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 50 cancer patients (Lymphoma(2 patient samples), Salivary(1 patient sample), Tongue(4 patient samples), Thyroid(1 patient sample), Buccal Mucosa(1 patient sample), Lung(3 patient samples), Breast(6 patient samples), Stomach (3 patient samples), Gallbladder(1 patient sample), Oesophagus (3 patient samples), Colon(5 patient samples), Pancreas (1 patient sample), Rectal(2 patient samples), Urinary bladder(2 patient samples), Prostate(4 patient samples), ovary(5 patient samples), endometrial(4 patient samples) and cervix(2 patient samples). Serum samples from normal subjects and cancer patients were accessed from cancer hospitals in Mysore, India (Preethi Centre for oncology, and KR Hospital, Logic and Clue Diagnostic center) and the protocols were approved by the ethical review committee (IHEC-UOM NO 35) and the patient's consent were taken. Blood samples were collected from each patient before any treatment. Samples were centrifuged at 2000xg for 30 min at room temperature and the separated sera were stored at -800C. The H&E stained tumour sections of patients were obtained from hospitals and were 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 were centrifuged at 10,000xg for 30 min at 40C. The supernatant was stored at -800C until further analysis. The protein estimation was done at UV 280 nm and Bradford reagent assay using Bovine serum Albumin (BSA) as standard.
Biotinylated hyaluronic acid was prepared according to Boregowda et al.2013 and Srinivas et al. 2016, ( 13, 24). In brief, HA dissolved in PBS-A was dialyzed in MES buffer and reacted with biotin_LC-hydrazide and EDC in DMSO. This was Incubated for 16hrs and then dialyzed against PBS-A and stored in glycerol at -200C.
Production of monoclonal antibody UNIVmAb
Hybridoma and the antibody were prepared according to Boregowda et al. (18, 22, 23). In brief, the hybridoma was grown in DMEM with human serum (pathogen and complement free)that were received from the hospitals. The antibody production in the presence of human serum (any blood groups) did not affect UNIVmAb recogntion of the human H11 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 was dissolved in PBS and dialyzed against PBS.
Statiscal Analysis:
Statistical differences between groups from ELISA were analyzed using graphpad prism version 5 software. Results are expressed as the mean±SD. A diiference with P values is defined as follows: P<0.001=extremely significant. For westerblot, image analysis was done using Image J software.
Methods
Detection of H11 antigen by ELISA using UNIVmAb
MaxiSorp flat-bottom high protein binding capacity polystyrene-96 well plates were used. Serum samples were diluted with 0.05M carbonate-bicarbonate buffer pH 9.6 to obtain a final concentration of 1µg/ml. 100µl of samples in triplicate were plated on to the 96well plate and incubated overnight at 40C. Following day the plate was blocked with skimmed milk (prepared in PBS) for 1 hour and incubated with UNIVmAb at 1:10000 overnight at 4oC. Following day the plate was washed with 0.2% Tween-PBS followed by incubation with b-goat anti-mouse antibody at 1:20000 for 1 hour and reacted with streptavidin-peroxidase at 1:50000 for one hour. Plate was washed with 0.2% Tween-PBS and 100 µl of ABTS (1.0 mg/mL) in 0.1M citrate buffer at pH 4.0 and 5%. Hydrogen peroxide. The reactions were stopped after one hour with 0.2M citric acid, and the absorbance was measured at 405nm. Figure 1. Experiments were repeated at least three times. Protein levels were measured by quantitative ELISA.
Western blot analysis of serum according to Boregowda et al, Fekry et al (15, 16)
50 μg proteins from serum lysate were resolved on 10% SDS-PAGE, transferred to PVDF membrane and reacted with UNIVmAb (1:1000 dilution) or anti HCAM mAb (1:1000 dilution) or with bHA probe (1:100 dilution) overnight at 4 0C. Following day, the blot was developed and the proteins were detected using Enhanced ChemiLuminescence (ECL). Since the isolation of antigen is vital to understand its property, we used purified circulating antigen by antibody conjugated CNBR activated sepharose and Cibacron blue affinity purification method. The purified H11 protein was reacted with UNIVmAb and was also cross reacted with bHA and HA-oligo (500ug Oligo) competition was also performed. Thisshowed that H11 antigen is a Hyaladherin.