The industry standard technology Enzyme-Linked Immuno-Sorbent Assay (ELISA) is widely used in biology and medicine for detection of viruses, bacteria, cancer markers, and other physiologically active substances. It is used routinely in research, clinical and pharmaceutical laboratories. However, in many cases the sensitivity of ELISA is inadequate. Thus, increasing the sensitivity, accuracy and general performance of these assays offers a significant opportunity to address unmet clinical needs. Our AmpliFlux technology improves the sensitivity and accuracy of numerous diagnostic assays.
Based on the combination of its patented and proprietary technologies, Allied Innovative Systems (ALLIS) has developed the AmpliFlux method for drastically increasing the sensitivity of ELISA-based assays. In conventional ELISA, antibodies for the analyte to be detected are attached to a solid phase, typically a polystyrene microtiter plate. A biological fluid sample with unknown concentration of the analyte and an enzyme-labeled antibody to the analyte are added. This results in formation of antibody-analyte- enzyme-labeled antibody complex on the surface of the solid phase. After incubation, the unbound analyte and enzyme-labeled antibody are removed from the solution by rinsing. Then, a substrate solution is added to the test tube or well and the amount of the bound enzyme-conjugated antibody is determined by detecting color, fluorescence or luminescence signals of the liquid phase in the final reaction mixture. The concentration of the enzyme-linked antibody is proportional to that of the analyte, and by utilizing appropriate standards, analyte concentration may be inferred. AmpliFlux is based on the use of a unique photochemical amplification reaction, and consists of two steps: The first step is a conventional ELISA. In the second step, AmpliFlux Reagent solution is added to the substrate solution and the mixture is irradiated by visible light. Illumination of the samples leads to a drastic increase in the final signal. The second step may take several minutes.
Example. Determination of HIV-1 p24 antigen by the AmpliFlux Method
In order to demonstrate that the AmpliFlux method allows one to increase the sensitivity of determination of HIV-1 p24 antigen, the reagents from the Alliance?HIV-1 P24 ELISA kit (Perkin Elmer Life Sciences (Boston, MA) for detection and quantitation of the major structural core component of HIV-1 virus were used. This kit is used to carry out p24 quantification assays in two formats: ICD and non-ICD. In the immune complex disruption (ICD) assay format, the immobilized monoclonal antibody captures both free HIV-1 p24 and that which has been released upon disruption of immune complexes in serum or plasma. In the non-ICD format, serum/plasma samples do not undergo immune complex disruption. The non-ICD assay format is used for detecting early infection during the earlier part of the window period due to its high analytical sensitivity, whereas the ICD assay format is used to detect cases during the later part of the window period.
The HIV-1 P24 ELISA kit contains a 96-well microtiter plate, the wells of which are coated with a highly specific mouse monoclonal antibody to HIV-1 p24. The captured antigen is complexed with biotinylated polyclonal antibody to HIV-1 p24 and streptavidin-HRP (horseradish peroxidase) conjugate. The concentration of the analyte is determined by measuring the signal (optical density) of th solution in microplate well in conjunction with a calibration curve. The detection limit of the tests was estimated as the analyte concentration corresponding to twice the value of the background signal.
Results. The calibration curves for conventional non-ICD and modified ICD ELISA formats for determination of HIV-1 p24 antigen are shown in Figures 1A and 1B, respectively. The corresponding calibration curves prepared using ELISA+AmpliFlux are shown in Figures 2A and 2B.
Perkin Elmer and our results (presented in Figure 1A) show that the limit of detection of HIV-1 p24 for the conventional non-ICD assay equals 3.3 pg/ml whereas the detection limit for the same non-ICD ELISA+AmpliFlux is approximately 0.08 pg/ml at 12 min illumination. Thus, the analytical sensitivity of the assay increases more than 40-fold using AmpliFlux. Even more impressive, a greater than 100-fold increase in the analytical sensitivity was achieved using AmpliFlux for the modified ICD assay format (see Figures 1B and 2B).
Conclusion: AmpliFlux technology allows one to increase sensitivity, accuracy, dynamic range, and signal-to-noise ratio of ELISA assays, while saving reagent costs and reducing time of analysis. Similar outstanding results have been achieved for other analytes, such as Hepatitis B surface antigen, Prostate specific antigen (PSA) and other physiologically active substances.
Fluorescence Enhanced Technique (FET)
In addition to the AmpliFlux technology ALLIS has developed an alternative method of enhancing the sensitivity of commercially available ELISA assays. Fluorescence Enhanced Technique (FET) is similar to AmpliFlux except that after performing of the conventional ELISA an enhancement reagent is added to the substrate solution, and the fluorescence of the sample is measured. Results obtained using FET for determination of HIV-1 p24 assay are presented in Figure 3.
The analytical sensitivity of ELISA + FET is approximately 10-fold higher than that for conventional ELISA.