Method for analyzing immunological difference of two kinds of states of individuals and method for assisting in determining states of individuals
A state and individual technology, which is applied in the fields of assisting in determining individual states, devices assisting in determining individual states, and devices for immune differences, and can solve problems such as patient discomfort and long duration
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Embodiment 1
[0052] General methods, including:
[0053] First, sequence and identify CDR3:
[0054] Use lymphocyte separation medium to separate peripheral blood T / B lymphocytes, extract DNA (or RNA), use multiplex PCR or 5'RACE to capture CDR3, and perform high-throughput sequencing through the Hiseq2000 or Hiseq2500 or Miseq platform.
[0055] After performing quality control on the measured data, they were compared to the IMGT database (http: / / www.imgt.org / ) to determine the CDR3 sequence.
[0056] Secondly, the analysis of immune results:
[0057] The high-frequency CDR3 sequence is a highly expanded clone (highly expanded clone), and the HEC rate is defined—the highly expanded clone-rate (HEC rate) is a frequency exceeding 0.05%, preferably, a frequency not exceeding 0.5% The ratio of the number of CDR3 species to the total number of CDR3 species.
[0058] PCA analysis was performed on differentially used V subtypes, V merged subtypes (Vmerge), and / or VJ combined subtypes.
[005...
Embodiment 2
[0075] Taking T lymphocytes as the research target, the optimized multiplex PCR technology is used to amplify the CDR3 region, the most diverse complementarity-determining region of the T cell receptor β chain, and the amplification primers, amplification methods, library construction and sequencing, etc. The method described in CN103205420A is carried out to obtain off-machine data, comprehensively analyze the TCR composition, evaluate the diversity of the immune system, and mine the relationship information between the immune repertoire and the occurrence and development of liver cancer, hepatitis, and rectal cancer.
[0076] The method comprises the steps of:
[0077] (1) According to the T cell receptor CDR3 sequence, design V segment and J segment primers such as CN103205420A, and reference sequence construction, including obtaining the known CDR3 sequence set from the database.
[0078] (2) Sample preparation
[0079] 1. Extract 5 mL of peripheral blood from the subject...
Embodiment 3
[0101] The peripheral blood samples of 10 cases of colorectal cancer and 20 cases of healthy people from the hospital were sequenced and tested for TCRβ chain CDR3. The PBMC isolated from peripheral blood was used as the research object for the sequencing of immune repertoire, and the content is as follows:
[0102] 1. Peripheral Blood Sampling
[0103] 1) Take 5ml of the peripheral blood sample from the patient and put it in an EDTA anticoagulant tube. Gently invert up and down 4-6 times and mix thoroughly, place at room temperature, and complete the separation of PBMC within 2 hours;
[0104] 2) Add 3 times the volume of sterile saline, and mix upside down;
[0105] 3) Take 3ml of cell layering solution in a 15ml centrifuge tube, and carefully pipette 4ml of whole blood cells diluted in step 2) along the tube wall and superimpose on the layering liquid surface, and divide into multiple tubes if the volume is greater than 4ml. Centrifuge horizontally at 400g for 30 minutes...
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