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One-step semi-double-antigen sandwich immunological detection method

A double-antigen sandwich and immunoassay technology, applied in the field of immunoassay, can solve the problems of false positives and poor specificity of the test results, and achieve the effects of simple operation, prolonged reaction time, and remarkable sensitivity

Inactive Publication Date: 2011-02-09
FAPON BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to alleviate the hook effect, the one-step method needs to increase the concentration of both the coated antigen and the labeled antigen, which may easily cause high false positives and background in the test results, resulting in poor specificity of the test.

Method used

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  • One-step semi-double-antigen sandwich immunological detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. One-and-a-half-step detection of HIV antibodies

[0040] 1.1 Antigen coating

[0041] Dilute HIV antigen H67 (product of Shenzhen Feipeng Biological Co., Ltd.) with carbonate buffer (50mM, pH9.51) in a certain proportion, add 100ul / well to the enzyme label plate (Shenzhen Jincanhua Industrial Co., Ltd. irradiated enzyme label plate), coated at 4°C for 18-24 hours; the next day, the plate was washed twice with PBST (10mM PB, 150mM NaCl, 0.05% Tween-20, pH7.4) washing solution, and patted dry; 20ul / well was added with blocking solution ( Yingke Xinchuang (Xiamen) Technology Co., Ltd.), sealed at 37°C for 2 hours or overnight at 4°C; shake off the liquid in the hole, pat dry, and place in a room with room temperature 20-25°C, humidity 55%-65%, and ventilation equipment Air-dried in the room; packaged in an aluminum film bag with a desiccant, and the coating is completed.

[0042] 1.2 Detection steps

[0043] In order to show the advantages of the one-step and...

Embodiment 2

[0066] Example 2. One-and-a-half-step detection of TP antibody

[0067] 2.1 Antigen coating

[0068] The TP antigens TP66 and TP67 (products of Shenzhen Feipeng Biological Co., Ltd.) were coated with the same coating method as the HIV antigen in 1.1.

[0069] 2.2 Detection steps

[0070] The one-and-a-half-step method, one-step method, and two-step method of TP are performed in parallel in the same way as the HIV detection steps in 1.2. The antigen-HRP-labeled conjugates used are TP47-HRP, TP48-HRP, and TP49-HRP (Shenzhen Feipeng Biological Co., Ltd. product).

[0071] 2.3 Test results

[0072] The coating antigen dilution ratio of the above three methods and the dilution ratio of the antigen-HRP labeled conjugate were respectively adjusted to their respective optimal conditions (those skilled in the art should be able to understand, so I won’t go into details here), and tested this method respectively. Laboratory treponema pallidum antibody diagnostic reagent quality cont...

Embodiment 3

[0084] Example 3. One-and-a-half-step detection of HTLV antibody

[0085] 3.1 Antigen coating

[0086] The HTLV antigen HTLV-Ag (product of Shenzhen Feipeng Biological Co., Ltd.) was coated with the same coating method as the HIV antigen in 1.1.

[0087] 3.2 Detection steps

[0088] The one-and-a-half-step method, one-step method, and two-step method for HTLV detection were performed in parallel in the same manner as the HIV detection steps in 1.2. The labeled conjugate used was HTLV-HRP (product of Shenzhen Feipeng Biological Co., Ltd.).

[0089] 3.3 Test results

[0090] Adjust the coating antigen dilution ratio of the above three methods and the dilution ratio of the antigen-HRP labeled conjugate to their respective optimal conditions (those skilled in the art should be able to understand, so I won’t go into details here), and detect the HTLV serum respectively. Plate PPHTLV0504 (Q panel company), including 7 positive sera, numbered P1-P7, and 13 negative sera, numbered ...

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Abstract

The invention discloses a one-step semi-double-antigen sandwich immunological detection method, which comprises the following steps of: adding a sample to be detected into a solid-phase support enveloped with an envelope antigen; keeping the temperature at the preset temperature, fully reacting for the first preset time, not removing the sample to be detected, and directly adding a labeled antigen; reacting for the second preset time, and completely washing an unbonded sample and the labeled antigen; and adding a substrate to react for the third preset time, detecting a display result and finishing the detection. Compared with the traditional one-step method, the detection method has the advantages of obviously improving the probability of forming an 'envelope antigen-antibody-labeled antigen' sandwich complex by bonding the labeled antigen and an 'envelope antigen-antibody' complex and effectively reducing adverse effect of the hook effect. Compared with the traditional two-step method, the method obviously improves the specificity. In the reaction of an antibody to be detected and a solid-phase antigen, multiple kinds of buffer solution and a certain inert protein are taken as diluted samples in the traditional two-step method, while the sample to be detected is used as a natural diluted sample in the invention, and the natural diluted sample can effectively eliminate non-specific binding.

Description

technical field [0001] The present invention relates to the field of immunoassay, in particular, the present invention relates to a novel one-and-a-half-step double-antigen sandwich detection method (hereinafter referred to as one-and-a-half-step method). Background technique [0002] Immunoassay is a method of applying immunological techniques to determine the substance to be tested in a sample. In clinical testing, antibodies or antigenic substances in samples are mainly detected through antigen-antibody reactions. According to different detection principles, immunoassays can be divided into indirect method, sandwich method (including double antigen sandwich method and double antibody sandwich method), capture method, competition method and so on. [0003] Antibody detection by double antigen sandwich method is a common method for detecting antibodies in immunoassay. The basic principle is to use the specific binding between antigen and antibody to form a sandwich comple...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N21/75G01N33/569
Inventor 何志强胡鹏范凌云王蓉娥郑焕巍王红颜文豪孙兴宝孙婧崔鹏
Owner FAPON BIOTECH INC
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