Antigen-Down ELISA Development Kit

1 Citation

This kit provides seven specially formulated ELISA solutions and a template for the initial development and optimization of antigen-down immunoassays by both novice and experienced users. The kit contains enough reagent for ten 96-well ELISA plates.

Figure 1. In Antigen-Down-ELISAs, a Target Antigen coated onto the ELISA plate surface is used to quantify specific antibody concentrations present within samples. When the sample is added, the antigen on the plate is bound by antibodies from the sample, which are then retained in the well. A species-specific antibody labeled with HRP is added, followed by substrate, a stop solution is added, and then the absorbance is read. The higher the signal, the more antibodies there are in the sample.
Figure 2. Initial Assay Feasibility Plate Map. Wells are coated with 100 µL per well of either 1 µg/mL (light gray) or 4 µg/mL (dark gray) antigen coating solution, or Antigen Coating Buffer blank well controls (white). A known Positive Control is diluted out via a series of 1:2 dilutions and added to six sections of the plate. After the antibody capture step, plates are incubated with three different dilutions of the HRP-IgG conjugate, washed, and developed with TMB substrate. See protocol.
Figure 3. Serial dilution of known positive control. See the protocol for details.
Figure 4. Serial dilution of conjugate. See the protocol for details.
Figure 5. Target Antigen Coating Optimization Plate Map. ELISA plate wells are coated with 0 , 0.25, 0.5, 1 , 2, and 4 µg/mL of the Target Antigen to assess optimal coating concentrations. A known Positive and Negative Control serum or plasma sample is diluted out according to the plate map pictured. Plate duplicates are exposed to a pre-determined dilution of the HRP-IgG conjugate. The plate is read at OD 450 nm. See protocol for details.
Figure 6. Establishing a Dilution Factor Range for Samples. ELISA plates were prepared according to the protocol. Known Positive Control serum or plasma samples were diluted according to the plate map using a 5-fold serial dilution scheme. An end-point analysis is conducted to determine whether or not this 5-dilution factor range resulted in stopped TMB OD 450 nm signals between 0.3 and 3.0 OD units in all the known Positive Control serum or plasma samples that were evaluated. See protocol.
Figure 7. Antibody-Specific versus Non-Specific Signal Over Time. This figure shows the two major signal generating processes: 1.) antibody-driven specific binding, and 2.) hydrophobic interaction-driven nonspecific binding, that occur within all ELISA formats. Specific signal levels off as the antibody-antigen binding process approaches equilibrium, but signal resulting from nonspecific binding of the HRP-conjugate (background signal) continues to accumulate. Signal:noise decreases with time.
Figure 8. Plate Set-up for Normalizing Plate-to-Plate Signal Variations by S/P Ratio. To normalize plate-to-plate variability during routine testing of unknown samples (6 unknown samples are shown in this example), include a Medium Titer and High Titer Positive Control, and a Negative Control on each ELISA plate. Run the controls in quadruplicate, at previously determined dilutions. All unknown samples will be tested (at least in duplicate) at 3 different dilutions. See protocol.
Figure 1. In Antigen-Down-ELISAs, a Target Antigen coated onto the ELISA plate surface is used to quantify specific antibody concentrations present within samples. When the sample is added, the antigen on the plate is bound by antibodies from the sample, which are then retained in the well. A species-specific antibody labeled with HRP is added, followed by substrate, a stop solution is added, and then the absorbance is read. The higher the signal, the more antibodies there are in the sample.
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Figure 1. In Antigen-Down-ELISAs, a Target Antigen coated onto the ELISA plate surface is used to quantify specific antibody concentrations present within samples. When the sample is added, the antigen on the plate is bound by antibodies from the sample, which are then retained in the well. A species-specific antibody labeled with HRP is added, followed by substrate, a stop solution is added, and then the absorbance is read. The higher the signal, the more antibodies there are in the sample.
Figure 2. Initial Assay Feasibility Plate Map. Wells are coated with 100 µL per well of either 1 µg/mL (light gray) or 4 µg/mL (dark gray) antigen coating solution, or Antigen Coating Buffer blank well controls (white). A known Positive Control is diluted out via a series of 1:2 dilutions and added to six sections of the plate. After the antibody capture step, plates are incubated with three different dilutions of the HRP-IgG conjugate, washed, and developed with TMB substrate. See protocol.
Figure 3. Serial dilution of known positive control. See the protocol for details.
Figure 4. Serial dilution of conjugate. See the protocol for details.
Figure 5. Target Antigen Coating Optimization Plate Map. ELISA plate wells are coated with 0 , 0.25, 0.5, 1 , 2, and 4 µg/mL of the Target Antigen to assess optimal coating concentrations. A known Positive and Negative Control serum or plasma sample is diluted out according to the plate map pictured. Plate duplicates are exposed to a pre-determined dilution of the HRP-IgG conjugate. The plate is read at OD 450 nm. See protocol for details.
Figure 6. Establishing a Dilution Factor Range for Samples. ELISA plates were prepared according to the protocol. Known Positive Control serum or plasma samples were diluted according to the plate map using a 5-fold serial dilution scheme. An end-point analysis is conducted to determine whether or not this 5-dilution factor range resulted in stopped TMB OD 450 nm signals between 0.3 and 3.0 OD units in all the known Positive Control serum or plasma samples that were evaluated. See protocol.
Figure 7. Antibody-Specific versus Non-Specific Signal Over Time. This figure shows the two major signal generating processes: 1.) antibody-driven specific binding, and 2.) hydrophobic interaction-driven nonspecific binding, that occur within all ELISA formats. Specific signal levels off as the antibody-antigen binding process approaches equilibrium, but signal resulting from nonspecific binding of the HRP-conjugate (background signal) continues to accumulate. Signal:noise decreases with time.
Figure 8. Plate Set-up for Normalizing Plate-to-Plate Signal Variations by S/P Ratio. To normalize plate-to-plate variability during routine testing of unknown samples (6 unknown samples are shown in this example), include a Medium Titer and High Titer Positive Control, and a Negative Control on each ELISA plate. Run the controls in quadruplicate, at previously determined dilutions. All unknown samples will be tested (at least in duplicate) at 3 different dilutions. See protocol.

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Data Sheet Safety Data Sheet

SKU: 9101

Volume: Reagents for 10 96-Well Plates
Price:
$590.00
Ships: 1-2 business days
Milestones Common to All AD ELISA Development Projects
  1. Define Project Scope/Objectives and Construct Assay Development Plan.
  2. Acquire Key Antigen-Down ELISA Assay Components
  3. Demonstration of Basic Assay Feasibility.
  4. Optimization of Assay Performance Parameters.
  5. Accounting for Natural Variation in Biological Samples and Assay Conditions.

Product Specific References

PMID Publication
36838675Ismail, M., et al. 2023. Experimental Validation of MHC Class I and II Peptide-Based Potential Vaccine Candidates for Human Papilloma Virus Using Sprague-Dawly Models. Molecules (Basel, Switzerland).

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