Preparation method and application of protein chip based on serological detection of syphilis

A protein chip and syphilis technology, applied in the field of biotechnology detection, can solve the problems of inability to make correct diagnosis alone, low sensitivity and specificity, and inability to distinguish recent or previous syphilis infection and treatment or no treatment, and achieve important Clinical value and social benefit, high specificity, high sensitivity

Inactive Publication Date: 2015-08-19
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Neither serological test alone can make a correct diagnosis, and the nontreponemal antigen test can be used as treatment monitoring, but its sensitivity and specificity are low, and it needs to be validated by the Treponema pallidum-specific antigen test
On the other hand, since the positive detection method of Treponemal pallidum specific antigen may last for a lifetime, it is impossible to distinguish whether syphilis infection is recent or previous infection and treatment or untreated, and a non-treponemal antigen detection method is required to confirm the diagnosis

Method used

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  • Preparation method and application of protein chip based on serological detection of syphilis
  • Preparation method and application of protein chip based on serological detection of syphilis
  • Preparation method and application of protein chip based on serological detection of syphilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, gold foil chip surface chemical modification

[0085] Step 1: Cleaning of Gold Foil Chips

[0086] Prepare TL1 solution (H 2 O: H 2 o 2 : NH 3 ·H 2 O=5:1:1, volume ratio) into a stainless steel box, put the gold foil chip into the box, bathe in 82°C water for 6 minutes, rinse with deionized water 4-5 times, ethanol twice, 3 minutes each time; air dry with nitrogen, Store dry.

[0087] Step 2: Chemically modify the surface of the cleaned gold foil chip to obtain a solid phase carrier

[0088] Spot the modification solution DSU on the washed gold foil chip (0.85 μl per spot), and incubate for 2 h in a humid chamber at room temperature in the dark. Wash 5 times with acetone, 4 minutes each time; then wash 3 times with PBS (PH7.4) solution, 2 minutes each time. Dry with nitrogen gas to obtain a solid phase carrier, which is ready for use.

[0089] figure 2 with image 3 The characterization of gold foil chip before and after DSU modification was ob...

Embodiment 2

[0090] Embodiment 2, quality control experiment

[0091] Preparation of incubation solution 1: Dissolve Treponema pallidum recombinant antigen rTpN15-17-47 in PBST-BSA solution, and configure a concentration gradient of 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13μg / mL, 1.56μg / mL, 0.78μg / mL, 0.39μg / mL, 0.19μg / mL solution.

[0092] Prepare incubation solution 2: dissolve the polyclonal rabbit anti-syphilis antigen (rTpN15-17-47) IgG antibody in PBST-BSA solution, and configure the antibody concentration gradient to be 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5μg / mL, 6.25μg / mL, 3.13μg / mL, 1.56μg / mL, 0.78μg / mL, 0.39μg / mL, 0.19μg / mL solution.

[0093] Prepare incubation solution 3: Dissolve Cy3-labeled goat anti-rabbit IgG antibody in PBST-BSA solution, and configure the fluorescent secondary antibody concentration gradient as 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.313 μg / mL mL, 0.156μg / mL, 0.078.μg / mL, 0.039μg / mL, 0.019μg / mL solution.

[...

Embodiment 3

[0116] Embodiment 3, repeatability experiment

[0117] Each 32 wells were regarded as one group, and there were three groups in total. According to the method steps of Examples 1 to 3, the concentration of Treponema pallidum recombinant antigen rTpN15-17-47 and the concentration of 50 μg / mL of rabbit anti-Treponema pallidum antigen IgG were incubated. Antibody and Cy3-labeled goat anti-rabbit IgG antibody at a concentration of 2.5 μg / mL. Repeatability experiment results, as shown in Table 1 and Figure 8 shown.

[0118] Table 1

[0119]

[0120] As shown in Table 1, SD is the standard deviation, and CV is the coefficient of variation. These two indicators can reflect the stability of the protein chip in the detection of anti-rTpN15-17-47IgG antibody and there will be no large deviation.

[0121] Such as Figure 8 Shown: The fluorescence results of the three groups are not limited by different sample wells, with good repeatability and high stability.

[0122] From Table...

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Abstract

The invention relates to the field of biotechnical detection, and in particular relates to a protein chip for detecting syphilis as well as a preparation method and application thereof. A surface dot matrix of a solid-phase vector of the protein chip is fixed with a treponema pallidum recombinant antigen rTpN15-17-47; and the protein chip disclosed by the invention can be used for respectively detecting IgG and IgM antibodies in blood serums of patients suffering syphilis, is high in sensitivity, ensures that the visual detection limit is as low as 0.19mu g / mL and the positive rate can reach 99.0%, and ensures that both the visual detection limit and the positive rate are higher than those of conventional TRUST and TPPA detection methods.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a protein chip for detecting syphilis and its preparation and application. Background technique [0002] Syphilis is a chronic, systemic sexually transmitted disease caused by Treponema pallidum (Treponema pallidum). Its transmission routes are mainly sexual transmission, mother-to-child transmission, and rarely through organ transplantation. Syphilis is divided into primary syphilis, secondary syphilis, latent period, and tertiary syphilis. Primary syphilis is usually acquired through direct sexual contact with another person's infected lesion. About 3 to 90 days after the initial contact (average 21 days), a skin lesion called a chancre appears at the point of contact. A chancre is a firm, painless, non-itchy skin sore with a well-defined base and well-defined edges. Secondary syphilis occurs about four to ten weeks after the first infection. A red to pink, non-itchy, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/571
CPCG01N33/571
Inventor 杜卫东黄娜丽叶雷
Owner ANHUI MEDICAL UNIV
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