Data Availability StatementAll data generated or analyzed during this study are included in this published article. UNIVmAb detected H11 protein, is usually a unique hyaluronan binding protein, that can be used as a common biomarker for all those cancers. for 30?min at room temperature and the separated sera were stored at ? 80?C. The H&E stained tumour sections of patients were obtained from hospitals and were graded using the TNM grading system. Serum samples treated with 4 lysis buffer, made up of 0.2?M TrisCHCl (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 10,000for 30?min at 4?C. The supernatant was stored at ? 80?C 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. [13] and Srinivas et al. [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 16?h and then dialyzed against PBS-A and stored in glycerol at C 20?C. Production of monoclonal antibody UNIVmAb Hybridoma and the antibody were prepared according to Boregowda et al. [18, 22, 23]. In brief, the hybridoma was produced 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 produced in DMEM made up of 10% (v/v) inactivated human serum. After 21?days, the media was collected and precipitated Ginsenoside Rg1 with cold saturated ammonium sulphate answer (final 50%) at 4?C overnight and centrifuged at 12,000for 30?min. Ginsenoside Rg1 The pellet was dissolved in PBS and dialyzed against PBS. Statistical 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 performed using Picture J software. Strategies Recognition of H11 antigen by ELISA using UNIVmAb MaxiSorp flat-bottom high proteins binding capability polystyrene-96 well plates had been used. Serum examples had been diluted with 0.05?M carbonate-bicarbonate buffer pH 9.6 to secure a final concentration of just one 1?g/ml. 100?l of examples in triplicate were plated to the 96well dish and incubated BGLAP right away in 4?C. Pursuing day the dish was obstructed with skimmed dairy (ready in PBS) for 1?h and incubated with UNIVmAb in 1:10,000 at 4 overnight?C. Following time the dish was cleaned with 0.2% Tween-PBS accompanied by incubation with b-goat anti-mouse antibody at 1:20,000 for 1?h and reacted with streptavidin-peroxidase in 1:50,000 for just one hour. Dish was washed with 0.2% Tween-PBS and 100?l of ABTS (1.0?mg/mL) in 0.1?M citrate buffer at pH 4.0 and 5%. Hydrogen peroxide. The reactions were stopped after one hour with 0.2?M citric acid, and the absorbance was measured at 405?nm Fig.?1. Experiments were repeated at least three times. Protein Ginsenoside Rg1 levels were measured by quantitative ELISA. Open in a separate windows Fig.?1 Detection of Ginsenoside Rg1 normal and Malignancy antigen by ELISA using UNIVmAb. a Lane 1, 2, 3 normal serum (each common of three determinations) Lane 4. Ca belly Grade 1. Lane 5. Ca tongue Grade 1. Lane 6. Ca Colon Grade 1. Lane 7.Ca belly Grade 2. Lane 8.Ca cervix Grade 2, Lane 9. Ca Cervix Grade 3. b 1C4, normal serum, 5 and 6 Grade 1, Tongue, 7C9 Grade 2, breast, (10C13 Grade 3 samples) 10: Colon, 11: Lung, 12: Oesophagus, 13: Ovary. (average of four samples from each serum). There is progressive over-expression of H11 in sera as the tumour progress Western blot analysis of serum according to Boregowda et al. [15] and Fekry et al. [16] 50?g proteins from serum lysate were resolved on 10% SDS-PAGE,.