Application of serum protein marker group in preparation of detection kit for identification of schistosomiasis and detection kit

A technology for detecting kits and serum proteins, which is applied in the field of diagnostic protein markers for advanced schistosomiasis japonicum, can solve problems such as inappropriate treatment plans, delayed optimal treatment time, and irrelevance, and achieve reliable results, large market prospects, and accurate identification. Effect

Active Publication Date: 2021-02-02
JIANGSU INST OF PARASITIC DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the current status of prevention and control of advanced schistosomiasis, new patients with late blood still appear in areas where the transmission interruption standard has been reached for a long time. Schistosomiasis infection has nothing to do with it, which brings a problem to the prevention and control of schistosomiasis, that is, whether the new "late blood" patients screened by the population belong to the classic late blood or the newly developed late blood.
Many patients may not be clearly identified due to limited detection methods, and they are all classified as advanced schistosomiasis, which not only easily delays the best treatment time, but also easily leads to inappropriate treatment plans

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Detection of differentially expressed proteins between patients with classic and newly developed advanced schistosomiasis and healthy controls by iTRAQ technology

[0018] 1. Test samples

[0019]Serum from late blood patients in Kunshan, Changshu, etc., 20 cases of serum from newly developed late blood patients, and serum from healthy people. Collect 2 mL of whole blood on an empty stomach in the morning, let it stand at 4°C for 1-2 hours until the blood coagulates and precipitate the serum, centrifuge at 3000g for 10 minutes, collect the supernatant, aliquot it on ice and store it at -80°C for later use.

[0020] 2. Detection method

[0021] (1) Use the Proteominer kit to remove high-abundance proteins in serum: add DTT with a final concentration of 10mM to the enriched protein sample, and bathe in water at 56°C for 1h; after cooling to room temperature, add IAM with a final concentration of 55mM, and place in a dark room for 45min Add 1 mL of cold aceton...

Embodiment 2

[0030] Example 2: Verification of mass spectrometry MRM in traditional late blood and new late blood

[0031] 1. Test samples

[0032] Serum from late blood patients in Kunshan, Changshu, etc., 20 cases of serum from newly developed late blood patients, and serum from healthy people. Collect 2 mL of whole blood on an empty stomach in the morning, let it stand at 4°C for 1-2 hours until the blood coagulates and precipitate the serum, centrifuge at 3000g for 10 minutes, collect the supernatant, aliquot it on ice and store it at -80°C for later use.

[0033] 2. Detection method

[0034] The establishment of the experimental method:

[0035] (1) Select relevant target proteins suitable for MRM detection and analysis;

[0036] (2) Quality assessment of the extracted protein;

[0037] (3) Select parent-child ion pairs suitable for MRM detection;

[0038] (4) Based on the analysis software Skyline, optimize the scanning parameters of the mass spectrometer—collision energy;

[0...

Embodiment 3

[0050] Example 3: The detection kit prepared by the serum protein marker group.

[0051] The composition of the kit:

[0052] Reagent A: polyclonal antibody for detection of apolipoprotein;

[0053] Reagent B: polyclonal antibody for detection of isopentenyl cysteine ​​oxidase;

[0054] Reagent C: phosphate buffer;

[0055] D: ELISA 96-well plate.

[0056] In a specific embodiment, the kit can be used to differentiate classic late blood from new-onset late blood using the following steps:

[0057] (1) Serum samples are freshly obtained and stored in a -80°C refrigerator;

[0058] (2) Serum samples were divided into three tubes according to the stock solution, 5-fold dilution, and 10-fold dilution;

[0059] (3) Add 1 μl of each of the two antibodies to a 96-well plate for coating;

[0060] (4) According to the ELISA method, add the serum sample to a 96-well plate, and calculate the OD value by color development, and the OD value = (sample OD-blank control OD) / negative contr...

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Abstract

The invention discloses the application of a serum protein marker group of isopentenyl cysteine ​​oxidase and apolipoprotein in the preparation and identification of classical advanced schistosomiasis and newly developed advanced schistosomiasis detection kits, using iTRAQ combined with MRM technology , to identify and verify the serum proteins in the serum of classic late blood patients, newly developed late blood patients and normal people. 2-3 times of those in the newly developed late blood group; in the newly developed late blood group, the expression level of apolipoprotein was 2 times that of the classic late blood group. The invention also discloses a detection kit for distinguishing classic advanced schistosomiasis from newly developed advanced schistosomiasis, which comprises isopentenyl cysteine ​​oxidase polyclonal antibody, apolipoprotein polyclonal antibody, sample loading buffer and ELISA 96-well plate. The kit of the invention can accurately distinguish classic type and newly developed advanced schistosomiasis, and has great market prospect.

Description

technical field [0001] The invention belongs to the field of diagnostic protein markers for advanced schistosomiasis japonicum, and specifically relates to the application of a serum protein marker group in the preparation and identification of classic advanced schistosomiasis and newly developed advanced schistosomiasis detection kits and the detection kit. Background technique [0002] Late schistosomiasis (referred to as late blood) is due to repeated or large-scale infection of Schistosoma japonicum cercariae, untimely treatment, and a long period of pathological development. It is the terminal form of schistosomiasis and poses a serious threat to the physiological health of patients. life threatening. With the continuous implementation of comprehensive prevention and control measures for schistosomiasis, especially the development of treatment and rescue projects for patients with late blood, the patient's condition has been better controlled. However, data show that n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
Inventor 华海涌黄玉政尤璐唐凤胡楠楠
Owner JIANGSU INST OF PARASITIC DISEASES
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