MPD^TM Enhanced Immunoassay (IA/MPD)

ABSTRACT: BioTraces has developed this significant new assay protocol, also called the super immunoradiometric assay, or SIRMA, which provides quantitative measurement of biological substances at levels as low as a few femtogram/ml. Unprecedented sensitivity of IA/MPDs for interleukins (IL-1 beta, IL-4, IL-6, IL-10, IL-11 and IL-12), p24 HIV antigen and human tropic hormones (TSH, LH, FSH and hCG) has been documented. Our data obtained in a clinical study of AML patients for a plurality of cytokines (IL-1 beta, IL-6, and IL-10) demonstrates a quantitatively accurate MPD immunoassays with sensitivity of about 5-10 fg/ml, i.e. lower limit of detection (LOD) of 0.2 attomole/ml. This sensitivity is about 500 times better than currently used ELISA. Also, for p24-antigen we achieved about 5 attomole/ml sensitivity. This permits immunological detection and quantitation of virial load down to about 20 virions per sample.

The need for better Immunoassays: The predictive power of serum levels of different proteins has been hindered by unreliable quantitative methods. Both polyclonal and monoclonal antibodies against a majority of important biomedical targets with molecular weight above 10,000 Daltons are available. With a variety of good antibodies to choose from, a number of ELISA, EIA and RIA kits are available. Unfortunately, the standard immunoassays are not sensitive enough to determine the normal serum levels of many important biologicals, e.g. cytokines, chemokines, and other signaling molecules. For example, the blood levels of many interleukins are less than 0.5 pg/ml. Our studies of IL-6 shows that 50% of healthy individuals have level of IL-6 below 0.2 pg/ml. The levels of many substances, e.g. cytokines are upregulated during conditions of exaggerated immune response and certain diseases. However, even when the level of this molecule is upregulated, it is still between 0.5 and 1 pg/ml for majority 50% of patients. For example our studies demonstrated that for a case of AML, only 30% of patients shows IL-6 levels above 1 pg/ml. The lower limit of detection (LOD) of typical, enzymatic immunoassay kits for many targets, e.g. cytokines is about 1 pg/ml, and is above the serum concentrations of both healthy individuals and patients. Thus, even the best EIA systems are already at the detectability limits and often exhibit large variation in assay signals majority (false positives). Errors of a given assay for the same sample can be greater than 50 percent.

MPD enhanced immunoassays: Coupling MPD technology to a sandwich immunoassay has resulted in the development of a new class of ultra-sensitive assays. IA/MPD is an antibody sandwich capture assay and uses similar reagents as, IRMA or ELISA.(see Figure 1)

1: Super IRMA Directly labeled antibody (like IRMA)
2: Enhanced ELISA Biotin/Avidin system (like ELISA)

Figure 1. Two implementations of MPD enhanced immunoassay. One antibody is immobilized and serves to capture a ligand and a second antibody, which binds to a different epitope on the ligand, is labeled and used to quantitate the ligand. The amount of bound tracer antibody is directly proportional to the amount of bound ligand. Because of the extraordinary sensitivity of the MPD instrument, it has been possible to miniaturize the IA/MPD format, reducing the assay volume and amount of reagents needed including the tracer antibody. The IA/MPD is designed to use 1,000-fold less radioisotope than IRMA. Additionally, specialized blocking and assay reagents have been developed to reduce nonspecific biological background (NSBB). The collective techniques used to optimize the IA/MPD, results in up to 500-fold greater sensitivity than a classical IRMA or ELISA.

The challenge - biological backgrounds elimination: An important task in the development of any IA/MPD is the reduction of nonspecific biological background (NSBB). We showed that biological background, which is generally believed to be an unsurmountable barrier, can be overcome through modifications of the assay protocol and using tailored reagents. The ultrasensitivity of MPD technology enables us to identify which components in the assay are responsible for the biological background, and thereby design strategies for their removal. NSBB reduction involves selecting the right microtiter plate, miniaturizing the assay format, selecting IA/MPD compatible antibody combinations, and using our proprietary blocking, assay, and washing buffers that are tailored to each specific assay. For example, we demonstrated that considerable reduction in NSBB follows modification of the blocking reagent. Additional reductions of NSBB were achieved after other assay components were also optimized. Using our novel NSBB reduction methods, we can reduce biological background about a hundred fold. Better blocking and washing procedures enabled considerable improvement of inter- and intra- assay variability.

In some cases, however, it seems that specific biological background (SBB) is more important than NSBB, e.g. for interleukines, not much change in the assay sensitivity has been observed without serum, with snake serum or chicken serum. Also, extreme dependence on the pH has been observed for IL-11 and Il-12. We hypothesize that this reflects interactions between the two used Ab's. Thus, a very important aspect of the development of any IA/MPD is a comparative study between different monoclonal capture antibodies. We observed that for a given amount of capture antibody, there is only a small difference between avidities of different antibodies, but a rather large difference in background. For example, for two different antibodies tested at 1 microgram/well for IL-11 IA/MPD the backgrounds are 38 and 44 dpm, respectively. When the amount of capture antibody is increased to 4 microgram/well there is small change of amount of bound IL-11 but background increases considerably to 115 and 213 dpm, respectively. These results suggest that in the case of IL-11, there is some cross-reactivity between the capture and detector antibodies. Thus, further diminishment of the amount of capture antibody may permit considerable elimination of the assay background. Herein, the superior sensitivity of MPD helps.

In our experiments we used a biotinylated antibody for detection and a cross-reactivity between capture antibody and ¹²⁵I-streptavidin which limits assay sensitivity. Our results suggest that optimal procedures for ELISA and IA/MPD are often different. In the first case, the need for a high level of signal dominates, wherein in the development of IA/MPD the use of highest specificity antibodies and better background elimination are crucial to reach sub-attomole/ml levels of detection.

Summary: The IA/MPD solves the problems of both signal to background ratio and radioactive waste disposal. The IA/MPD consists of the same components as a standard sandwich immunoassay: antibodies, ligand standards and antibody tracers. However, the format, volumes, and amount of tracer antibody are scaled down to reduce nonspecific sticking and the amount of radioisotope needed. The IA/MPD is designed to use 1,000-fold less radioisotope and 10 times lower sample volume while providing a 500 times better sensitivity than ELISA or IRMA. We demonstrated that even at the 10 fg/ml level, the CV's of the assay are better than 15%.

For additional information about MPD enhanced immunoassay please contact:

Dr. A.K.Drukier, 703-7931550 x 108 e-mail: akd@biotraces.com
Dr. E. Schors, 703-7931550 x 112 e-mail: lenas@biotraces.com