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ELISA protocol for HMGB1

ELISA protocol for HMGB1 - ELISA Assay Forum

ELISA protocol for HMGB1 - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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  #1  
Old 11-22-2010, 01:44 PM
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Default ELISA protocol for HMGB1



Hi!

I am struggling in optimizing ELISA for HMGB1.

Does anyone have a good protocol?

Is it best to block with milk, or is BSA good enough?

Thanks!
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  #2  
Old 12-02-2010, 10:16 AM
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Default 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.
Principe
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.
Reagents
Coated plate : dispense 100 μl of 5 g/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 l of aliquote with 9.99 ml of SPSD. From this, for to realize series standards calibration, dilute 320 l with 680 l 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 g/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.
Procedure
Dispense in each specific wells, coated MTP, 50 μl of SPSD, followed by 10 l 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 l substrate and incubate between 45-55 C for 10 min., then pipet 50 l of color reagent, mix gently on shaker plate, read Abs at 630 nm, and calculate against calibration curve.
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  #3  
Old 03-11-2011, 05:57 PM
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Default Re: ELISA protocol for HMGB1

Hi there

I am not sure about the blocking, but I assume you are developing an in-house ELISA for HMGB1 detection. I am trying to validate a commercial assay and running into major problems with my recovery. It seems that HMGB1 can bind to Ig (specifically IgG). I am not sure how to get around this but I am fairly certain that it's interfering with my recovery. Also, the commercial kit has very low absorbance (0.7 at the high std). I am not sure if your home brew assay would be better, if it is i may convince my boss to let me try to make one.

There is a good paper out about interfering factors in plasma and serum with HMGB1. It might answer your blocking question.
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  #4  
Old 03-13-2011, 03:38 AM
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Default Re: ELISA protocol for HMGB1

In my experiments I have also encountered anther problem - I am workin with a monoclonal HMGB1, and I believe it is forming aggregates, causing my triplicates onthe ELISA plate to have a very high variance.
I have to perform quadriplicates in order to get a good statistical significance.
Besides that, HMGB1 binds easily to anything, it even has some binding to blocking buffer .
The best simple protocol to assess binding is using a slot blot.
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Old 03-16-2011, 11:17 AM
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Default Re: ELISA protocol for HMGB1

In all ELISA capture methodology, choice of antibodies for wells coat, is driver for good performance analysis, which between immunochemical characteristic their capability of binding versus specific antigen is fundamental. Such as says in my precedent post, monoclonal antibodies from different mammalian showed not very high capability of binding against human HMGB1, and for to clear a hurdle, its preferable dilute 1:1 serum sample in sample diluents (or SPSD) and to dispense 50 l into MTP wells, then incubate for 24 h at 37 C. After washing, pipette 50 l monoclonal antibody (label or unlabeled) in each wells and incubate at room temperature for 2 h. In all package for HMGB1 determination is writing : "This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the immunoassay, the possibility of interference cannot be excluded". Therefore, several serum components bind to HMGB1, between them anti-HMGB1 antibodies (IgG and IgM) represent a significant factor that is found in most sera and plasma samples investigated, including those from healthy individuals. Moreover, the titers of antibodies against HMGB1 in serum correlated inversely with the ELISA detection of rHMGB1 added to the serum samples. These data imply a role of autoantibodies to HMGB1 for interfering with HMGB1 detection by ELISA. (Vilma Urbonaviciute V., et al. : (2007) Factors masking HMGB1 in human serum and plasma. J.of Leuk. Biol. 81:67-74. [Only registered users see links. ]). At today not is clear, considered the "sticky" nature of the HMGB1, as says well Eitan, if is it a specific antibody reaction (autoantibody) or cross-reaction (for sequential or conformational homology), or non-specific bond (sticky bond). You ask to me if “my home brew assay would be better”, I don't know if it is, but I think that in the life we all search the best and when we get it, really we sure that is improvement or this is just one step for a better in future? You don't have to convince your boss to try do in the house one, but to involve him to make, and my protocol could be a good base for to start. If you wish even evaluate an immunoblotting method in home made, this is the basis road. Equal amounts of diluted serum samples is subjected to sodium-dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), and electroblotted in transfer buffer (25 mm Tris, 40 mm glycine, 0.05% SDS, 20% methanol) to nitrocellulose membranes (Bio-Rad or Schleicher & Schuell) or electrophoretically transferred onto polyvinilidene difluoride (PVDF) membranes (Bio-Rad). Membranes were subsequently blocked for nitrocellulose with Blok A (PBS containing 0.1 % Tween-20, 5 % nonfat dry milk) or Blok B (TBS containing 5 % defatted dried milk, 0.05 % Tween-20 ) for PVDF and incubate for 1–2 h at room temperature. Probed with 1 : 1000 dilute anti-human HMGB1 mouse monoclonal antibody (Sigma) or chicken anti-HMGB1 antibody. Bound antibodies were visualized with 1 : 2000 dilute HRP-conjugated anti-mouse IgG (Sigma) or anti-chicken IgY-HRP antibody and immunoreactivity assessed by chemiluminescence reaction using the ECL Western blocking detection system. Densitometric scanning analysis was performed on Mac OS 9.0 version, using NIH Image 1.62 software, developed at the U.S. National Institutes of Health [[Only registered users see links. ].
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