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Liquichip for parallel detection of colorectal cancer protein marker, preparation and application thereof

A technology of protein labeling and parallel detection, which is applied in the field of immunology technology and clinical detection, can solve the problem that only one can be detected at a time, which is inconvenient for clinical diagnosis, and achieve the effect of saving detection cost and improving sensitivity and specificity

Inactive Publication Date: 2006-05-03
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the inconvenience of the existing tumor marker ELISA detection, which can only detect one marker at a time and the needs of clinical diagnosis, the problem to be solved by the present invention is to provide a liquid phase chip for parallel detection of colorectal cancer protein markers and its preparation method And application, to provide a non-invasive, convenient and accurate clinical detection method and detection kit for early detection, early diagnosis and early treatment of colorectal cancer

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Coupling of capture antibodies to known numbered microspheres

[0027]1. Select No. 26, 36, 46, 56, 66, 76, and 86 carboxyl microspheres (Luminex Company) respectively, and oscillate the microsphere suspension with a vortex shaker for 20 seconds to mix the microspheres evenly.

[0028] 2. Take about 2×10 carboxyl microspheres of each number above. 3 Transfer each to a centrifuge tube and centrifuge at ≥8000×g for 2 min to precipitate carboxylated microspheres.

[0029] 3. Remove the supernatant and add 100μl dH 2 O, use a vortex shaker to resuspend the microspheres for 20 seconds, centrifuge at ≥8000×g for 2 minutes, and precipitate the carboxylated microspheres.

[0030] 4. Remove the supernatant, add 80 μl, 100 mM, pH=6.2 sodium dihydrogen phosphate salt solution, and resuspend the washed carboxy microspheres with a vortex shaker for 20 seconds.

[0031] 5. Add 10μl, 50mg / ml Sulfo-NHS (with dH 2 O dilution), oscillate gently with a vortex shaker.

[00...

Embodiment 2

[0043] Example 2: Conjugation of detection antibody to biotin

[0044] 1. Dissolve activated biotin (Sigma company) in dimethyl sulfoxide at a concentration of 1 mg / ml.

[0045] 2. The purified detection antibodies to be coupled (respectively: MAb mouse anti-IgGlclone #M111146(10-c10), Mab clone M8073021(10-c04), clone #CA72-4M, MAb-c004-10 mouse anti-IgG1, clone 242 IgG, MAb mouse anti-IgG1 clone #M94193 (10-P15), N53378-08D: NP-6 Pab rabbit-anti-Hu E B, CCSP-2 antibody) were dissolved at a concentration of 1mg / ml In 0.1mol / L pH9.0 sodium bicarbonate solution.

[0046] 3. Mix the activated biotin solution and the antibody solution to be coupled at a volume ratio of 1:8, and incubate at room temperature for 4 hours.

[0047] Under the condition of 4.4°C, dialyze in 0.05mol / L, pH7.2 PBS for 24h, and change the medium 4 times to remove unbound free biotin.

[0048] 5. Add 0.02% NaN3 to the biotin-bound antibody solution, aliquot them separately, and store them in the dark at ...

Embodiment 3

[0049] Example 3: Application of the liquid chip for parallel detection of colorectal cancer protein markers described in the present invention in clinical detection

[0050] (1) The technical process of using the colorectal cancer protein markers of the present invention to detect protein tumor markers in parallel with liquid-phase chips:

[0051] 1. Take out 500 capture antibody-conjugated microspheres prepared above for each tumor marker, mix them in equal proportions, and distribute them in 96-well plates. Each well contains various capture antibody-conjugated microspheres. For each 500 cells, add 50 μl of serum to be tested and incubate at 37°C for 2 hours.

[0052] 2. Centrifuge at ≥8000×g for 2 minutes, remove the supernatant, add 300 μl of 1% PBS-BSA, vortex for 30 seconds, and seal in a 37°C incubator for 1 hour.

[0053] 3. Centrifuge at ≥8000×g for 2 minutes. Remove the supernatant, add 300 μl PBS-TBN, and vortex for 30 seconds. Centrifuge at ≥8000×g for 2 minute...

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Abstract

The invention discloses a colon cancer protein mark parallel test liquid phase chip, which is mainly formed by: micro ball, capture antibody, test antibody and streptomycin-phycoerythrin, wherein the capture antibody with the corresponding micro balls form coupling conjugated, which uses red laser to active the red categorizing fluorescence of the sphere base material and ascertains the type by the different color of the sphere base material; the test antibody is a skin factor mark antibody; the capture antibody and the test antibody can combine with the colon cancer protein mark; the test antibody combines with the streptomycin-phycoerythrin and uses green laser to active the phycoerythrin to measure the report fluorescence molecular number of the sphere base material, which can indirect ascertain the colon cancer protein mark content combines with the sphere base material. The invention also discloses the colon cancer protein mark parallel test liquid phase chip applied in preparing the test agent.

Description

technical field [0001] The invention relates to a liquid phase chip and its preparation method and application, in particular to a liquid phase chip for parallel detection of colorectal cancer protein markers and its preparation method and application, belonging to the technical fields of immune technology and clinical detection. Background technique [0002] The number of new cases of colorectal cancer worldwide reaches 940,000 every year, and nearly 500,000 people die of colorectal cancer every year. Colorectal cancer is the third leading cause of cancer death. Colorectal cancer is also one of the most common malignant tumors in my country, and currently ranks fourth in the incidence of malignant tumors. To improve the survival rate and quality of life of patients with colorectal cancer, early detection, early diagnosis and early treatment are the key. [0003] At present, the main methods for early detection and diagnosis of colorectal cancer are fiber endoscopy, fecal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/531
Inventor 高雪芹张华宁韩金祥
Owner SHANDONG MEDICAL BIO TECH RES CENT
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