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New HIV infection detection kit and preparation method

A technology for infection detection and kits, which is applied in the field of HIV new infection detection kits and preparations, can solve the problems of changes in detection sensitivity, reduction of antigen activity, false positives and misjudgments, etc., to reduce the amount of addition, avoid activity loss, and specificity strong effect

Active Publication Date: 2019-07-19
北京新创生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When lysine is near the active center of the antigen, the steric hindrance generated by the combination of lysine and HRP will reduce the activity of the antigen or even lead to the inactivation of the antigen. When reacting with the sample, it is easy to produce no reaction or weak reaction, and false Positive leads to misjudgment
[0007] In the prior art, when the detection sensitivity of a certain antigen in HIV-1 decreases, the method of recombining and fusing multiple antigens is often used to increase the content of multiple antigens in the original recombinant antigen in equal proportions, which will lead to other unidentified antigens. The detection sensitivity of the intervened polypeptide changes, which brings troubles to the detection

Method used

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  • New HIV infection detection kit and preparation method
  • New HIV infection detection kit and preparation method
  • New HIV infection detection kit and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Mouse anti-Trx antibody indirectly labeled with horseradish peroxidase (HRP)

[0048] Prepare antigen:

[0049] The nucleotides of the three peptides of HIV-1 B, E and D were synthesized for the whole gene to obtain the three target genes of HIV-1 B, E and D, and the nucleotide sequence 1 used for synthesizing the connecting peptide was combined with HIV -1 The three target genes of B, E and D were connected, and after NcoI and XhoI double digestion and DNA ligase connection, the target gene was connected into the pET30a-Trx expression vector, transformed into Escherichia coli BL21 (DE3), induced by IPTG, and finally Three target proteins were obtained. The three antigens are all expressed in inclusion bodies in Escherichia coli. After the bacteria are sonicated, the precipitate is collected by centrifugation, lysed by inclusion bodies, and purified to obtain three target proteins Trx-B, Trx-E and Trx-D, the three target proteins can be used to directly coat the plate...

Embodiment 2

[0068] Kit preparation

[0069] The kit mainly includes the following ingredients in Table 1:

[0070] Components in the kit in Table 1

[0071]

[0072]

[0073] Prepare enzyme-linked plates

[0074] 96-well plate, blocking solution, sample diluent, enzyme diluent, strong positive control (HPC), weak positive control (LPC), calibrator (CAL), negative control (NC), sealing film, TMB chromogenic solution A Solution, TMB Chromogenic Solution B, and Stop Solution were purchased from Yingke Xinchuang (Xiamen) Technology Co., Ltd.

[0075] Dilute and mix the mouse anti-goat IgG antibody at a ratio of 1:2500 with coating buffer (50mM carbonic acid buffer at pH 9.6), add 100ul / well into a 96-well plate, wrap with plastic wrap, and keep the temperature at 4°C Place in the box for 16 hours, dry the coating solution directly, then dilute and mix the goat anti-human IgG antibody at a ratio of 1:6000, add 100ul / well into a 96-well plate, wrap it in plastic wrap, and place it in a...

Embodiment 3

[0078] Detection method of the kit

[0079] 1) Numbering: Take the required number of coated microwell strips, place them on the plate rack, and mark them.

[0080] 2) Adding samples: first dilute and mix the sample, calibrator (CAL) and negative and positive controls 100 times with the sample diluent, and then add 100ul diluted samples and calibrator (CAL) to the corresponding wells in order And negative and positive controls.

[0081] 3) Incubation: incubate the enzyme-linked plate in step (2) for 60 min in a 37° C. incubator.

[0082] 4) Plate washing: fully wash the enzyme-linked plate incubated in step (3) 5 times with washing buffer, and then buckle dry (the soaking time in washing solution is 60 seconds).

[0083] 5) Add enzyme: add enzyme conjugate to the wells after washing the plate in step (4), add enzyme conjugate 100ul to each well.

[0084] 6) Incubation: place the enzyme-added enzyme-linked plate in a 37° C. incubator and incubate for 30 minutes (the enzyme-lin...

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Abstract

The invention discloses a new HIV infection detection kit and a preparation method, and relates to the technical field of biology. According to thenew HIV infection detection kit, a one-step enzyme reaction method is used, detection time is greatly shortened, sensitivity is high, specificity is high, and stability is good; andall three antigens of B, E, and D of HIV subtypesin China can be simultaneously detected, and the detection accuracy rate of a new HIV infection is increased. Asecondary coating method is used for coatingan enzyme-linked plate, the amount of adetection raw material is greatly decreased, and indirect labeling is used for improving the activity, reaction consistency, and accuracy rate of the antigen; and detection of trace samples is facilitated, steric hindrance is reduced, and the reaction efficiency, sensitivity, and specificity are improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for emerging HIV infection and a preparation method. Background technique [0002] Human immunodeficiency virus (Human immunodeficiency virus, HIV) new infection detection is an important part of AIDS surveillance. It is necessary to understand the spread of the virus in a timely manner, find high-risk groups, and effectively evaluate disease interventions, so as to achieve a reasonable allocation of health resources. . Currently, there are three main methods for estimating the rate of new infections: cohort studies, mathematical models, and laboratory tests. Although cohort studies are the gold standard for obtaining new infection rates, they require huge manpower, material and financial resources due to the long observation and follow-up time, and also have defects such as loss of follow-up and selection bias, which prevent them from being widely used; Although th...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/569G01N21/79
CPCG01N33/535G01N33/56983G01N21/79
Inventor 刘兴旺景滢滢
Owner 北京新创生物工程有限公司
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