MPD enabled Fast Immunoassay (Fast-IA/MPD)

Abstract: BioTraces has developed an MPD enhanced immunoassay which is up to 500-times more sensitive than prior-art immunoassays. This sensitivity can be trade-off for shorter measurement time. The MPD enhanced fast immunoassay (Fast-IA/MPD) enables for the rapid quantitative measurement of biological targets, e.g. cytokines. We developed a fast (5-10 min) immunoassay with a sensitivity of a few pg/ml. A faster method of quantitation may enable physicians to rapidly identify the early stage of an acute allograft rejection or septic shock so that immediate counter measures can be taken. Wherein "state of art" ELISAs takes a few hours, the Fast-IA/MPD permits about a twenty-fold greater speed with a similar level of sensitivity (10 pg/ml).

Introduction: In immunodiagnostics, one may trade-off sensitivity for speed. Fast-IA/MPD permits quantitation of multiple targets from the same sample within a 5-10 minute time frame. Fast immunoassays are an emerging technology and early detection of acute allograft rejection and subsequent organ rescue is clearly needed. In the USA, there are 2,000 heart, 3,000 liver and 10,000 kidney transplans annually. The rejection rate varies between the different organ types, but on average it is about 20%. Fast-IA/MPD could be used to quickly and simultaneously measure the key cytokines in serum. It may also identify other types of immune disorder anomalies such as septic shock or acute inflammation due to bacterial infections. Fast-IA/MPD would enable emergency medical professionals to make a rapid diagnosis of an immune disorder so that immediate, often life saving counter measures can be taken.

Currently, using separate "state of art" ELISAs, a few hours for each ELISA would be required to determine the levels of targets proteins. Using the Fast-IA/MPD we expect to measure three different targets simultaneously in about 5-10 minutes. Fast-IA/MPD will be developed into a commercially viable format, consisting of an assay card that inserts into an MPD instrument.

MPD Enhanced Fast Immunoassay: Coupling the MPD technology to paper immuno-chromatography has resulted in the development of fast yet very sensitive immunoassays. Combining these two different technologies is highly synergistic. Prior-art paper immunochromatography provides the means to quickly separate and capture specific antigens, but is restricted to qualitative analysis. MPD technology, permits the accurate quantification of bound antigens with sub-pg/ml sensitivity.

Even at very level, the antigen (Ag) and antibody (Ab) create Ag/Ab complexes. Brownian movement and molecular diffusion determine the chance of Ag and Ab encounters leading to binding. In the sandwich immunoassays, e.g. ELISA, an excess of Abs (capture and detector) are used which leads to a greater chance that the Ag will encounter an Ab and bind. Molecular diffusion remains a limiting factor, so that ELISAs require about an hour to ensure Ag/Ab binding. In paper chromatography, in contrast, fluids are drawn through the paper by a wicking action rather than diffusion. The paper provides a matrix in which Ags in solution, as they travel through the paper, can come in close contact with immobilized Abs favoring binding. The interaction between Ag and Ab can be quick (seconds) since it's not dependent on diffusion.

The paper immunochromatography is exemplified with the home pregnancy tests, consisting of a strip with an immobilized anti-hCG monoclonal Ab1. In the load zone, a Ab2 conjugated to chromophore, will bind to hCG that may be present in a urine sample. hCG/Ab2-chromophore complex is wicked up the strip, and some of it bind to the immobilized Ab1 band. This would be seen as a color band and would indicate the presence of hCG, or in other words, pregnancy. The test is fast and can generate a qualitative yes answer within several minutes. An advantage of paper immunochromatography, besides its great speed, is its ability to measure multiple samples simultaneously. Different kinds of capture Abs can be easily immobilized on different bands of the immunochromatographic strip. A signal at each band corresponds to a different kind of Ag and multiple samples can be read simultaneously.

A problem with paper immunochromatagraphy, however, is that this technology provides only qualitative not quantitative data. Paper immunochromatography generally has poor sensitivity because the signal generated are very weak - below the quantitation level of colorometric or fluorometric instrumentation. For this reason, paper immunochromatography has been restricted for use in measuring Ags of high concentrations with visual inspection. Coupling the paper immuno-chromatography technology to the MPD instrumentation, allows accurate and reliable quantitation of bound Ags. A radiotracer (Tr) is used in place of a chromophore, leading to Ag/Ab-Tr complexes. Although the radiosignals generated would be small, the MPD instrumentation, with its hundred-fold greater sensitivity than other types of detectors, permits measurement of the samples. For example, the level of cytokines (IL-1 , IFN, TNF, IL-2) become elevated during the onset of an acute allograft rejection, and will be reliably and quickly measurable with this new technology.

Fast-IA/MPD consist of a specially designed immunochramtographic card to (see Figure 1.) to be read by an spatially resolved MPD instrument. The card consist of three paper immunochromatographic strips in parallel (one sample, two reference). Each strip contains a patch made of lyophilized mixture of tracer antibodies (¹²⁵I-anti-IL-1 , ¹²⁵I-anti-IFN- , ¹²⁵I-anti-TNF- , ¹²⁵I-anti-IL-2) and four separate bands containing immobilized capture Abs, anti-IL-1 , anti-IFN- , anti-TNF- , and IL-2, respectively. The two reference strips each contain an additional patch with lyophilized cytokine standards. The card is enclosed in a protective plastic sheath. The ImmunoCard will be inserted in between two detectors of SR-MPD instruments. Each of the instrument's detectors is covered by a thin tungsten mask with holes that are alignment with the expected band locations.

The sample (plasma or urine) is added to the loading reservoir at the top of the card for simultaneous distribution into the three separate immuno-chromatographic strips . As the sample is drawn through each strip by the wicking action of the paper, it will pass through the tracer antibody patch forming cytokine/¹²⁵I-Ab complexes, which will be carried away and deposited on the appropriate capture bands downstream. The two reference strips also have an extra patch upstream which contain free cytokines that serve as reference standards. The reference strips permit reliable measurement of the level of antigens.

Speed and Sensitivity of the Fast-IA/MPD: Detection is remarkably fast. The binding chemistry takes from 30 seconds to a few minutes, which is basically the time it takes for the fluid to be wicked through the paper. The actual speed can be adjusted simply by changing the type of paper used and blocking conditions, which will affect the paper's wicking speed. Our experimentations suggest that 2-4 min is the optimum time needed for Ag/Ab complex capture. The next time constraint is the counting time required by the MPD instrument to accurately measure the bound tracer within each of the multiple bands in the card. A specially configured MPD instrument consist of two SR-MPD detectors that are in perfect alignment with each other, as well as with, the bands in the ImmunoCard. SR-MPDs concurrently count all twelve bands and improve the counting efficiency, and thereby, shorten the counting time. Based on our previous experience, and the amount of Ag/Ab/Tr complex expected to be captured at each band on the card, only about 1-2 minutes of counting time would be required using the MPD device.

The Fast-IA/MPD card permits simultaneouse and accurate quantitation of the plasma levels of IL-1 , IL-2, IFN- , TNF- , within 5 minutes. The assay sensitivity should be similar to that of the ELISA, at about 10 pg/ml limit of detection, which is sufficient to measure the elevated expected levels of target cytokines produced during the onset of an acute allograft rejection (e.g. both, IL-2 and TNF are at ng/ml level). The heart of the Fast-IA/MPD is the MPD instrumentation, which has hundreds of times greater sensitivity than other commercially available detectors.

Fast-IA/MPD for TSH and hCG: The Fast-IA/MPD have been demonstrated for the tropic hormones, TSH and hCG. A test strip contained two different bands of immobilized capture Abs, one with an anti-FSH(alpha subunit) and a second with an anti-mouse IgG. Note that the pituitary tropic hormones (TSH, FSH, LH) and placental hormone (hCG), all are comprised of two subunits, an identical alpha and a characteristic beta. Therefore, the anti-FSH (alpha subunit) capture Ab, would bind all four of these different tropic hormones.

In a prototype fast immunoassays for TSH, a radiolabeled (¹²⁵I) anti-TSH(beta) Ab was used as the tracer. For this assay, a set of TSH standards was prepared by serial dilution, Concentrations ranged from 1.7 - 42 pg/ml (0.375-15 µI.U.). Each TSH standard was used in a separate fast immunoassay. At the start of an assay, 2 µl of ¹²⁵I -anti-TSH(beta) (about 2,000 dpm) was added to 10 µl of a TSH standard. The Ag/Ab mixture was added to the load zone on the immuno-chromatographic strip. Immediately, a PBS soaked cotton pad was applied upstream to serve as a source of fluid to be wicked up through the strip carrying the antigen/¹²⁵I-antibody complexes with it. The fluid front reached the end of the strip in about 3-4 minutes. A new immunochromatographic strip was used with each TSH standard. The strips were dried and the background reference, Ag capture and tracer Ab reference zones were measured in an appropriate MPD instrument. Figure 2 shows performance of a Fast-IA/MPD for TSH. We demonstrated good linearity of the assay. The lower limit of detection in these prototype assays is 10-20 pg/ml. A similar Fast-IA/MPD was also performed for hCG. The same methodology was used, except an anti-hCG(beta) ¹²⁵I-Ab was used in place of the anti-TSH tracer Ab and hCG standards were prepared ranging from 81 to 3,225 pg/ml.

Although the sensitivity of this Fast-IA/MPD was no better than the classical ELISA, the actual time for its completion was a remarkable 5 minutes whrease ELISA takes a few hours. Note that this prototype assay was not optimized. Better types of chromatographic paper could have provided for a matrix with faster binding kinetics. Also, better antibodies and blocking procedures would lower the nonspecific biological background. In the future, Fast-IA/MPD will be fully optimized to be about ten times more sensitive without sacrificing assay speed.