Sequence and application thereof of anti-tumor monoclonal antibody
A monoclonal antibody and sequence technology, applied in the field of biomedicine, can solve the problems of hidden symptoms, difficult early diagnosis, no early diagnosis method, etc., and achieve the effect of high efficiency and broad spectrum application value
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Embodiment 1
[0030] Example 1. Preparation of Colon Cancer Cell Membrane Antigen Crude Extract
[0031] Take fresh surgical resection specimens of gastric cancer: the pathological diagnosis is poorly differentiated gastric cancer, and the method of cracking potassium at a high concentration is used to extract cell membrane antigens and prepare crude extracts of membrane antigens. Wash the blood with sterilized saline at 4°C, carefully cut and grind in an ice bath, add 3M KCl solution while grinding, grind until homogeneous, pass through a sieve, stir overnight at 4°C for 3 hours, and centrifuge at 10,000 rpm at 4°C After 20 minutes, absorb the supernatant, add an equal volume of saturated ammonium sulfate to precipitate, and centrifuge at 10,000 rpm at 4°C for 20 minutes. After the precipitate was completely dissolved with 10mMPBS, it was dialyzed against 10mMPBS. After the dialysis was completed, it was aliquoted and stored at -80°C for later use.
Embodiment 2
[0032] Embodiment two, Panning screening
[0033] The phage surface display library is a non-immune murine ScFv expression library previously constructed by the patent applicant. This patent is to screen antibodies that can specifically recognize colon cancer from this library. After the plastic culture dish was coated with the colon cancer cell membrane antigen, the supernatant of the recombinant phage was poured on it, cultured at 37°C for 2 hours, and the plate was washed 20 times with PBS and PBST successively. Then, the TG1 cultured to the logarithmic growth phase was poured onto the immunoadsorbed petri dish, and incubated at 37° C. for 1 hour. Such a process of "adsorption-elution-propagation" is Panning screening, and then superinfection with M13K07 can generate a phage surface display library enriched in clones. Thereafter, 7 rounds of screening were carried out as described above.
Embodiment 3
[0034] Example 3. Identification of antigen-binding activity of a single recombinant phage clone
[0035] 1. Preparation of monoclonal recombinant phage
[0036] After Panning screening, the TGI bacterial solution was diluted according to the original solution; 1:10; 1:100; 1:10004 dilutions, spread on SOBAG agar plates, and cultured overnight at 30°C. Pick 90 single colonies and inoculate them in 100ul of 2×YTAG respectively, culture overnight at 30°C, take 20ul of the culture, add it to 200ul of 2×YTAG containing 5×10pfu / ml M13K07, culture with shaking at 37°C for 2h, centrifuge, and use Resuspend the pellet in 200ul of 2×YTAK and incubate overnight at 30°C. The supernatant after centrifugation is a single recombinant phage.
[0037] 2. ELISA detection of antigen-binding activity (Douillard, et al., Enzyme-linked Immunosorbent Assay for Screening monoclonal antibody production using enzyme-labeled second antibody, Meth. Enzymol, 92:168-74, 1983)
[0038] Set up 1 negativ...
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