Protein chip for detecting blood and cerebro spinal fluid pathogen antibody, and its preparing method and use

A pathogen antibody and protein chip technology, applied in the field of biochips and their preparation, can solve the problems of large differences in specificity, sensitivity, stability, threat to the health of experimenters, serious cross-reaction, etc., so as to retain a high degree of specificity, improve Detection speed and efficiency, the effect of low sample consumption

Inactive Publication Date: 2005-07-20
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), rubella virus (RV), Japanese encephalitis virus (JEV), mumps virus (MV), Mycobacterium tuberculosis, and Leptospira to detect ten kinds of pathogens, it takes 20 kits to detect all IgG and IgM, Each reaction can only detect one indicator, which is slow, inefficient, expensive, and requires a large amount of samples, which is actually impossible; moreover, the antigens in the currently used detection kits are crudely extracted antigens from pathogen cultures. The virus content is low, the composition is complex, the background is high, and the cross-reaction between various pathogens is serious, and the specificity, sensitivity, and stability vary greatly; in addition, the substrates used in the experiment are also highly toxic chemicals. There is also a potential threat to the health of personnel

Method used

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  • Protein chip for detecting blood and cerebro spinal fluid pathogen antibody, and its preparing method and use
  • Protein chip for detecting blood and cerebro spinal fluid pathogen antibody, and its preparing method and use
  • Protein chip for detecting blood and cerebro spinal fluid pathogen antibody, and its preparing method and use

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Modification of slide and fixation of antigen, such as figure 1 The appearance of the protein chip for detecting serum cerebrospinal fluid pathogen antibodies, such as figure 2 Spotted array diagram.

[0046] Put the slides on a slide rack, put them into a glass jar filled with 350ml of cleaning solution (NaOH 100g, ethanol 600ml, water 400ml), place on a shaker at 60 rpm, and shake flatly for 2 hours; pour off the cleaning solution, fully Wash 4 times with water, 3 minutes each time; soak the slides in a glass jar filled with 350ml polylysine PBS solution (polylysine 35ml, PBS 35ml, water 280ml), and place on a shaker at 60 rpm , shake flatly for 1 hour; immerse the slide in water and wash it up and down 5 times; put it in a centrifuge, centrifuge at 800 rpm for 5 minutes, put it in a clean plastic box, place it vertically for 2 weeks or apply after baking in an oven use.

[0047] For 2 serum samples, choose a 2×2 microarray chip, (such as figure 1 ) wh...

Embodiment 2

[0049] Embodiment 2: Antigen-antibody reaction and detection

[0050] Add blocking solution (1% BSA, 0.2g / L KCl, 1.44g / L Na2HPO4, 0.24g / L KH2PO4, 8g / L NaCl, 0.1% Tween-20,) to seal on the chip where the antigen has been spotted, at 37°C hour, block the non-specific sites on the surface of the substrate; wash 3 times with PBST (0.2g / L KCl, 1.44g / L Na2HPO4, 0.24g / L KH2PO4, 8g / L NaCl, 0.1% Tween-20), each 10 Seconds after seeding, rinse with PBS (0.2g / L KCl, 1.44g / L Na2HPO4, 0.24g / L KH2PO4, 8g / LNaCl), and centrifuge at 800 rpm for 3 minutes to remove excess blocking solution; After diluting 10 times with PBS, take 3 μL and add it to the array. The first sample is added to the upper and lower arrays of the first column; the second sample is added to the upper and lower arrays of the second column, and placed in a hybridization box at 37°C 30 minutes to fully react the antigen and antibody; wash 3 times with PBST, 10 seconds each time, rinse with PBS, and centrifuge at 800 rpm for...

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Abstract

The invention discloses a protein chip for detecting pathogen antibodies of blood and cerebrospinal fluid and its preparing method, and the protein chip includes substrate and peculiar-culture or shape protein or polypeptide antigen of array distributed pathogen and contrasted dot coating, where the substrate is a glass substrate; and the antigen and contrasted dot coating refers to 13 antigens with ten indexes including HSV-I, HSV-II, VZV, CMV, EBV, RV, JEV, MV, MT and LP, uniformly distributed on the glass substrate in a dot matrix form, and positive contrast, negative contrast and blank contrast. The protein chip of the invention can obtain multiple-index reacting result only by one reaction, judges infection of different pathogens and has the characters of quickness, high efficiency, accuracy and parallel diagnosis.

Description

technical field [0001] The invention relates to a biological chip and its preparation method and application, in particular to a low-density protein chip and its preparation method and application, especially to a blood cerebrospinal fluid pathogen antibody detection chip and its preparation method and application. Background technique [0002] In recent years, the incidence of infectious diseases in the central nervous system (CNS) has increased. The pathogens include various bacteria, fungi, viruses, spirochetes, etc. Etiological diagnosis is the gold standard for diagnosis. Once the etiological diagnosis is confirmed, there is no need to mention the differential diagnosis. At present, pathogen diagnosis still relies on morphological examination, pathogen isolation and culture, and enzyme-linked immunosorbent assay (ELISA) to detect antibodies; while for Mycobacterium tuberculosis, spirochetes, and various viral infections, including herpes simplex virus type I ( HSV-I), ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/552G01N33/569
Inventor 刘毅韩金祥朱波高雪芹潘继红黄海南黄海燕鲁艳芹
Owner SHANDONG MEDICAL BIO TECH RES CENT
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