 minutes in the dark to avoid foaming;

[0046] 2) Dissolve 16.8mg of SPDP in 0.5mL of DMF in a 1.5mL centrifuge tube;

[0047] 3) Add all the solution prepared in 2) into the solution in 1), place in an incubator at 37°C and mix for 30 minutes;

[0048] 4) Add 230ul of 1M DTT solution, continue to place in the incubator at 37°C and mix for 30min;

[0049] 5) Use the pretreated P6-DG desalting column to desalt with 0.15M, pH6.0 PBS, collect the liquid of the first peak, and obtain the sulfhydryl-modified horseradish peroxidase solution, and store it at 4°C for later use ;

[0050] (2) Amino modified antibody:

[0051] 1) Prepare a SMPB solution with a concentration of 0.1mM using DMF as a solvent, add the antibody solution wit...

Embodiment 3

[0057] Example 3: Preparation of an enzyme stabilizer used in conjunction with antibody horseradish peroxidase markers

[0058] Take the prepared antibody horseradish peroxidase marker as the matrix, add bovine serum albumin (BSA) to make the final concentration 10mg / ml, add kathon to make the final concentration 10ul / ml, add the matrix volume One-third glycerin makes the enzyme stabilizer. The usage amount of the enzyme stabilizer is 2-3 times of the volume of the antibody horseradish peroxidase marker.

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PUM

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Abstract

The invention discloses an antibody horseradish peroxidase marker, which is formed by cross-linking an amino-modified antibody (NH2-Ab) and a sulfydryl-modified horseradish peroxidase (SH-HRP), and the antibody can be any antibody suitable for an antigen enzyme-linked immunosorbent assay kit or a chemiluminescence detection kit. The invention also discloses an application of the marker in preparation of an antigen enzyme-linked immunosorbent assay kit or a chemiluminescence detection kit. Experiments prove that when the antibody horseradish peroxidase marker is combined with an enzyme stabilizer and a diluent for use, the sensitivity and the stability of the antibody enzyme marker can be remarkably improved, the detection background value is greatly reduced, the specificity is better, and the application prospect is great.

Description

technical field [0001] The invention relates to an antibody horseradish peroxidase marker and its preparation and application, belonging to the technical field of enzyme immunity. Background technique [0002] ELISA (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay kit) is the most widely used technique in enzyme immunoassay technology. The basic method is to adsorb known antigens or antibodies on the surface of a solid phase carrier (polystyrene micro-reaction plate), make the enzyme-labeled antigen-antibody reaction on the solid phase surface, and wash the free components in the liquid phase by washing method. remove. [0003] Antigen or antibody enzyme markers play an important role in the sensitivity and specificity of immunoassay, and are key reagents in enzyme immunoassay detection. Horseradish Peroxidase (HRP) has high specific activity, stability, small molecular weight, easy preparation of pure enzyme, and is currently the most commonly used ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/53
CPCG01N33/535G01N33/5306
Inventor 欧兰香陈振寇宗阳丁兴龙
Owner 山东莱博生物科技有限公司
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