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Method for detecting biological sample

A biological sample and detection method technology, applied in the field of medical testing, to achieve the effects of avoiding damage, fast and simple detection, and low cost

Inactive Publication Date: 2018-08-17
祝胜郎 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, clinical practice shows that the positive rate of serum PLA2R antibody in patients with spontaneous membranous nephropathy is only 68.5%, and 31.5% of patients with idiopathic membranous nephropathy are still negative for serum PLA2R antibody, so it is difficult to effectively detect

Method used

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  • Method for detecting biological sample
  • Method for detecting biological sample
  • Method for detecting biological sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 Separation

[0110] like figure 1 As shown, the urine samples collected from the subjects were separated as follows:

[0111] A 40 ml fresh urine sample from the subject was collected, centrifuged at 1000-4000 rpm for 10 minutes, and the sediment (containing cells and cell debris) was collected. The pellet was resuspended in PBS and centrifuged again at 1000-4000 rpm for 10 minutes to wash off residual soluble protein and collect the pellet. The sediment was resuspended in PBS and divided into three test samples I, II and III.

[0112] Wherein, the sample I to be tested was suspended and fixed with 0.5 ml of 4% paraformaldehyde for 30 minutes for immunohistochemistry and immunofluorescence. Observational tests such as electron microscopy. Gel electrophoresis buffer was added to test sample II for western blot detection (WesternBlot). Protease inhibitors were added to the sample III to be tested and stored at -80°C for detection by proteomics chips and the...

Embodiment 2

[0113] Example 2 Immunofluorescence detection

[0114] like figure 2 As shown, the samples to be tested were subjected to immunofluorescence detection, as follows:

[0115] Using anti-human IgG4 antibody, anti-human PLA2R antibody, anti-human NPHS2 antibody and anti-human IgA antibody as primary antibodies, the source of antibody serum is mouse, rabbit or sheep, monoclonal antibody or polyclonal antibody;

[0116] Use donkey anti-mouse, donkey anti-rabbit or donkey anti-goat antibodies labeled with FITC, Cy3 and Cy5 as secondary antibodies;

[0117] The detection method is single staining (one antibody primary antibody), double staining (two primary antibodies) or triple staining (three primary antibodies);

[0118] The immunofluorescence primary antibody was diluted 100-200 times with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), and incubated with PBS containing 0.2% Triton and 1% BSA (PBST) for 15- 30 minutes, primary antibody incubation over...

Embodiment 3

[0124] Example 3 Urine IgG4 immunofluorescence detection of patients with different renal diseases and normal subjects

[0125] According to the non-invasive detection method described above in the present invention, the samples collected from normal healthy subjects, patients with membranous nephropathy, patients with focal segmental glomerulosclerosis (FSGS), patients with diabetic nephropathy (DN), minimal change nephropathy (MCD) patients and patients with IgA nephropathy were subjected to IgG4 immunofluorescence detection in the urine. The detection images are shown in Figure 5-11 .

[0126] Figure 5 For the immunofluorescence detection image of normal healthy subjects, as shown in the figure, normal human urine sediment IgG4 signal is negative.

[0127] Figure 6-7 For the immunofluorescence detection images of patients with membranous nephropathy, Image 6 is the IgG4 signal on intact cells, Figure 7 is the IgG4 signal on cell debris. As shown in the figure, th...

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Abstract

The invention provides a method for detecting a biological sample. The method comprises the following steps: (1) separating the biological sample collected from a subject to obtain a sample to be detected; and (2) detecting one or more of IgG4, PLA2R, Nephrin and NPHS2 in the sample to be detected, and adopting the obtained detection result as a preliminary basis for judging that whether the subject needs renal biopsy or not. The detection method can be used to perform primary screening on membranous nephropathy, and provides reference data for whether renal biopsy puncture in the later stageis performed or not; and the detection method is noninvasive, and has the advantages of simplicity in operation and low detection cost.

Description

technical field [0001] The invention relates to the field of medical testing, in particular to a method for detecting biological samples. Background technique [0002] The prevalence of chronic kidney disease (CKD) in my country is 10.8%. Membranous nephropathy (MN) is a group of diseases characterized by the deposition of immune complexes under the glomerular basement membrane epithelium with thickening of the basement membrane. Common causes of symptoms. Membranous nephropathy accounts for about 6.0%-28.8% of primary glomerular diseases, becoming the second cause of primary glomerular disease after IgA nephropathy, and also an important cause of end-stage renal disease (ESRD). . A large-scale study by the team of Academician Hou Fanfan of Nanfang Hospital in 2016 showed that the proportion of membranous nephropathy was 23.4%, second only to IgA nephropathy (28.1%). [0003] Membranous nephropathy is divided into idiopathic membranous nephropathy (IMN) and secondary membr...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/68
CPCG01N21/6428G01N21/6486G01N33/6854G01N33/6893G01N2800/34G01N2800/347
Inventor 不公告发明人
Owner 祝胜郎
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