TB-SPECIFIC BIOMARKERS – DISCOVERY AND DEVELOPMENT OF A DIAGNOSTIC PLATFORM

<b>Project Methods</b><br> SELEX selection of TB biomarker aptamers. A random oligonucleotide ssDNA library (ROL30) will be synthesized as described by Nutiu and Li (36) but with some modifications. The library will consist of the five regions: (i) a forward primer binding region followed by an AAGCTT extender sequence, (ii) a 10-mer randomized region (N10), (iii) a 15-bp segment designed to bind to a biotinylated, ATCG extender, (iv) a 20-mer random region (N20) and finally (v) a reverse primer binding region at the 3' end. A biotinylated probe, complementary to region (iii) of the sequences in ROL30, will be synthesized to aid in library capture during SELEX. This probe will be tethered to a streptavidin coated magnetic beads. All oligos will be synthesized by Integrated DNA Technologies.For SELEX, the combinatorial DNAlibrary is mixed with the biotinylated-DNA probe and incubated in a thermal cycler from 95°C to 41°C in a gradual linear ramp, and then incubated at room temperature for about 45 min to complete probe-library annealing. This library-probe complex is incubated with streptavidin coated magnetic beads for 1 hour with shaking and then gently washed five times to remove excess biotinylated probes as well as some unbound library. Two peptides each of pks5, MB1895c, and MB2515c will be used to select structure-switching aptamers. These peptides will be incubated for 1 hour at room temperature with the immobilized library on a rotary shaker. Subsequently, the supernatant (enriched with peptide-specific structure switching aptamers) is collected and amplified by HotStart Taq polymerase, with subsequent analysis by gel electrophoresis. The amplicons from this PCR step are then used as a template for asymmetric-touchdown PCR (25:1 ratio of forward:reverse primers, where the reverse primer has a biotin tag), which is then purified by a MiniElute PCR kit. Since the next round of SELEX requires only the sense strand, the biotin-tagged DNA strands will be removed by denaturing at 95oC (10 min), quickly chilling on ice for 10 min, and then incubating with prewashed Dynabeads M-280 Streptavidin for 15 min on ice. The sense strands will then be used for the next round of SELEX. We expect to use a total of 16 iterations of SELEX. Selected DNA molecules with high affinty to peptides will be cloned, sequenced and reconfirmed using DNase footprinting assay.Lateral flow assay development:The LFA is a point-of-care dipstick test that uses gold-conjugated, anti-mycobacterial, monoclonal antibodies or aptamers impregnated onto an immunochromatographic test strip, to detect circulating M. bovis peptides from. If mycobacterial antigen is present in the specimen, suspended, gold-conjugated, anti-mycobacterium antibodies or aptamers bind to the antigen. The gold-antibody or aptamer-M, bovis complex migrates, by capillary action, up the test strip where it interacts with immobilized anti-mycobacterial, monoclonal antibodies causing a visible, red line to develop. The LFA kit consists of immunochromatographic test strips, positive control, and assay diluent, which can be stored at room temperature for up to two years. To perform the LFA, one drop of diluent (~40μL) is added to a container with 40μL of the serum from suspect animals. The dipstick will be inserted into the container and incubated at room temperature for 10 minutes.First, experimentally infected calf and deer sera will be prospectively collected and sent from NADC to Dr. Sreevatsan lab for testing with LFA. All previous and new experimental infections with sera representing baseline to 60 days post infection collected at 3 week intervals will be tested. A total of 4 separate experimental infection sera and their contemporary controls will be used. These samples from experimental infections are already available for testing in Sreevatsan lab. For field samples, deer and cattle serum samples (-80oC) collected from bovine TB outbreaks and cryopreserved at NVSL, NADC and MSU-VDL (previously DCPAH) will be retrospectively tested by LFA. All field samples will be tested in a blinded fashion.Thermal Contrast Measurement of Mycobacterial peptide titers:We will test a novel method to provide quantification of LFAs in comparison to semi-quantitative LFA titers, using the heat signature of laser irradiated gold present in the LFA. To detect the presence of gold nanoparticles conjugated to monoclonal antibodies or aptamers on the LFA test line, the line will be irradiated with a 0.01W laser (532nm, Millennia Vs, Diode pumped, Santa Clara, CA) for 30 seconds and the temperature change (i.e.thermal contrast) will be recorded with an infrared camera (FLIR ThermoVision™ A20, Portland, OR). Three spots on each horizontal LFA test line will be irradiated and the average maximum temperature change is calculated. An antigen titer is calculated from the thermal contrast using a calibration curve established by 2-fold serial dilutions of three specimens in triplicate with known mycobacterial peptides spiked into negative sera. Thermal contrast quantification will be performed on 200 positive and 100 visually negative LFA dipsticks.All positive, suspect and negative test lines will be extracted and confirmed for the presence of mycobacterial peptides by tandem mass spectrometry. We have recently performed this analysis in collaboration with Drs. Lyaschenko and Waters from their recent study samples (38) and demonstrated the appearance of >20 mycobacterium-immune complexes in the serum of experimentally infected animals (unpublished).?