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Process for preparing protein, DNA and lipid microarray by using APES modified glass slide

A microarray and protein technology, which is applied in the field of biochip/microarray preparation, can solve the problems of parallel detection of autoantibodies, increase the physical and mental burden of patients, and inconvenience clinicians, and achieve high sensitivity, good fixation effect, and coincidence rate high effect

Inactive Publication Date: 2006-04-26
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rheumatism is difficult to diagnose in the early stage, but in the late stage of the disease, multiple organ systems in the whole body will be involved, causing great pain to the patient, so a highly sensitive and comprehensive diagnostic method is needed, and the current autoantibody detection mainly uses Immunological methods, commonly used immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay, etc., the results of these methods are generally incomparable, and these methods can only detect a single antibody, in the actual application process, cannot A large number of parallel detection of multiple autoantibodies, and for the diagnosis of rheumatism, multiple antibodies need to be detected, so the existing detection methods require a relatively large number of reagents and clinical specimens, which increases the physical and mental burden of the patient and also imposes a burden on the clinic. physician inconvenience

Method used

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  • Process for preparing protein, DNA and lipid microarray by using APES modified glass slide
  • Process for preparing protein, DNA and lipid microarray by using APES modified glass slide
  • Process for preparing protein, DNA and lipid microarray by using APES modified glass slide

Examples

Experimental program
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Effect test

Embodiment 1

[0060] (1) Rinse the slides with 1N NaOH and concentrated sulfuric acid respectively, rinse with double distilled water until the slides are clean, and spin dry in a centrifuge;

[0061] (2) Put the above-mentioned cleaned glass slides into APES diluted with acetone with a volume ratio of 1:47, and stay for 30-40 seconds;

[0062] (3) Take out the slide, stop for a while, control the dry droplets, and then rinse with pure acetone solution to remove unbound APES;

[0063] (4) Using the APES-modified slide as a substrate, immobilize proteins to prepare a microarray.

[0064] Human IgG starting from 50mg / mL, with PBST (each liter containing NaCl 8.0g, KCl 0.20g, Na 2 HPO 4 1.44g, KH 2 PO 4 0.24g, Tween-20 1ml) for serial dilution, the concentrations are 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.05mg / ml, a total of ten gradients. On the above APES-modified slides, 5 replicates per row for each concentration point; positive control uses IgG diluted in the same spotting solution...

Embodiment 2

[0066] (1) Rinse the slides with 1N NaOH and concentrated sulfuric acid respectively, rinse with double distilled water until the slides are clean, and spin dry in a centrifuge;

[0067] (2) Put the above-mentioned cleaned glass slides into APES diluted with acetone with a volume ratio of 1:50, and stay for 20 to 30 seconds;

[0068] (3) Take out the slide, stop for a while, control the dry droplets, and then rinse with pure acetone solution to remove unbound APES;

[0069] (4) After recovery of the vector plasmid pcDNAII frozen at -70°C, inoculate LB (LB-Amp) agar solid medium containing 50 ml of ampicillin, and overnight at 37°C. Pick a single colony and inoculate it in LB-Amp liquid medium, shake the plasmid overnight at 37°C, extract the plasmid with a plasmid extraction kit, verify it by agarose gel electrophoresis, and measure its concentration to be 300 μg / ml.

[0070] (5) Using the above-mentioned APES-modified glass slide as a substrate, the extracted plasmid DNA was...

Embodiment 3

[0072] (1) Rinse the slides with 1N NaOH and concentrated sulfuric acid respectively, rinse with double distilled water until the slides are clean, and spin dry in a centrifuge;

[0073] (2) Put the above-mentioned cleaned glass slides into APES diluted with acetone with a volume ratio of 1:48, and stay for 32 to 42 seconds;

[0074] (3) Take out the slide, stop for a while, control the dry droplets, and then rinse with pure acetone solution to remove unbound APES;

[0075] (4) The cardiolipin solution purchased from Sigma was serially diluted with dimethyl sulfoxide (DMSO), and the concentrations were respectively 4000, 2000, 1000, 500, 200, 100 μg / ml, and sampled with a Cartisian sample spotter, Prepare microarrays.

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Abstract

The invention discloses a protein, DNA and lipid microarray preparation method of APES (3-amino propyl ethoxy silane) ornament slide, which comprises the following steps: (1) soaking the slide through strong alkali and concentrated sulfuric acid separately; cleaning the slide through double-distillation water; drying the slide through centrifuger; (2) putting the slide in the APES diluted by acetone with the bulk ratio between 1:45 and 1:55 for 20-50 sec; (3) fetching the slide for a while; rinsing the unconnected APES in the pure acetone; (4) choosing the relating APES ornament slide as substrate to fix protein, DNA and lipid; preparing microarray.

Description

technical field [0001] The present invention relates to a kind of preparation method of biological chip / microarray, relate in particular to a kind of glass slide modified with 3-aminopropyltriethoxysilane (APES), immobilize protein, DNA, and lipid, to prepare protein, DNA 1. A lipid microarray method for detecting multiple autoimmune antibodies, belonging to the technical fields of biochips, diagnostic reagents, and the like. Background technique [0002] The incidence rate of autoimmune diseases in the population is 3-5%, especially the highest incidence rate among the elderly. The most common feature of these diseases is the presence of autoantibodies in the serum, and different autoantibody profiles can distinguish the various diseases in this group. In addition to the corresponding clinical symptoms, the current international diagnostic criteria for autoimmune diseases are mainly based on the detection of autoantibodies in the patient's serum. Rheumatism is difficult t...

Claims

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Application Information

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IPC IPC(8): G01N33/531G01N33/53
Inventor 韩金祥徐华高雪芹刘毅
Owner SHANDONG MEDICAL BIO TECH RES CENT