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Application of pamam in preparation of reagents for immunoassay

An immunodetection and reagent technology, applied in the field of immunity, can solve the problems of limited signal amplification effect, expensive kits, complicated reagent preparation, etc., and achieve the effects of simple detection, good detection sensitivity and specificity, and simple preparation method.

Active Publication Date: 2021-12-28
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this biotin-avidin system is susceptible to the influence of endogenous biotin and has limited signal amplification because there are only 4 sites for avidin to bind biotin, and the enzyme and avidin The combination also requires a special process. In addition, the system also requires a linker to connect two or more biotins as an enzyme-avidin cross-linking agent. The preparation of the reagent is complicated and the kit is expensive.

Method used

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  • Application of pamam in preparation of reagents for immunoassay
  • Application of pamam in preparation of reagents for immunoassay
  • Application of pamam in preparation of reagents for immunoassay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] The preparation of embodiment 1 high-sensitivity enzyme-labeled reagent, flow process is as follows figure 1 shown.

[0075] (1) Partial carboxylation of PAMAM

[0076] Get 0.5mL PAMAM dendrimer, polymer ethylenediamine core, 5.0 generation solution (PAMAM dendrimer, ethylenediamine core, generation 5.0solution, 5wt%, purchased from Sigma Company) and dissolve in 2mL acetic acid, then add 0.005g butyl For dianhydride, the ratio of PAMAM dendritic polymer to succinic anhydride is 1:0.2 by mass, heated to 60° C. and reacted for 6 hours. After the reaction is cooled, the solution is transferred to a dialysis bag (molecular weight is 2000Da), dialyzed with 2L of pure water, and repeated 3 times to obtain partially carboxylated PAMAM (concentration is 10mg / mL), and then use nuclear magnetic resonance hydrogen spectrum and infrared spectrum Conduct potential and particle size detection, the results are as follows figure 2 and 3 shown.

[0077] From figure 2 It can be ...

Embodiment 2

[0090] Example 2 ELISA detection

[0091]The PAMAM-HRP-IgG prepared in Example 1 was filtered through a GE 200 gel filtration column, the filtrate was diluted to 1 / 50, and then diluted 2, 4, and 8 times in equal proportions, 0.8-0.9 mL per tube. Carry out ELISA detection then, specific ELISA operation is as follows: first with 3% (w / v) skimmed milk powder solution blocking IgG coated ELISA plate (Thermo company), then wash enzyme mark with PBST (1×, pH7.5) Plate four times, the IgG coating concentration is 2μg / mL, and the plate should be tapped after each washing. Add 100 μL of IgG antigen with a concentration of 200 ng / mL (IgG is the CD151 antibody purified from mouse ascites, produced by Guangzhou Jingda Biology) into the well plate, and the incubation time of the antigen and IgG antibody at room temperature is 45 min, and then PBST (1× , pH7.5) was washed four times, and clapped after each wash. Dilute the enzyme-labeled reagent (PAMAM-HRP-IgG) prepared in Example 1 and t...

Embodiment 3

[0092] Example 3 Immunohistochemical (IHC) detection

[0093] The steps of performing IHC detection on the PAMAM-HRP-IgG prepared in Example 1 are as follows:

[0094] (1) Immerse the paraffin sections of kidney tissue in the following solutions to dewax in turn: xylene I 10min, xylene II 5min, absolute ethanol I 10min, absolute ethanol II 5min, 95% ethanol I 5min, 95% ethanol II 5min, 90% ethanol I for 5 minutes, 90% ethanol II for 5 minutes, 80% ethanol for 5 minutes, and distilled water for 2 minutes.

[0095] (2) drying excess water, adding concentration is 3% H 2 o 2 Incubate at room temperature for 10 min, wash with PBS buffer (pH 7.4) 3 times, 5 min each time.

[0096] (3) The slices were submerged in PBS buffer (pH 7.4), and heated in a microwave oven (Galanz, P70D20P-TD(W0)), high-grade for 5 minutes, and medium-grade for 10 minutes. Wash with PBS buffer (pH 7.4) 3 times, 5 min each time.

[0097] (4) Dry the excess liquid, add BSA with a concentration of 5% by m...

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Abstract

The invention discloses the application of PAMAM in preparing preparations for immune detection. The present invention is based on the discovery by the inventors of the present invention that after PAMAM is modified, it is covalently linked to activated enzymes and specific antibodies, and the obtained enzyme-labeled reagents are used for immunoassays, which have the advantages of high sensitivity and the ability to retain the activity of enzymes and antibodies , the inventions and creations made. The high-sensitivity enzyme-labeled reagent provided by the present invention is obtained based on the aforementioned application, which can achieve the purpose of signal amplification, and the preparation method is simple and stable. When it is applied to immunoassay, the biotin-avidin system is not affected by endogenous Influenced by biotin, etc., the detection sensitivity is higher, but the specificity of detection will not be reduced, and the activity of antibodies and enzymes can be retained to the greatest extent. Applying it to immunoassay kits can provide a reliable means for clinical detection.

Description

technical field [0001] The invention relates to the field of immunity, in particular to the application of PAMAM in the preparation of reagents for immunological detection. Background technique [0002] Enzyme linked immunosorbent assay (ELISA for short) is a commonly used immunoassay method, which refers to the binding of soluble antigens or antibodies to polystyrene and other solid phase carriers, and the specificity of antigen-antibody binding. Methods for qualitative and quantitative detection of immune responses. The basic principle of this method is: ① Make the antigen or antibody bind to the surface of a certain solid phase carrier and maintain its immunological activity. ②The antigen or antibody is connected with an enzyme to form an enzyme-labeled antigen or antibody, which not only retains its immune activity, but also retains the activity of the enzyme. During the determination, the test specimen (to determine the antibody or antigen in it) and the enzyme-labele...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/545G01N33/535G01N33/58C08G73/02
CPCG01N33/535G01N33/545G01N33/581C08G73/028
Inventor 陈填烽刘宏星
Owner JINAN UNIVERSITY
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