Contact Us: www.pall.com/contact The Procedure for Optimized ELISA Protocol The Procedure for Optimized ELISA Protocol Background Methods Results Discussion Conclusions Summary Background The membrane used in an ELISA test can have a large effect on assay performance. Optimum reagent concentrations, blocking conditions, exposure times and signal development will all differ as membrane chemistry changes. This assay was designed to be used as a starting point for customers interested in evaluating performance of different membrane types with a widely recognized group of reagents. A customer should be able to repeat the results in this report exactly, and then adapt conditions and reagents for his own application. Methods Materials Pall Nylon 6,6 membranes, including Immunodyne ABC, Biodyne A, Biodyne B, Biodyne Plus, Biodyne C, and LoProdyne LP membranes Goat anti-rabbit IgG (primary antibody): Sigma R2004, Sigma Chemical Company, St. Louis, MO 63178 Rabbit anti-goat IgG (acts as an antigen in this assay): Sigma G 4018 Goat anti-rabbit IgG conjugated to horseradish peroxidase (secondary antibody): Sigma A 0545 Hammersten casein: BDH 44020, Gallard Schlessinger, Carle Place, NY 11514 Tween 20: Sigma P7949 Diaminobenzidene: Sigma D9015 Imidazole: Sigma I 0125 H 2 O 2, 30% Solution: Sigma H 1009 Magnetic stir plate with heating element Micropipettors Orbital shaker or nutator Solutions Required Phosphate buffered saline (PBS): 20mM Phosphate, 0.15M NaCl, ph 7 Blocking solution: 0.5% Hammersten casein, 0.05% Tween-20 in PBS. Heat on stir plate until casein is dissolved. This should occur at about 90 Celsius. Do not boil. Substrate solution (in water): Procedure 0.1 mg/ml diaminobenzidene 1 mg/ml imidazole 1 ul/ ml 30% H 2 O 2 (add just before use) Membranes should be agitated throughout incubation and wash steps. All steps to be performed at room temperature. 1. Application of Primary Antibody. Cards are cut from each membrane type, approximately 3 x 6 cm.
Primary antibody is spotted onto each membrane card with a micropipettor: 1ul of PBS solution containing 0.25, 0.1, 0.05, 0.025, 0.01 and 0.001ug per ul is applied with a micropipettor. Membranes are then air dried for 15 minutes. 2. Block. Membranes are placed in individual sealed bags with 2ml of blocking solution. Membranes are incubated with blocking solution for 30 minutes. 3. Antigen. Blocking solution is replaced with 1ml of PBS containing 10-250ng/ml rabbit anti-goat IgG. Membranes are incubated for 10 minutes. 4. Wash. Membranes are washed for 2 minutes with 5ml of blocking solution. 5. Conjugate. Membranes are incubated for 10 minutes with conjugate diluted in PBS, 1/300-1/500. 6. Wash. Membranes are washed three times, 2 minutes each, with 5ml of 0.1% Tween 20 in water. 7. Develop color. Membranes are incubated with 1ml of substrate solution for three minutes. Rinse in water, air dry. Biodyne membranes are designed to bind all types of protein molecules by passive adsorption. Evidence points to hydrophobic interactions as the primary binding modality. Immuodyne ABC membrane is a product from Pall which binds proteins covalently to the surface. LoProdyne LP membrane is designed for low protein binding. LoProdyne LP membrane has hydroxyl surface chemistry which tends to stabilize water molecules around the membrane, decreasing opportunities for hydrophobic binding. The best membrane to use for a given ELISA test is difficult to predict without empirical testing. Our experience shows that: Biodyne A membrane typically provides a good combination of high signal and low background with most assay systems. Biodyne B membrane produces the highest signal level, although background is sometimes higher than on the other membrane types. Biodyne Plus membrane produces results that are intermediate between Biodyne A and Biodyne B membranes, and will have a higher affinity than Biodyne A for negatively charged molecules. Biodyne C membrane has the lowest background, while still effectively binding proteins. Immunodyne ABC membrane produces high signal and low background. This membrane is particularly useful for assays in which small proteins or polypeptides are immobilized on the membrane. Smaller molecules have less tertiary structure than large proteins, and do not provide as many sites for hydrophobic interaction. LoProdyne LP membrane is designed to have very low protein binding. This membrane has very low background in ELISA tests, and often does not require any blocking agent. Proteins can be forced to bind to LoProdyne LP membrane by drying. Results Experiment 1: Matrix Variables Membrane type, primary antibody concentration and antigen concentration were all varied as shown below:
Sandwich ELISA test on different Pall nylon membranes. First antibody was spotted onto all membranes; concentrations per one ul spot are shown on the left. All membranes were blocked with casein/tween 20, incubated with antigen and 1/500 dilution of conjugate. Signal was developed with diaminobenzidene and peroxide. Signal was produced on all of the membranes with as little as 3 ng of primary antibody and 25 ng/ml of antigen. Highest signal intensity was generated on Immunodyne ABC membrane; the lowest on Biodyne C and LoProdyne LP membranes. Biodyne C and LoProdyne LP membranes also had the lowest background in this test. A closer look at signal and background can be seen in Experiment 2. Experiment 2: Sandwich ELISA test on different Pall nylon membranes. First antibody was spotted onto all membranes; concentrations per one micron spot are shown on the left. All membranes were blocked with casein/tween 20, incubated with 1ml of 250 ng/ml antigen, 1/300 dilution of conjugate. Signal was developed with diaminobenzidene and peroxide. Signal was produced with 1 ng of primary antibody on Biodyne A, Biodyne B, Biodyne C and Biodyne Plus membranes. LoProdyne LP membrane required 10 ng of primary antibody for a positive response. Color density within spots on Biodyne and Immunodyne membranes is very even. The highest concentrations of primary antibody resulted in lighter areas in the center of the spots, probably resulting from pro-zone concentration effects between antibody and antigen (excess antibody). Discussion Membrane Choice
The Biodyne membranes, which all have relatively high affinity for proteins, are widely used for ELISA systems. The surface chemistry on the different membranes affects performance to a controlled extent, so that Biodyne B membrane (positive surface charge) binds slightly more protein than Biodyne C membrane (negative surface charge). Biodyne A membrane is used most often because it provides a good combination of high sensitivity and low background. Biodyne B membrane is sometimes chosen for applications that require the highest sensitivity, and where background is less of a problem, while Biodyne C membrane is chosen when low background is most important. Immunodyne ABC membrane is gaining popularity for ELISA systems in a wide variety of applications, due to the advantages afforded by covalent attachment to the membrane. LoProdyne LP membranes are also used for ELISA applications. These membranes can often be used without a blocking step. Color Density: Color density within spots is quite even on Biodyne and Immunodyne membranes, although spots with light centers are sometimes observed at the highest antibody concentration. This is probably caused by the presence of excess antibody (prozone effect). Spots on the LoProdyne LP membrane have a different appearance, with dark centers and a light halo. Since proteins do not adsorb readily to LoProdyne LP membrane, more protein migrates with the solvent. The dark centers correspond to the area under the applied bead of liquid. The lighter outer edge is due to migration of unbound protein, following the solvent front. Spot Size: While size of spots in this test was determined by the primary antibody concentration, size can also be regulated with a slot blotter. If the same amount of protein is loaded in a larger volume (generally between 10ul and 100ul), the spot will cover the entire area of the slot blot aperture. This technique also works well on LoProdyne LP membrane: In addition to controlling the size of the spot, the biphasic appearance of the spot will also be eliminated by loading protein with a slot blotter. Optimization Once a membrane is chosen, assay parameters should be manipulated for best results, as follows: Primary antibody: Adjust concentration for highest sensitivity. Blocking agents: Adjust to find the minimum concentration which reproducibly results in acceptable background. The surfactant concentration is generally kept constant, while we have found that Hammersten casein can be effective at concentrations as low as 0.03%. Secondary antibody-conjugate: Adjust concentration for highest sensitivity. Substrate: Alternative substrates may also provide higher sensitivity or lower background. All of these steps are interdependent; a change in concentration may affect optimum concentrations of other reagents. Increasing exposure time of incubation steps can also be used to increase signal and sensitivity. Configuration and Pore Size Most ELISA assays are formatted in a flow through configuration: The membrane is placed at the bottom of a sample reservoir, and on top of an absorbant pad. Reagents added to the sample reservoir flow through the membrane into the pad, allowing discrete steps without having to use bags or other containers. In a flow through system, exposure times for all reagents are determined by the flow rate through the membrane. This flow rate will be determined by the viscosity of the fluid, thickness of the membrane and the pore size rating of the membrane. For example, exposure times with sample can be increased by using a membrane having a lower pore size rating. Pall nylon membranes are manufactured in a wide range of pore size ratings, from 0.04um to 5.0um, so that flow rate can be easily controlled. 0.45um membrane was used in this report as a typical starting point. The model assay format, however, is not very sensitive to changes in pore size as incubation times are defined by the soak times, rather than flow through the membrane. Conclusions Pall Biodyne, Immunodyne ABC and LoProdyne LP membranes are ideal materials for ELISA systems, with defined protein binding characteristics that can be matched to specific applications. All Pall nylon
membranes are white and have high tensile strength, so that they are readily adaptable for manufacturing processes. Optimum membrane choice, reagent concentrations and incubation times have to be determined empirically for each test system. This report details a model assay which can be used as a starting point for assay development. Summary An ELISA test method has been developed to demonstrate membrane performance in a typical sandwich assay format. The assay can be completed in 90 minutes, using separate antigen and conjugate steps. Results show detection of IgG protein with 1 ng of primary antibody, with sensitivity of lower than 25 ng/ml antigen. The assay can be performed simultaneously on multiple membrane types, with a wide range of antibody and antigen concentrations.