| | Re: ELISA protocol for HMGB1
As request you on the quality of the blocking solution, both of the ingredients are acceptable, using at 2-5 % for skimmed milk powder, and non greater of 1.5% for BSA. On the protocol, today, many approaches exist to realize a optimized ELISA method for HMGB1 determination. For following considerations the develop to ELISA sandwich indirect method, is the one which furnishes at the same time, good quality and lower cost. As capture, substance with good affinity bound to HMGB1 are RAGE-Fc (Receptor for Advanced Glycated End-products) and chicken polyclonal specific antibody (IgY). In vitro, high affinity human anti-HMGB1 antibodies block the binding of recombinant HMGB1 to RAGE. Anti-human HMGB1 antibody specifically binding to human HMGB1 by immunizing mammals such as rabbit, sheep, goats, muse, rat, or cattle with the whole or part of human HMGB1 obtain extremely small amounts of such antibody having high capability of binding to human HMGB1 and this result is correlate to extremely high molecule homology, difference 2-3 amino acid residues only, with human HMGB1. In contrary, the amino acid sequence (primary structure) of avian HMGB1 has low homology with same human molecule. The avian-derived anti-human HMGB1 antibody show high-titer antibody having high capability of binding to human HMGB1 and the birds are chickens. The avian antibody versus HMGB, obtained immunizing chickens with human HMGB1, can be such as polyclonal (IgG, from peripheral blood, or IgY, from egg yolks) or monoclonal (IgG from chicken immunized splenic B cell immortalized through fusion with chicken lymphoblastoid cell, and hybridomas-transfectoma technology). For second antibody and anti-species conjugate against second antibody no particular difference, using antibodies from various animal species versus whole molecule, has been observed.
Chicken polyclonal antibody (IgY) specific for HMGB1 coated onto the wells of the microtiter strips bind specifically HMGB1 in sample. After addition of a mouse anti-HMGB1 monoclonal antibody to form antigen-antibody complex. This complex are detect by addiction goat anti-IgG mouse antibody alkaline phosphatase conjugated. Thereafter substrate solution is added to the wells and revealed a visible reaction, through added colour reagent. The color develops is proportion to the amount of HMGB1 in the sample, and the color intensity is measured at 630 nm.
Coated plate : dispense 100 μl of 5 µg/ml anti-HMGB1 chicken (IgY) polyclonal antibody (SHINO-TEST Corporation cod. 326052233) in PBS (5.59 mM Na2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, and 2.68 mM KCl, at pH 7.2). Remove the unbound antibodies by washing the plate 3 times with wash solution (PBS containing 0.05 % Tween-20). Block the remaining binding sites in the wells, incubating the plates for 2 hr with 200 μl/well of blocking solution (1 % BSA in PBS). After washing plate are dry and put in aluminum package, with desiccant agent and store at 4 °C.
Serum/plasma standard diluents (SPSD) : 20 mM HEPES, 150 mM NaCl, 1 mM Mn2+, pH 7.2 containing 0.3% BSA, 0.05% Tween-20, 0.01 % NaN3
Standard human HMGB1 : dissolve 1 mg human HMGB1 (Sigma cod. H4652) in 1 ml dH2O, aliquote and store at -20 °C, stable for 6 months. For obtain 1000 ng/ml solution dilute 10 µl of aliquote with 9.99 ml of SPSD. From this, for to realize series standards calibration, dilute 320 µl with 680 µl of SPSD obtaining solution starter for standard series at 320 ng/ml. Further dilute 100 μl of 320 ng/ml solution with 300 μl of SPSD obtaining 80 ng/ml solution. From this last, to made doubling dilution up to 2.5 ng/ml. All the dilutions are stable for 24 h at 4 °C. For 0 ng/ml value to use only SPSD.
Second antibody : suspend 1 µg/ml mouse monoclonal anti-HMGB1 antibody (IgG1) clone HAP 46.5 (Sigma cod. H9537) in PBS containing 3 % FCS, 1 % pooled mouse normal serum, 0.01 % thimerosal and 10 mg gentamicin.
Conjugate : dilute 1:1000 goat anti-mouse IgG antibody alkaline phosphatase-conjugated (Sigma cod. A7434) in PBS containing 1 % BSA and 0.3 % Tween-20, 0.01 % di NaN3.
Substrate : dissolve in Tris-HCl 0.1 M a pH 9.0 buffer, 5 mM MgCl2, e 0.05 mM di sodium pyrophosphate.
Color reagent : dissolve in 800 ml 5 N sulfuric acid, 1.5 g/L ammonium molybdate, 0.085 g/L malachite green and 0.2 g/L di Tween-20, and up to 1 liter with 5 N sulfuric acid.
Dispense in each specific wells, coated MTP, 50 μl of SPSD, followed by 10 µl serum/plasma or 10 μl HMGB1 standards series solutions, for to realize calibration curve, mix carefully and incubate at 37 °C for 2 hrs. Washing 3 times with wash solution and dispense 50 μl of second antibody and incubate for 1 h at 37 °C. Wash again such as above and dispense 50 μl of conjugate and incubate for 1 h at room temperature. Wash once with washing solution and twice with dH2O. Dispense 50 µl substrate and incubate between 45-55 °C for 10 min., then pipet 50 µl of color reagent, mix gently on shaker plate, read Abs at 630 nm, and calculate against calibration curve.