We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0 parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35 min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5–8 h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous.

Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein).

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PCR is the most sensitive molecular diagnostic available for infectious diseases. The goal for low-resource settings is a simple, inexpensive instrument. Toward this goal, we previously published a self-contained sample preparation instrument that uses magnetics and prearrayed reagents in thin tubing to extract nucleic acids and perform isothermal amplification and detection of extracted biomarkers. To incorporate PCR thermal cycling, after biomarker is magnetically extracted from a patient sample, the section of tubing containing the extracted biomarker and PCR reagents is alternately positioned within two constant temperature blocks. This instrument was evaluated initially by extracting and amplifying a 140 bp fragment of the IS6110 sequence of tuberculosis from TE buffer. The mean cycle threshold for 5 × 10 6 copies of IS6110 was 25.5 ± 1.5 cycles (n = 4), which was significantly different from negative control samples (34.0 ± 2.6 cycles; n = 3). Using a more clinically relevant sample, we extracted and amplified Plasmodium falciparum DNA from malaria-infected human blood cultures. The average cycle threshold for 1% parasitemia samples was 24.7 ± 1.5 cycles (n = 3) and significantly different from negatives (31.5 ± 2.1 cycles; n = 3). This approach integrates biomarker extraction, PCR amplification, and detection in a simple, linear tubing design with potential for use as a low-resource instrument.

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy) 2 (H 2 O) 2 + (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/ Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 µm magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

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OBJECTIVES/SPECIFIC AIMS: High-sensitivity diagnostics for early infant diagnosis (EID) of HIV at the point of care (POC) are not widely available. Lateral flow immunoassays (LFA) can detect HIV-p24, but are not sensitive enough in practice. With improvements, LFA are a compelling platform for POC in EID. We used functionalized magnetic beads and immunocomplex dissociation to improve sensitivity of HIV-p24 LFA. Here, we evaluate the utility for LFA to quantitatively report HIV-p24 concentration and estimate HIV viral load. Using purified p24 protein and virion constructs, we determined the limits of detection for HIV-p24 using LFA rapid tests. Using measurements from HIV-p24 ELISA, laboratory-developed RT-qPCR, droplet digital PCR, and gold standard clinical viral load, we further characterized the relationship between HIV-p24 concentration, HIV genomic RNA, and LFA test line signal. METHODS/STUDY POPULATION: We measured HIV-p24 concentration by ELISA (R&D Systems) and LFA (Alere Determine HIV-1/2 Ab/Ag Combo). An LFA reader instrument was used to image test lines and measure test line signal on the LFA. HIV viral loads were measured using RT-qPCR and droplet digital RT-PCR protocols adapted in our lab. We obtained gold standard viral load measurements using the Roche Cobas TaqMan system at Vanderbilt University Medical Center. Data analysis was performed using Prism 7 and Stata 14. RESULTS/ANTICIPATED RESULTS: LFA test line signal increases in a predictable, dose-dependent manner and correlates with concentration of purified HIV-p24 with a linear range between 50 and 1000 pg/mL (Spearman r=1; p=0.0004). We compared p24 concentration (ELISA). We evaluated the utility of LFA to quantify HIV-p24 from virions suspended in human plasma, which increased the limit of detection for HIV-p24 to 100 pg/mL and shifted the linear range 100–10,000 pg/mL (Spearman r=0.77; p<0.001). To evaluate the relationship between HIV-p24 concentration and concentration of HIV RNA, we employed 3 molecular techniques. The LFA is capable of detecting HIV-p24 concentrations that correspond to a range of viral loads between 653,000 and 1655 copies of viral RNA/mL. DISCUSSION/SIGNIFICANCE OF IMPACT: Our preliminary results are very promising, indicating that commercially available LFA can quantitatively measure HIV-p24 concentration to low levels. When coupled with our analysis of the relationship between HIV-p24 concentration and HIV RNA concentration, LFA may be a potential platform allowing us to estimate HIV viral burden at clinically relevant levels. Our next steps will be to evaluate this relationship in primary, clinical specimens in collaboration with the Tennessee Center for AIDS Research. We will incorporate technologies to improve the sensitivity of these LFA and evaluate their performance in field settings in Zambia. Our findings are broadly applicable for use in HIV care and treatment programs and early infant diagnosis programs around the world.