Sandwich ELISA (“multiple antibodies with antigen trapped in between”) has several advantages over Direct and Indirect ELISA.
Primarily, there is no need to pre-purify the sample for detection and specificity of antigen recognition is greatly enhanced by combining the selectivity of two antibodies, even in the presence of some contamination. Both polyclonal and monoclonal antibodies can be used and should be of very high purity. The key requirement of the system is that the primary “capture” antigen epitope (binding site) not interfere with or overlap the “secondary” or detection antibody epitope.
A combination that frequently works well is to coat the microtiter wells with a well-defined monoclonal antibody. This antibody will capture the intended antigen taking only one epitope out of the assay. The secondary antibody, a conjugated polyclonal, now has the possibility of multiple binding epitopes per antigen, thus increasing the signal intensity.
As in the comparison of Direct and Indirect ELISA, if a conjugated secondary (“detection”) antibody is not available, a third antibody, conjugation of choice, is targeted against the species and isotype of the secondary. The principle disadvantage of Indirect ELISA is the increase in assay time. Customers should consider the extra labor hours involved when deciding between the less expensive option of a third antibody against the more expensive but timing saving notion of custom conjugation of the existing secondary. Zytomed GmbH offers Custom Antibody Conjugation for all forms of detection.
Direct Sandwich ELISA
Solutions:
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Coating Solution: Antigen standard, samples and antibody are diluted in coating solution; typically 10 mM PBS, pH 7.2-7.6; 20 mM Tris-HCl pH 8.0-8.5; and more rarely 50 mM sodium carbonate, pH 9.5. All solutions are made with distilled or deionized water and 0.1% sodium azide may be added. 10 mM PBS is formulated as: 8.0 g NaCl, 1.42 g Na2HPO4.2H20, 0.2 g KH2PO4 , 1.42 g Na2HPO4.2H20, 0.2 g KCl to 1 liter with distilled water, pH 7.4
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Blocking Solution: Application of a non-reactive protein solution to occupy protein-binding sites not bound with antigen, which might lead to antibody binding and high background signal. Frequently used blocking agents at typically 2-5% are BSA, non-fat dry milk, casein, gelatin and some commercially available blocking kits. Blocking conditions need to be optimized for any individual assay. PBS based blocking solution is formulated as: add to Coating Buffer 5.0 g BSA (fraction V) per liter.
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Primary Antibody Solution: Primary antibody should be diluted in Blocking buffer to prevent non-specific binding. Dilute antibody to the concentration recommended by the manufacturer: typically 0.5-1.0 ug/ml. Some manufacturers recommend including 0.02% Tween 20 or NonIdet P-40.
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Wash Solution: Typical wash solutions include 100 mM PBS or 100 mM Tris-buffered saline at neutral pH (7.0-7.4) with a detergent added such as, most frequently, Tween 20 or Nonidet-40 at 0.02% to 0.1%.
Protocol:
Plate Coating with Primary (Capture) Antibody
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Dilute Coating Antibody (primary) in Coating Buffer to the concentration recommended on the Technical Data Sheet. Avoid polystyrene containers when storing or diluting antibody. If the standard curve signals are not as high as desired, increase Coating Antibody concentration.
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Add 100 μl of diluted Coating Antibody per well to polystyrene microtiter plate.
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Cover the plates and incubate 2 to 4 hours at RT or overnight (12 to 18 hours) at 4oC with gentle agitation (e.g., rocker plate).
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Aspirate the Coating Antibody from the wells and tap on absorbent paper to remove excess liquid.
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Add 400 μl per well Wash Buffer, incubate 30 seconds to 1 minute and aspirate. Invert plate on a clean tissue and tap to remove excess fluid.
Blocking the Plate
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Add 300 μl per well of Blocking Solution to each well. Cover plates and incubate 1-2 hours at RT. Excessive blocking should be avoided.
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Aspirate Blocking Solution and tap on absorbent paper. Plates are ready for use, but must not be allowed to dry out. Keep in a moist environment or in sealed foil bags.
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Prior to starting the assay, wash the microplate 3-6 times with 400 uL per well of Wash Buffer and tap on absorbent paper to remove all excess liquid. After this point, do not allow to dry completely at any time.
ELISA Procedure (Biotin conjugation example)
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Dilute standards and samples in the Coating Buffer most relevant to your samples
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Add 100 μl of standards, samples and controls to appropriate wells. Controls should include a reagent blank and a substrate blank. Incubate now for the time determined during assay optimization. As a starting point, try 30 minutes, which extend as long as 2 hr.
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The microtiter plate is inverted on a clean tissue and tapped to remove liquid. Wash step is repeated 3 times with fresh Wash Buffer.
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Dilute Biotinylated (example) Detection Antibody in Coating Buffer to the manufacturer’s recommended dilution.
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Add 50 or 100 μl of diluted Detection Antibody to each well except chromagen blank, cover the plate and incubate for the time and temperature determined during optimization. For a starting point, use 1 hour at RT or several hours at a cooler temperature (4-8oC).
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Aspirate solution from wells and tap excess fluid onto a clean tissue.
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Wash each well 3-6 times with 400 μl Wash Solution, removing excess liquid.
If a conjugated secondary was unavailable, continue with Indirect Sandwich ELISA.
Indirect ELISA
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The choice of a third antibody requires that it recognize the secondary antibody’s host immunoglobulin isotype and species, and that primary and secondary antibodies be of different species origin. Dilute in Coating Buffer to approximately 1.2 time the secondary antibody’s concentration
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Incubate wells with the conjugate-bearing antibody following the protocol for secondary antibody above.
Development of Biotin Staining for both Direct and Indirect ELISA
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Dilute Streptavidin-HRP according to manufacturer’s instructions in Standard Diluent.
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Add 100 μl of diluted Streptavidin-HRP per well, cover the microplate and incubate at RT for 15 to 45 minutes.
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Aspirate solution from the wells.
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Wash the microplate 3-6 times with 400 uL per well of Wash Solution, removing all solution inverting the plate on a clean tissue and tapping out residual fluid.
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Prepare TMB (tetramethylbenzidine) according to manufacturer’s instructions. Zytomed GmbH′sready-to use TMB solution is an excellent substrate for detecting horseradish peroxidase (HRP) labeled probes. TMB produces a soluble end product which is blue in color and is quantitated at 650 nm, providing an ultrasensitive quantitative substrate system.
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Add 100 μl of TMB to each well and incubate in the dark at RT 10-60 minutes (generally 30 recommended).
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The reaction can be monitored as a function of time for kinetic assays or stopped with 100 μl 0.1% sodium fluoride and read at 650 nm within 30 minutes of adding sodium fluoride.
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Read the microplate at 650 nm.
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If the procedure demands conversion to the yellow diimine, add 100 μl of either 0.5M/Liter sulfuric acid or 0.25M/Liter hydrochloric acid and read the absorbance within 5 minutes.
Calculations for Direct/Indirect Sandwich ELISA
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Calculate the average OD (optical density) at 450 or 650 nm for all standards, controls and samples.
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Construct a standard curve by plotting each standard optical density (ordinate) vs. the Standard concentrations (abscissa) on semi log graph paper.
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Determine the concentration of each unknown sample from the standard curve.
