Hyaluronan (Na salt) (Acros Organics, New Jersey, USA). Guanidium hydrochloride, bovine testicular hyaluronidase type I S, EDC [1, ethyl 3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, MES buffer (2-N-morpholino ethane sulfonic acid) Protease inhibitors cocktail and biotinylated goat anti-mouse IgG’s HRP conjugate were procured from Sigma chemicals, USA. EZ-Link Biotin LC hydrazide purchased from Pierce, Rockford, USA. Anti-human CD44 (H-CAM, Clone 1M7.8.1) antibody was purchased from Fisher Scientific, USA. Streptavidin-peroxidase conjugate (HRP) obtained from Invitrogen, USA.
Sample collection and preparation
The study consisted of 70 normal subjects and 45 cancer patients (Lymphoma,(2) Salivary(1), Tongue(4), Thyroid(1), Buccal Mucosa(1), Lung(1), Breast(6), Stomach (3), Gallbladder(1), Oesophagus (3), Colon(3), Pancreas (1), Rectal(2), Urinary bladder(2), Prostate(4), ovary(5), endometrial(3) and cervix(2). Serum samples from normal subjects and cancer patients accessed from cancer hospitals in Mysore, India (Preethi Centre of oncology, and KR Hospital, Logic and Clue Diagnostic center) and the protocol approved by the ethical review committee (Mys–00340-AA-NH,an d IHEC-UOM NO 35) and the patient’s consent was taken. Blood samples were collected from each patient before any treatment. Samples centrifuged at 2000xg for 30 min at room temperature for 1 hr, and the separated sera were stored at –800C. The tumor sections from patients after H&E stain was graded using the TNM grading system. Serum samples treated with 4X lysis buffer, containing 0.2 M Tris-HCl (pH 8.0), 80 mM EDTA, 4 mM PMSF, 4 mM Benzamidine-HCl and 2% Triton X 100 plus protease inhibitor cocktails and centrifuged at 10,000xg for 30 min at 40C. The supernatant was stored either at –800C until further analysis. The amount of protein estimated at UV 280 nm and Bradford reagent using BSA as a standard.
Preparation of biotinylated hyaluronic acid according to Boregowda et al.2013 and Srinivas et al. 2016, ( 13,24) In brief, HA dissolved in PBS-A dialyzed in MES buffer and reacted with biotin_LC-hydrazide and EDC in DMSO. Incubated for 16hrs, dialyzed against PBS-A and stores in glycerol at –20C
Production of monoclonal antibody UNIVMab
Hybridoma and the antibody were prepared according to Boregowda et al. (18, 22, 23). In brief, the hybridoma grown in DMEM under pathogen and complement free human serum received from the hospitals. The antibody production in human serum of any blood groups did not affect UNIVMab in recognizing the human antigen. The clones were grown in DMEM containing 10% (v/v) inactivated human serum. After 21 days, the media was collected and precipitated with cold saturated ammonium sulphate solution (final 50%) at 40C overnight and centrifuged at 12000xg for 30 min. The pellet dissolved in PBS and dialyzed against PBS.
The statistical evaluations from Western blots were analyzed using the two-tailed Student-test. Statistical P values defined as follows: P<0.05 = significant; P<0.01 = highly significant; and P<0.001 = extremely significant. Data are presented as means±SEM