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.
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:
$572.00

An Antigen-Down (AD) format ELISA refers to an ELISA plate configuration whereby the plate wells are pre-coated with a highly purified target antigen (hereafter called Target Antigen) that will subsequently be used to quantify specific antibody concentrations that are present within serological or cell culture origin samples (Figure 1). Having the ability to detect and quantitate a population of specific antibodies to a particular pathogen, allergen, or self-antigen within serological samples is essential for any successful epidemiological, allergic hypersensitivity, or autoimmune focused monitoring study.

Briefly, the specific antibody detection and quantification attributes of the AD ELISA format result from its simplistic design. Commercial ELISA plate manufacturers each utilize their own in-house polystyrene plastics modification process. Inner surfaces of each sample well possess the modified plastics surface chemistry to enable passive binding of the Target Antigen to the individual plate well surfaces. Examples of different types of Target Antigens include oligopeptides, polypeptides, and certain polysaccharide molecules. After the Target Antigen has been adsorbed to the ELISA plate wells, the entire well surface is treated with a blocking agent that will minimize unwanted non-specific binding signal. Reduction of non-specific binding signal is a universal goal of all types of ELISA format.

Once the Target Antigen-coated plate has been created (blocked and stabilized), screening of serum, plasma, or cell culture samples for the presence of a Target Antigen-specific poly or monoclonal antibody concentration or titer can begin. To do so, samples are incubated in the coated wells, after which the ELISA plates are washed to remove unbound sample. Next, anti-animal isotype specific IgG-HRP conjugate is added to each well and incubated. After the conjugate incubation period has ended, ELISA plate wells are washed to remove all non-specifically bound HRP conjugate. The next step is to add a chromogenic HRP substrate, such as TMB, to the plate wells. This will allow for the visualization and electronic quantitation of how much Target Antigen-specific antibodies were present within the samples. In the presence of the HRP-IgG conjugate (which is specifically bound to Target Antigen that was previously absorbed onto plate well surfaces), the horseradish peroxidase (HRP) will cause a color change in the initially clear TMB substrate solution. In general, the more intense the TMB color present within a respective sample well, the greater the amount of Target Antigen-specific antibody in that particular sample.

To summarize, the basic AD ELISA format is capable of providing a relative estimation of the Target Antigen-specific antibody titer/concentration present within the serological test samples, as well as a means by which to determine the identity of the isotype(s) that are participating in the humoral immune response. This Antigen- Down ELISA Development Kit was created to help guide end users through the common steps and frequently encountered issues associated with building an AD ELISA. The guide describes the assay performance milestones that must be achieved to enable progression through the various development stages of the antigen- down format ELISA (Table 1). The finalized ELISA should efficiently detect isotype-specific, low to high positive titer, humoral antibody responses in serum or plasma samples.

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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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