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Reagent kit and method for detecting digestive system tumor biological mark group by immunomic mass spectrometry

A technology of biomarkers and kits, applied in the field of disease detection, can solve the problem that disease markers cannot be detected at the same time

Inactive Publication Date: 2008-06-04
许洋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using antibodies and three-color immunofluorescence, up to three disease markers can be detected, but more than three disease markers cannot be detected at the same time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Application of immune group mass spectrometry to detect variant expression of digestive system tumor biomarkers

[0066] (1) Experimental method

[0067] 1. Materials

[0068] 1. Specimen sources: A. Serum from 920 normal controls; B. Serum from 208 patients with gastric cancer; C. Serum from 226 patients with liver cancer; D. Serum from 182 patients with colorectal cancer.

[0069] 2. Quality control: A. Human standardized quality control serum B. Mass spectrometer laser energy regulation: Before each test, use the above-mentioned standardized quality control serum.

[0070] 3. The matrix contains the same amount of labeled antibodies: anti-FPA (fibrinopeptide A), anti-C3a (complement C3a), anti-ApoA-I (apolipoprotein A-I), anti-ApoA-II (apolipoprotein A-II) .

[0071] 2. Method

[0072] 1. Collection of samples: After the whole blood is collected, draw the serum and store it at -80°C; take out the serum sample in the refrigerator at -80°C, and put it on ...

Embodiment 2

[0085] Example 2 Sorting and Identification of Digestive System Tumor Biomarkers

[0086] The 1465±15Da, 8938±15Da, 28078±15Da, and 8707±15Da biomarkers were sequenced by MS / MS, post-source dissociation (PSD) and protein ladder sequencing. By breaking molecules into pieces, protein ladders can be generated. This gradient is then analyzed by mass spectrometry. Biomarkers identified as variantly expressed biomarker clusters:

[0087] Experimental results

[0088] (A) 1465±15Da: FPA-degAla (fibrinopeptide A with alanine removed from its amino terminal) sequence FPA fragment (1465.7Da, DSGEGDFLAEGGGVR)

[0089] (B) 8938±15Da: N-terminal of C3a-degArg (complement C3a whose C-terminal has removed arginine) sequence

[0090] Ser-Val-Gln-Leu-Thr-Glu-Lys-Arg-Met-Asp-Lys-Val-Gly-Lys-Tyr-Pro-Lys-Glu-Leu-Arg-Lys-Cys-Cys-Glu-Asp- Gly-Met-Arg-Glu-Asn-Pro-Met-Arg-Phe-Ser-Cys-Gln-Arg-Arg-Thr-Arg-Phe-lle-Ser-Leu-Gly-Glu-Ala-Cys-Lys- Lys-Val-Phe-Leu-Asp-Cys-Cys-Asn-Tyr-Ile-Thr-Glu-Leu-Arg-A...

Embodiment 3

[0103] Example 3 Preparation of mutated or modified biomarker antibodies

[0104] Synthesis of mutated or modified biomarkers: Synthesis of FPA-degAla, C3a-degArg, mutated ApoA-I, mutated ApoA-II. The synthetic mutated biomarkers were immunized into mice, and after the immune response appeared, B cells were isolated from peripheral blood. Single lymphocytes secreting the desired antibody were selected and isolated using a hemolytic plaque assay. Expand single cells to 1 x 10 7 More than one, the mRNA was extracted with the Quick mRNA Purification Kit. Using the extracted mRNA as a template, a cDNA strand is synthesized. Using this cDNA as a template, add the mouse antibody heavy chain variable region (V H ) universal primer, light chain variable region (V L ) universal primer, carry out polymerase chain reaction, obtain the amplified V H gene fragment and V L Gene fragment. The amplified products were identified and separated by 15g / L agarose gel electrophoresis. Reco...

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Abstract

The invention relates to a biomarker captured on a surface matrix adsorbed with an antibody group, which is detected by a quantitative mass spectrum analysis under the control of standard quality control serum. The definition of immunomic mass spectrometry analysis, namely IMS, is a method which combines a group of various antibodies with a spectrometry to accurately identify a variant or modified biomarker group. A plurality of biomarkers can be captured on one antibody group matrix at the same time, and a spectrometry accurate analysis is processed to the captured variant or modified biomarker. The invention can detect a plurality of biomarker groups at the same time. The method of the invention can be used for detecting a biomarker combination in fluid which is separated from a human body. The biomarker combination is applicable to the detection method of a kit which identifies in vitro fluid of a normal person and patients with different types of tumours of the digestive system. The method is accurate, convenient and rapid.

Description

technical field [0001] The present invention relates to a novel method for protein analysis in biological samples, a method for capturing biomarkers through a matrix that can be combined with antibodies, and using mass spectrometry with quantitative control to detect the biomarkers. The invention mentioned here relates to the field of disease detection and is a new non-invasive in vitro detection method. More specifically, this invention relates to biomarkers, and these antigens or biomarkers can be used to distinguish multiple diseases at once with higher specificity and sensitivity of antibody panels and quantitatively controlled mass spectrometry. The present invention can be applied to the detection method or kit development of the digestive system tumor biomarker combination in the body fluid that has been separated from the human body. Background technique [0002] With the implementation and completion of the Human Genome Project, scientists proposed the concept of t...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/53G01N30/02
Inventor 许洋
Owner 许洋
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