Keywords: HIV load test, p24 antigen assay, colloidal magnetic nanoparticles, agglutination test, and micro fluidic chip
A new platform and assay for the detection of the HIV p24 antigen in serum or blood is to be developed. The advantages of a p24 test are that it can detect HIV at an early stage of the infection, before antibodies develop, and that it is quantitative. The DETECTHIV project aims at developing an extremely simple viral load test with only one reactant (grafted colloids). In a first phase, a magnetic nanoparticles assay will be developed for use in a microtiter plate with the goal to detect concentrations as low as 1 ng/ml. In a second phase, the use of nanoparticles on a microfluidic chip will allow the detection of p24, to levels as low as 0.1 picog/ml, one to two orders of magnitude more sensitive than classical Enzyme Linked Immuno-Sorbent Assay (ELISA) p24 tests. Validation of the platform will be done with bothrecombinant p24 samples and patient samples.
The test consists principally of optically detecting the formation of a colloidal gel of magnetic nanoparticles (‘agglutination test’). The gel forms in a magnetic field under the presence of antigens that are capable of linking irreversibly two colloidal particles. Therefore, the latter are grafted with antibodies that are specific to the p24 antigen. The detection is achieved and through simple optical absorbance measurements, owing to the strong optical scattering modification when passing from nanometric colloids to the gelled state.
In the microfluidic chip test, a sample solution of serum or blood is transported through a suspension of magnetic nanoparticles that are magnetically retained within a microfluidic channel. When brought into a magnetic field, the particles will be able to approach each other, form chains and will be irreversibly linked if the p24 antigen is present. Subsequently, on-chip light scattering techniques will be used to quantify the concentration of permanent chains or clustered beads, which is proportional to the p24 antigen concentration.
Given that immunoassays are the most widely used assays in clinical diagnostics, the impact of lowering their cost while enhancing their efficiency would provide a much-needed contribution towards lowering healthcare costs. The chosen assay, i.e. the p24 antigen-based viral test, would be of great benefit in the global battle against HIV infection and AIDS, especially in the developing world that constitutes over 70% of the affected world population. An arsenal of laboratory methods is available to screen blood, diagnose infection and monitor disease progression in individuals infected by HIV. ELISA is the most commonly used test to screen for HIV infection.
One advantage of the use of magnetic colloids arises from the fact that the particle surface has a very strong colloidal stability in any type of buffer, serum or plasma and has functional groups or receptors so that the particles can be further grafted with the biomolecules of interest. After trapping molecules (present in small amounts in solution) on the nanoparticles, the latter can be handled with applied magnetic fields. There is a net advantage due to faster reaction rates, trapping of the analyte and separation without losses.
With this innovative, sensitive p24 assay, the aim is to approximate the sensitivity of the current viral load assays with the added advantage of being more robust (because of the superior physical stability of the p24 antigen). Although measurement of HIV-1 RNA (viral load) is the acknowledged "gold standard" marker for monitoring disease activity in patients receiving highly active antiretroviral therapy, it remains a very expensive test in resourceconstrained settings. As the DETECTHIV project promises to establish a more sensitive, faster and simpler test than a conventional ELISA p24 test and a more affordable test than an HIV-1 RNA viral load test, it has full potential to set a new standard in the diagnosis and follow-up of HIV infected and AIDS patients during the complete duration of the infection.
The final goal of this project is the development of an agglutination test for the HIV p24 antigen with a detection sensitivity that is one to two orders of magnitude better (0.1 pg/ml) and up to an order of magnitude faster than standard ELISA p24 tests. A single platform that handles magnetic actuation, microfluidics, optical detection and signal processing on a disposable microfluidic chip will be provided. Magnetic nanoparticles with appropriate surface chemistry for selective recognition of the p24 antigen will be the first to be developed. Clinical validation of the assay for the detection and quantification of p24 antigens is an important milestone. For ultimate sensitivity, magnetic retention and actuation will be used to separate nanoparticle clusters from nonreactive nanoparticles in a microfluidic chipbased system. By means of an optical detection system with integrated polymer-based waveguides, it will be possible to exploit the scattered light to retrieve information from the nanoparticles.
With the recent availability of low-cost/free anti-HIV drugs, a remaining major hurdle is the availability of an affordable viral load test. Viral load tests help determine the correct regimen of anti-HIV drugs. The current gold standard for viral load testing (the Polymerase Chain Reaction-based test) is unfortunately out of reach for the budgets of developing nations. Consequently, most of these countries commence treatment without viral load monitoring. The new p24 assay platform developed within the DETECTHIV project will be of considerable benefit to the development of a low-cost viral load test for HIV. The aim is to use South Africa as the beta test site. The total number of HIV infections in South Africa is currently estimated at around 4.7 million people. South Africa is the ideal pilot site because it has conditions that are reminiscent of both, the developed and developing world, as well as an established medical infrastructure.
1 - Martin Gijs
Ecole Polytechnique Fédérale de Lausanne (EPFL) (CH)
Swiss Federal Institute of Technology Lausanne (EPFL)
Institute of Microelectronics and Microsystems
Tel.: +41 21 693 67 34
Fax.: +41 21 693 59 50
|Official Address||Other Information|
|2||Marc Van Ranst||
Katholieke Universiteit Leuven (KUL) (B) |
Clinical and Epidemiological Virology
Aids Reference Lab, UZ-Sint Rafaël,
Microtoren 7de verdiep
|Tel: +32-16-347.908 |
Ayanda Biosystems (Ayanda) (CH) |
Ayanda Biosystems SA
EPFL - PSE Parc Scientifique
|Tel/Fax: +41 21 693 8631 |
Bertin Technologies (Bertin) (F) |
Biotech Systems Department
Parc d'activités du Pas du Lac
10 bis avenue Ampère
|Tel: +33 1 39 30 60 70 |
Fax: +33 1 39 30 61 85
Ecole Supérieure de Physique et de |
Chimie Industrielles de la ville de Paris
Laboratoire Colloïdes et Matériaux
ESPCI, 10 Rue Vauquelin
F-75231 Paris cedex 05,
|Tel: +33 1 40 79 52 19 |
|6||Jörg P. Kutter||
Denmark Technical University (DTU) (DK) |
MIC - Dept. of Micro
Technical University of Denmark
DK 2800 Lyngby,
|Tel: +45 4525 6312 |
Fax: +45 4588 7762
ADEMTECH SA |
Parc Scientifique Unitec 1
4, Allée du Doyen Georges Brus
|Tel: +33 686 57 32 23 |
Fax: +33 5 57 02 02 06