Gastrointestinal cancer determination method
A technology for gastrointestinal cancer and determination methods, applied in biochemical equipment and methods, microbiological measurement/inspection, instruments, etc., can solve problems such as unestablished early diagnosis and detection methods for pancreatic cancer
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experiment example 1
[0132] Experimental Example 1 Recognition site of anti-human haptoglobin polyclonal antibody (rabbit)
[0133] Using 50mM phosphate buffer, prepare 100μg / mL haptoglobin phenotype 1-1 (Hpt1-1, manufactured by Sigma-Aldrich) and haptoglobin phenotype 2-2 (Hpt2-2, manufactured by Sigma-Aldrich) company), 3:1 mixed sample buffer 1 (0.25M Tris-HCl (tris-hydrogen chloride), pH 6.8, 8% SDS, 40% glycerol, 0.02% BPB, 20 % 2-mercaptoethanol) to make samples.
[0134] Thereafter, 4 μL of the sample was subjected to electrophoresis on a 12.5% polyacrylamide gel. The obtained electrophoresis gel was semi-dry blotted to PVDF membrane using Bio-Rad's blotting system according to the usage protocol. The transferred PVDF membrane was blocked with a 4% phosphate buffer solution containing Block Ace (manufactured by DS Pharma Biomedical Co., Ltd.).
[0135] Prepared by diluting anti-human haptoglobin polyclonal antibody (rabbit) [anti-human Hpt antibody (Poly), manufactured by Immunology ...
experiment example 2
[0140] Experimental Example 2 Acquisition of anti-human haptoglobin monoclonal antibody (10-7 antibody) reactive to α chain
[0141] (1) Production of haptoglobin
[0142] By adding 10% fetal bovine serum (FBS, Biological Industries (Biological Industries), Israel), 100 U / mL penicillin and 100 μg / mL streptomycin in RPMI and L-glutamine and NaHCO 3 (manufactured by Sigma-Aldrich) at 37°C, 5% CO 2 The human colorectal cancer cell line HCT116 (ACTT) was cultured under certain conditions. The culture plate used is IWAKI culture plate 10cm and 15cm (IWAKI company, Tokyo, Japan). The obtained culture solution was divided into two parts, one part was used to obtain fucose-free haptoglobin (Hpt(-)) for non-fucose culture, and the other part was used to obtain fucose-containing haptoglobin (Hpt(-)). Globin (Hpt(+)) was cultured with the addition of fucose. That is, one part was recovered after culturing the cells for 96 hours using RPMI without adding FBS when recovering the cult...
experiment example 3
[0157] Experimental Example 3 Anti-Human Haptoglobin Monomer Reactive to β Chain and Unreactive to Human Haptoglobin with S-S Bond Severed Acquisition of cloned antibodies (3-1 antibody and 3-5 antibody)
[0158] Using the various anti-human haptoglobin monoclonal antibodies obtained in Experimental Example 2 (3), perform the following immunoblotting treatment, and sort out human haptoglobin that is reactive to the β chain and has a cleaved S-S bond. Reactive antibodies.
[0159] That is, first, use 50mM phosphate buffer to prepare 100 μg / mL of Hpt1-1 and Hpt2-2 respectively, and mix the sample buffer 2 (0.25M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.02% BPB), make the sample. Also, similarly, Hpt1-1 and Hpt2-2 prepared at 100 μg / mL were mixed with the same sample buffer 1 as in Experimental Example 1 at a ratio of 3:1 to prepare a sample.
[0160] Thereafter, 4 µL of the samples prepared in the sample buffer 2 were subjected to electrophoresis on a 7.5% polyacrylamide ...
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