Technologies such as ELISA, PCR and western blot are now front and center for HIV diagnosis and research.
In January 2013, an assoc. prof of biomedical engineering at Columbia Univ., Samuel K. Sia, developed a lab-on-a-chip technology that not only checks a patient’s HIV status with just a finger prick, it also synchronizes the results automatically and instantaneously with central health care records. The technology, developed in collaboration with OPKO Diagnostics and called mChip, performs all essential enzyme-linked immunosorbent assay (ELISA) functions, and produces results within 15 min—10 times faster than clinical benchtop ELISA diagnostics. The microfluidic-based diagnostic device, field-tested in Rwanda, may provide people in remote, developing areas of the world with laboratory-quality diagnostic services traditionally available only in centralized health care settings.
In assessing the device’s abilities in the field, Sia’s research team tested more than 200 serum, plasma and whole-blood samples, in which all results were synchronized in real time with the patients’ electronic health records. The device also transmitted all whole-blood test results from a Rwandan clinic to a medical record database stored on the cloud. The mChip produced results in agreement with a leading ELISA test, including detection of weakly positive samples that were missed by existing rapid tests.
ELISA diagnostic tests comprise an analytical biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of an antigen in a blood sample. The first screening test used for HIV because of its high sensitivity, ELISA, even in first-generation form, could accurately detect HIV antibodies at 50 days, as many HIV-positive victims will seroconvert within 25 days of infection. In an ELISA diagnostic test, a patient’s serum is diluted and applied to a plate to which HIV antigens are attached. If antibodies to HIV are present in the serum, they may bind to these antigens. The plate is then washed to remove all other components of the serum. A secondary antibody that is chemically linked in advance to an enzyme is then applied to the plate, followed by another wash. As a result, the plate contains enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence.
Since its inception in the 1960s, ELISA technology has undergone evolutions that drive point-of-care solutions to people around the world, that have permitted the introduction of low-cost approaches—such as the mChip—to deliver sensitive results at a rapid pace. Even in clinical laboratory settings, while first-generation technology could only detect HIV-1 antibodies of the IgG isotope, current fourth-generation ELISA technology has increased the sensitivity of the assay with the ability to test for both HIV-1 and HIV-2 antibodies of the IgM, IgG and p24 isotopes and antigen. Current fourth-generation ELISA diagnostics can present accurate results within one week of infection.
Western blot confirms diagnosis
For highly variant viruses, like HIV, a follow up to ELISA testing is required to ensure a positive result. If an ELISA test is negative after three months of potential HIV exposure, the patient is deemed, with 99.999% accuracy, HIV negative. While ELISA technology is highly sensitive, false positives do occur. This is where western blot technology excels as a common follow up diagnostic test, offering a 100% accurate result.
Like ELISA, western blot is an antibody detection test. However, unlike the ELISA method, the viral proteins are separated first and immobilized. In subsequent steps, the binding of serum antibodies to specific HIV proteins is visualized through gel electrophoresis. This occurs when possible HIV-infected cells are opened and the proteins within are placed into a slab of gel, to which an electrical current is applied. Different proteins will move with different velocities in this field, depending on their size, while their electrical charge is leveled by sodium lauryl sulfate. Once the proteins are separated, they are transferred to a membrane and the procedure continues similar to ELISA.
A recent technology for disease research introduced by Molecular Devices, called the ScanLater western blot detection system, combines western blot membrane detection with a multimode microplate reader platform, eliminating the need to acquire a separate dedicated western blot system. The system is a time-resolved fluorescence-based western blot detection assay, a detection method that reduces stray excitation light, resulting in lower background noise and higher sensitivity. It comprises the ScanLater western blot detection cartridge, ScanLater western blot kit and image acquisition software powered by SoftMax Pro. The kits contain europium-labeled secondary antibodies designed to work with existing primary antibodies without further optimization. The substrate-free method of western blot detection not only outperforms traditional chemiluminescence and fluorescence-based western blot detection, it also allows continuous membrane detection.
PCR drives diagnosis, research
Time is of essence when it comes to a disease like HIV. The quicker a patient is aware of their status, the sooner they can be started on life-prolonging antiretroviral therapy. The most accurate diagnostic tool to detect HIV in blood is the HIV PCR test. Aside from being considered more accurate than assay-based tests, HIV PCR is also one of the only procedures for early detection.
Unlike the fourth-generation ELISA and western blot, HIV PCR does not rely on the presence of antigens or antibodies in the blood. Instead, it identifies genetic material by highlighting sequences of HIV within a patient’s DNA. This is done through nucleic-acid amplification testing to observe the resulting polymerase chain reaction (PCR). The HIV PCR process is then further divided into two subcategories that determine viral load in a serum sample: RNA PCR and DNA PCR. To perform the test, an enzyme called DNA polymerase is used to pursue a pro-viral genome sequence and amplify it. In addition to the enzyme, the PCR-inducing cocktail also contains oligonucleotides and deoxynucleotide precursors, as well as a cofactor called MgC12.
Trends toward more point-of-care PCR technologies have pushed boundaries at universities. California Institute of Technology has developed one such technology. The new PCR machine is small enough to stow in a backpack and operates at the push of a button. The diagnostic prototype runs off a rechargeable battery and comprises a chip that can analyze blood sample to spot different pathogens. The cost: under $1,000 and one scan only costs $5.
However, PCR doesn’t just serve as a diagnostic tool. The need to find a cure for HIV has spurred demand for new assays that can more precisely quantify reservoir HIV DNA. Bio-Rad’s QX100 Droplet Digital PCR (ddPCR) system has been used in a research setting for this purpose.
The ddPCR is a nucleic acid molecule counting method that works by partitioning the sample into droplets to an extent that either zero or only a small number of DNA molecules of interest (targets) are present in each partition, with thousands of reactions running in parallel. The result gives users an absolute measurement that doesn’t require the need for a standard curve. Eliminating the standard curve reduces error and improves precision, enabling reproducibility. Because samples are partitioned and an end-point analysis performed, the background DNA is reduced, lessening competitive and inhibitor effects and allowing for greater discrimination between similar nucleic acid quantities.
A recent study in PLOS ONE led by Dr. Matthew Stain and Dr. Douglas Richman of the Center for AIDS Research at the Univ. of California, San Diego found that Bio-Rad’s ddPCR can be used to create a standardized, accurate assay for persistent HIV DNA in infected patients. “This is attributed to the fact that ddPCR is able to reliably detect HIV DNA targets well below the limit of quantification of qPCR,” says Bio-Rad’s GXD Div., Hercules, Calif. When using identical quantities of clinical samples from peripheral blood, ddPCR’s precision was between four-fold to more than 20-fold better than qPCR. Moreover, ddPCR reaction conditions can be tuned to make it less sensitive to unknown mutations within an HIV-positive sample, thereby increasing its ability to detect and quantify the presence of the virus.
These results suggest the ddPCR assay could prove useful for clinical studies aimed at eradication of HIV from infected patients. In fact, Dr. Richman worked with a team of researchers, led by Dr. Deborah Persaud of Johns Hopkins Children’s Medical Center, that showed, for the first time, the functional cure of an HIV-infected infant. The ddPCR assay was used to confirm the findings, determining there was in fact no detectable virus in the baby.