MPDTM Enhanced Immunoassay for HIV-1
ABSTRACT: BioTraces, Inc. developed ultra-sensitive assays for detection of human immunodeficiency virus (HIV-1). The two complementary assays are an IA/MPD for detection p24 antigen of HIV-1, and an ultrasensitive MPD enhanced quantitative PCR (qPCR/MPD). MPD is a promising new diagnostic method for screening blood products and monitoring retroviral therapy. MPD increases the sensitivity and enables reduction of non-specific biological background (NSBB). We have developed a MPD enhanced immunoassay, which provides quantitative measurement of proteins of a few femtogram/ml. We have conducted pilot studies comparing the IA/MPD to ELISA for p24 HIV antigen. Our data demonstrate the unprecedented sensitivity of the IA/MPD for p24-antigen. At present, its sensitivity is below an attomole per ml, i.e. about 500-fold better than prior art assays. This strongly suggest the ability to detect about 20 HIV-1 virions per sample by a low cost immunoassay.
New Immunoassays for HIV-1: Our immunoassay for p24 antigen is about 1000 times more sensitive than prior-art immunoassays. This assay is being tested on clinical research samples to demonstrate both improved diagnostic capability , and quantitation of p24 antigen reliable and sensitive enough for predicting disease progression. The IA/MPD provides a robust early detection of HIV-1 and can be used to considerably shorten the "detection window" due to limitations of current immunoassays. We are currently developing a p24 antigen detection assay with a further improved limit of detection (LOD) of 2 femtogram/ml. The development of such a diagnostic assay with 5,000-fold greater sensitivity than conventional tests will:
The worldwide epidemiological data regarding the AIDS pandemic shows the clear need for the most sensitive screening and diagnostic tests which detect the virus at the earliest possible time following infection. Therapies using triple combination of anti-retroviral agents increased the value of early detection and ultra-sensitive assays for HIV-1. The virus should be detected before it destroys the immunological system. First generation clinical screening tests were based on the presence of the p24 antibody, a relatively late marker of HIV infection. Many HIV-1 infected individuals (termed "slow progressors") may not test positive for p24 antibody for months or years after infection. This has prompted the development of a new generation of immunoassays based on direct detection of the p24 antigen, a product of the HIV envelope core. The p24 antigen assays have the potential to greatly shorten the "window" of detection. Unfortunately, these new assays are still limited by their detection limits of a few pg/ml.
Need for low cost HIV-1 assays: Virologists have long recognized the insensitivity, unreliability, and of lack of prognostic value for tests using HIV-1 antibodies. Detection schemes based on antibody detection rely on host response and are extremely variable. For example, there are some individuals who remain with an undetectable level of p24 antibodies for up to 10 years following infection . Thus, only methods based on ultrasensitive detection of p24 antigen and viral nucleic acids may be useful during early infection or in long-term asymptomatics. Several commercial firms have developed HIV-1 assays based on the presence of p24 antigen (viral core proteins). These ELISA assays offer limits of detection (LOD) at 5-12 pg/ml sera. It was demonstrated that the viral load of about 2,000 HIV-1/ml of serum leads to about 1 pg/ml of p24-antigen/ml. For reliable early detection and blood screening, it is necessary to use assays with the LOD better than 0.05 pg/ml, and a dynamic range of several orders of magnitude. This would permit the reliable, low cost assay of viral load at the level of below 100 virions/ml. The IA/MPD for p24 antigen achieved this goal, and we expect that a further tenfold increase in sensitivity can be achieved by optimization of assay conditions.
Benefits of early detection include protection of blood recipients and infected individuals who can benefit from early intervention. Clinical trials of a "triple combination" therapy have shown that early diagnosis with immediate intervention can stop the progress of HIV infection. Early detection can also be critical to the prevention of related opportunistic infections (ROI), which can be treated prophylactically before the immunological system of the patients is destroyed. Another aspect of early detection is the impact on prevention and the continued spread of the disease. In addition, more sensitive diagnostics are needed to aid clinicians who monitor therapy monitoring. In this case, however, one needs to measure both the free p24 and p24 bound to antibodies.
IA/MPD vs. ELISA for HIV-1 p24 Antigen: An HIV-1 p24 antigen IA/MPD was developed by modifying a commercially available ELISA, the Retro-Tek (Cellular Products Inc., Buffalo, NY). The same capture and detector antibodies as well as antigen standards were used in both assays. However, a set of proprietary reagents that were tailored for this IA/MPD was required in order to reduce biological background. Both assays were performed in serum and in quintuplets. Figure 1 shows a comparison of standard curves for the ELISA and the IA/MPD. In the ELISA (left plot), absorbance values ranged from 0.795 down to 0.052, corresponding to p24 antigen concentrations of 125 pg/ml to 7.8 pg/ml, respectively. In the IA/MPD (right plot), SR-MPD counter reads from 146 dpm down to 1 dpm, corresponding to p24 concentrations of 30 pg/ml to about 10 fg/ml, respectively. The computer analysis of the standard curve suggests the LOD of 5 fg/ml. Thus, the sensitivity of this commercial ELISA kit was improved about 1000 fold by using MPD technology (ELISA, 7 pg/ml vs. IA/MPD, 0.005 pg/ml).
 Figure 1. Comparison of the sensitivity of IA/MPD(type 2) and ELISA for p24 anrigen. Note the approximate 500-fold better lower limit of detection for IA/MPD as compared to the ELISA (in buffer) |
The power of the IA/MPD becomes evident when the coefficients of variance of the standard curves for these two immunoassays are examined, (see Figure 2). For the ELISA the CVs rapidly worsen at antigen concentrations below 5 pg/ml, demonstrating the uselessness of this assay when trying to measure trace amounts of the antigen. In contrast, the IA/MPD, maintains a good CV profile throughout the entire standard curve down to about 10 fg/ml level.
 Figure 2. Coefficients of variance (CVs) for IA/MPD and ELISA for p24 antigen. Note the good reproducibility for the IA/MPD (CV<20%) even at antigen concentrations below 0.02 pg/ml. |
 Figure 3. Comparison of sensitivity of IA/MPD and ELISA for virial load measurements. |
We improved the IA/MPD by optimization of assay reagents, use of better blockers and more stringent wash. We expect that the p24 antigen IA/MPD can be improved even further; the assay format, volumes and protocol are currently being optimized. At the lowest point of the IA/MPD standard curve, the signal to instrument background ratio is better than 20, i.e. another 10 to 20-fold improvement in assay sensitivity is possible by further reduction of NSBB. We achieve a similar assay sensitivities (sub-attomole/ml) as already achieved with the IA/MPD for TSH and some interleukins. Taking into account that clinically proven correlation (20,000 HIV-1 = 1 pg/ml of p24 antigen) we reached a sensitivity of 10 copies of HIV-1 per sample, or about 50 copies/ml, i.e. sensitivity characteristic for quantitative PCR tests (see Figure 3). We also demonstrated using a CPI panel, that IA/MPD is more reliable than ELISA (see Figure 4).
Towards further improvement of the p24 antigen IA/MPD: An important task in the development of any IA/MPD is the reduction of nonspecific biological background (NSBB). Our experience has shown that biological background, which is generally believed to be an unsurmountable barrier, can be overcome through modifications of the assay protocol and by using tailored reagents. The p24 immunoassay sensitivity can be improved by increasing the specific activity of the tracer which at the same time will reduce NSBB. The radioactive signal generated in samples with trace amounts of p24 antigen can be greatly amplified using BioTraces' SuperTracer. The SuperTracer is a large complex of streptavidin and a proprietary macromolecule that can be labeled with up to several hundred 125I atoms. Use of our general SuperTracer in the place of 125I-streptavidin will boost assay signals by at least 100 fold. This new technology therefore, offers a means to greatly enhance the sensitivity of the existing IA/MPD for p24 antigen.