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Coating antibody quantitation

Coating antibody quantitation - ELISA Assay Forum

Coating antibody quantitation - 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 11-10-2009, 10:26 AM
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Default Coating antibody quantitation



I am in the process of developing my own ELISA, and want to quantitate the amount of primary antibody that has bound to the microplate well after the initial coating step. Is anybody aware of an assay that can be used to measure this?

Thanks in advance!
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Old 11-10-2009, 07:00 PM
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Default Re: Coating antibody quantitation

Some rough ideas:

1. Manufacturers usually state the binding capacity of polypropylene as ng of protein/mm^2
2. You can bind anti-rabbit-HRP to detect the capture antibody, then add color reagent. Extrapolite Abs to binding efficiency. Should block first, of course.
3. Re-use the solution containing coating Ab to coat a 2nd, 3rd plate etc..until you see the re-use/dilution have a negative effect.
4. Before and after protein quantitation assays of coating Ab solutions.
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Old 11-16-2009, 04:46 PM
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Default Re: Coating antibody quantitation

Method that used by me to evaluate efficiency coating, is micro determination of proteins bound to well surface through colorimetric BCA (bicinchoninic acid), which has a sensibility between 0.2 to 50 g/500 l (Zagami F.: On type-labda micromolecular mieloma case. Technical considerations. Bioch. Clin. Vol. 16, n 3:237-241, 1992). Reagent is composed by 3 components : a) Reagent A ( dissolve in 80 ml dH2O, 8 g sodium carbonate monohydrate, 1.6 g sodium tartrate, adjuste pH to 11.25 with 10 M of NaOH and bring to 100 ml with dH2O. Stable 1 year at room temperature); b) Reagent B (dissolve in 80 ml dH2O, 4 g bicinchoninic acid sodium salt [BCA] and bring to 100 ml with dH2O. Stable 1 year at room temperature); c) Reagent C : dissolve 0.4 g cupric sulfate.5dH2O in 10 ml of dH2O. Stable 1 year at room temperature); d) Work solution (mix 1 volumes reagent C with 25 volumes reagent B, and then to add at mixture, 26 volumes of reagent A, that from a green color solution that it's stable for 1 week); e) Standards BSA or bovine gamma globulin stock solution (2 m mg/ml), before to use realize serial standards from 1, 3, 4, 6, 8, 12, 16 and 20 g/ml in PBS pH 7.4. Procedure : After coating step, wash the wells with appropriate washing solution, presence of the detergents don't influence the BCA reaction. For to realize standard curve dispense in uncoated strips 50 l of every standards, while in coated wells dispense 50 l of PBS pH 7.4, and to shake, avoiding squirts, for 10 mins. Distribute in all wells 100 - 150 l of work solution, cover sealing with adhesive plastics, and incube, in continuous shaking, at 37 C for 2 hrs. Read, within 1 hr., Abs at 562 nm (540-570) nm. Employ for the calculation Excel software. Optimal coating has been considered when protein concentration result inclusive between 50 and 500 ng, depending from molecular characteristics of the proteins used. In every case, for values below this concentration the coating results less efficient.
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Old 12-30-2009, 02:05 PM
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Default Re: Coating antibody quantitation

Quote:
Originally Posted by danfive View Post
Some rough ideas:

1. Manufacturers usually state the binding capacity of polypropylene as ng of protein/mm^2
2. You can bind anti-rabbit-HRP to detect the capture antibody, then add color reagent. Extrapolite Abs to binding efficiency. Should block first, of course.
3. Re-use the solution containing coating Ab to coat a 2nd, 3rd plate etc..until you see the re-use/dilution have a negative effect.
4. Before and after protein quantitation assays of coating Ab solutions.
It's good!
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Old 09-13-2013, 10:12 AM
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Default Re: Coating antibody quantitation

For the characterization of polystyrene (PS), polypropylene (PP) and glass materials aminated solid surfaces use a simple colorimetric method called “Amino Density Estimation by Colorimetric Assay (ADECA) that is based on the reversible complexation of the coomassie brilliant blue (CBB) with the N+ groups (protonated) on solid surfaces quantifying them. The assay measures the released dye thanks to the use of a unique sodium carbonate-methanol buffer. The method ADECA is said to be “reversible” since the solid surface functional groups remain unchanged after characterization. After fixation of the CBB dye to the aminated substrate and washing steps to remove unfixed dye, the release of CBB is performed using an appropriate organic-aqueous solution.
Procedure
Wells of the MTP polystyrene (PS) solid surfaces was filled up with 200 μl of a glutaraldehyde solution (5% in 3 M sodium acetate buffer) from 25% solution (Sigma cod. G5882). After 10 min reaction time at room temperature in the dark, the wells were emptied, and 200 μl of BSA in 3 M sodium acetate was added. The BSA coupling reaction takes place within 10 min at room temperature in the dark. The plates were emptied by inversion before adding 200 μl of NaBH4. After 10 min reaction time, the wells were rinsed five times with PBST solution (PBS 10-2 M, adjusted to pH 7.35 with 1N NaOH, contained 0.05% Tween-20). Blanks were prepared using sodium acetate solution instead of BSA solution. Dispense in continue wells 250 l of CBB dye (CBB stock solution : dissolve 50 mg [0.05 % - w/v] of CBB G250 in 10 ml [10% - v/v] of methanol and 5 ml [5% - v/v] of glacial acetic acid and stirred vigorously. Then, after the dissolving is complete, dH2O purified [85% - v/v] was added up until the final volume becomes 100 ml) dilute solution in detection buffer (mixing 250 ml of carbonate buffer 0.25 M at pH 11.25 with 250 ml of methanol) at different concentrations between 0.01 and 15 μg/ml. Incubate for 30 min. Wash twice the wells with 300 l of washing solution (10 ml methanol, 5 ml glacial acetic acid and 85 ml dH2O) to remove unfixed dye, then dispense 250 l of detection buffer and incubate for 1-2 min. add 20 l of 3 N HCl and read Abs at 620 nm that correspond to free blue dye in solution and the available N+ onto the solid surface is 1 CBB/ N+.
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Old 01-28-2014, 10:30 AM
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Default Re: Coating antibody quantitation

Efficiency targets in capture ELISA method
Microtiter plate wells are coated with 100 l of 20 g/ml streptavidin solution in NaHCO3 50 mM at pH 8.5, and incubated for 16 h at 4 C. Drain wells and dispense 200 l of blocking buffer (2% BSA in PBS) for 1 h at room temperature. Wells washed 10 times with 0.2% PBST (135 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 at pH 7.4, containing 0.2% Tween 20), and eluated using either protocol (*): a) neutral-pH (PBS) as control in quadruplicate, and b) low-pH (glycine-HCl buffer 0.2 M at pH 2.2) for evaluate desorption first target in quadruplicate. After three wash cycle with 0.1% PBST, 100 l of rabbit polyclonal antibody against cathepsin B, diluted 1:1000 in blocking buffer, is adsorbed to a microtiter plate, to which cathepsin B (67.2, 38.1, and 19.05 ng/ml), is added. Cathepsin B is eluted either with: a) neutral-pH (PBS) as control in duplicate, and b) low-pH (glycine-HCl buffer 0.2 M at pH 2.2) for evaluate antigen-antibody complex disruption second target. Residual cathepsin B is subsequently detected in a sandwich ELISA assay with secondary sheep anti-cathepsin B antibody HRP conjugated. After 2 h of incubation at 37 C, peroxidase substrate 3,3,'5,5-tetramethylbenzidine in peridrole buffered is added. The amount of degraded substrate, as a measure of bound immuno-complexed cathepsin B, is determined by absorbance at 450 nm.
(*) For each PBST washing, 200 l of 0.2% PBST buffer is added to the well and shaken for 5 min at 50 rpm and then discarded. Dispense 100 l of PBS or glycine-HCl buffer in corresponding wells and shaken for 5 min at 100 rpm, removed eluate from the microtiter well, and repeat it for several cycles. Neutralize each eluate with 100 l of phosphate buffer 200 mM at pH 7.5. The same amount of this buffer is used to stabilize the microtiter well surface between glycine-HCl elutions and then added to the eluates, followed, at the end of eluate process, by once wash with PBST.
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