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Method for preparing enzyme labelling antibody

An enzyme-labeled antibody and labeling technology, which is applied in the field of immunoassay to achieve the effect of increasing the connection amount, improving the signal amplification ability, and increasing the concentration

Inactive Publication Date: 2011-08-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the colloidal gold complex enzyme-labeled antibody has higher sensitivity, it is still based on the prepared traditional enzyme-labeled antibody, and the coupling process of antibody and enzyme in the traditional sense has not changed.

Method used

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  • Method for preparing enzyme labelling antibody
  • Method for preparing enzyme labelling antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Take 2.5mL of 1% sodium citrate aqueous solution and add it to the boiling 100mL of 0.01% chloroauric acid solution, and at the same time perform magnetic stirring to make it evenly mixed. After reacting for a period of time, the solution turns from yellow to blue, and finally turns into wine red. Stop heating and keep cooling under the condition of magnetic stirring to obtain colloidal gold with a diameter of about 18nm.

[0024] Take a certain volume of colloidal gold solution, centrifuge at 15000g and 4°C for 15min, suck off the supernatant, and redissolve with half the original volume of deionized water. with 0.2M K 2 CO 3Adjust the pH value of the concentrated colloidal gold to about 8.2-8.5, and then add a certain concentration of horseradish peroxidase (HRP, isoelectric point PI = 7.2-8.0), so that the final concentration of HRP is 50 μg / mL. Rotate and mix at room temperature or 4°C for 1-2 hours, then centrifuge the reaction solution at 15,000g and 4°C, absorb...

Embodiment 2

[0027] Get 1.5mL of 1% sodium citrate aqueous solution and add to the boiling 100mL containing 0.01% chloroauric acid solution, and colloidal gold with a diameter of about 25nm can be obtained according to the steps in Example 1.

[0028] Concentrate the colloidal gold with 0.1M hydrochloric acid to adjust the pH value to about 5, then add a certain concentration of alkaline phosphatase (AP, isoelectric point PI = 4.5), so that the final concentration of AP is 50 μg / mL. . Except that N,N'-disuccinimide carbonate is used as the bifunctional reagent (the final concentration of N,N'-disuccinimide carbonate in the solution is 0.2% to 1%), the rest of the steps are the same as in Example 1.

Embodiment 3

[0030] Add 1.2mL of 1% sodium citrate aqueous solution into 100mL of boiled 0.01% chloroauric acid solution, and follow the steps in Example 1 to obtain colloidal gold with a diameter of about 40nm.

[0031] Concentrate the colloidal gold with 0.1M hydrochloric acid to adjust the pH value to about 5 with the steps in Example 1, and then add a certain concentration of galactosidase (β-Gal, isoelectric point PI =4.6) to make the final concentration of β-Gal 100μg / mL. The remaining steps were the same as in Example 1, except that the final concentration of the added antibody was changed to 50 μg / mL.

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Abstract

The invention provides a method for preparing an enzyme labelling antibody. The method comprises the following steps of: concentrating a colloidal gold solution, and then adjusting the pH value of the concentrated colloidal gold solution to be slightly greater than the pH value of an isoelectric point of an enzyme for labelling; adding an aqueous solution of the enzyme for labelling into the colloidal gold solution, mixing the solutions, and then connecting the antibody to molecules of the enzyme on the surface of colloidal gold by a bifunctional reagent; and carrying out centrifugation to obtain an enzyme labelling colloid gold compound antibody. In the method, enzyme labelling on the colloidal gold is accomplished in a direct-absorption simple mode; the separation of the enzyme and a coupling agent and separation purification after connection of the antibody can be finished only by the simple centrifugation without the complicated and high-loss expensive steps of gel filtration, dialysis or affinity chromatography; by concentration operation prior to enzyme absorption by the colloid gold, the connection amount of a connected reagent can be increased, and the concentration of gold colloid particles in the enzyme labelling colloid gold compound antibody can be increased simultaneously; and the enzyme labelling colloid gold compound antibody is accordant with the characteristic.

Description

technical field [0001] The invention belongs to the technical field of immune analysis, and in particular relates to a method for preparing an enzyme-labeled antibody. Background technique [0002] Enzyme immunoassay technology has developed rapidly since its inception, and has been widely used in many fields such as drug screening, medical diagnosis, and food safety testing. It not only inherits the advantages of high sensitivity of radioimmunoassay, but also overcomes the disadvantage of using radioactive labels. Enzyme immunoassay organically combines the high efficiency of enzymatic reactions with the high specificity of immune reactions, and can quantitatively detect various analytes such as proteins, toxins, pesticides, microorganisms, etc. It is an immunoassay technology that has been promoted in production and clinical practice. In addition, the use of enzymes as labels in immunoassays also lies in the fact that the enzymes can maintain long-term stability, are not...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/532
Inventor 吴坚李冬阳应义斌
Owner ZHEJIANG UNIV
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