Background

Contamination has been an issue plaguing the original PCR experiments. After ordering new solutions and still seeing contamination, we concluded that the problem did not lie with the reagents, the equipment or the graduate students techniques. The problem must have thus been associated with the oligo-primer set developed. There must have been a problem with nonspecific binding of the primers allowing for 75 bp sections of other DNA sequences to be amplified. Also, it was possible that the materials received from the vendors weren’t as specific as we wanted. The primers could have had some additional oligo contained in them, since theoretically one extra sequence present could yield 2n times more products, more than enough to pose PCR contamination problems. 

The final target of these studies has always been to develop a highly sensitive assay for the trace detection of prion proteins in body fluids. iPCR, which combines the specificity of immunoassays and the amplification power of PCR, is an ultrasensitive technique for trace analysis of proteins and antigens. iPCR usually lowers the LOD for protein detection about three (3) orders of magnitude, when compared to conventional ELISA. This approach requires: (1) immobilizing a prion protein to a solid support surface; (2) linking thru the detector Ab with a DNA label; (3) amplifying the DNA label by PCR; and (4) subsequently detecting amplification products. The procedure has been been illustrated in Figure 1 (and variations thereof).

One critical aspect of iPCR is the efficient coupling of the target-specific Ab (detector) with the oligo (to form the immunocomplex or chimera). Typically, this was achieved by successive coupling steps of several components. This strategy needs a large number of incubation steps, and usually entails an incomplete interphase coupling of the reagents, occurring with an efficiency of only ~10% for each step. In order to circumvent this problem, we tried to use synthetic strategies based on self-assembly of molecular building blocks to get the immunocomplex (Ab-oligo chimera) for iPCR. This method seemed to be an effective approach to couple several components for iPCR. Its advantage includes high sensitivity, high reproducibility, high linearity, time savings, single step protocol, minimization of signal loss due to inefficient coupling in many incubation step, and an improved ease of handling. We are not the first to consider using a chimera, but it requires the in-house synthesis, purification, isolation, and characterization before it can be routinely applied in a iPCR format. 

Another common problem in immunoassays is nonspecific binding. We performed a series of comparison studies to understand where this came from, and found it mainly occurred between the capture Ab and detector Ab in the immunocomplex formation step. In order to decrease this unwanted binding, we took several steps, such as changing the concentration of capture and detector Ab, trying different blocking buffers and different wash procedures, and so forth. Further studies are in progress. 

The stated objectives for this work were:

1. We have been engaged in the development of ultrasensitive, microtiter-based immunoassays for the infectious agent (PrPres, enzyme resistant or diseased prion protein) responsible for chronic wasting disease (CWD).
2. A second, initially proposed objective in 2002 was the development of an expression immunoassay (ExIA) for PrPc (and PrPres), Figure 1B. In ExIA, the sequence of the DNA label in the sandwich ELISA format encodes for a reporting enzyme.

Methodology

The first experiment of using the original template/primers suggested something was wrong since it showed contamination after ordering and using all new solutions. These solutions were used very carefully, all dilutions were made in the UV hood, with freshly prepared 1X TE buffer in new tubes with new pipette tubes. The experiment was done on the Eppendorf thermocycler, and detected using Agarose gel and ethidium bromide staining. Figure 2 shows a picture of the gel, indicating a band in lane 4, the no template control (NTC) lane. A series of blank solutions were run, using freshly diluted primers and dNTPS with the new primers, as well as the old primer set. These studies also showed that either all of the solutions were contaminated or the problem was elsewhere (faulty template). A melting curve analysis was done on the products to see if whatever was being amplified in the NTC tube was really the same product. The melting curve analysis showed all amplified products melting at about 80.1C, suggesting that all products amplified were of the same length and similar sequences. The NTC tube showed real amplification around cycle 25, when its signal surpassed the reference signal (Rox) incorporated into the Sybr Green Master Mix from ABI. From the first experiment onwards, six additional real time experiments were run showing very similar results each time concerning the NTC tube and the linear range determined. Each time the samples were linear from 4 x 10-7 g/ml to 4 x 10-12 g/ml. These calibration plots were very linear. It may well be possible to lower the LODs, with additional efforts to control the NTC problems. 

We then combined the optimized, as above, PCR results with the ELISA conditions, as optimized by Neil. The first assay gave favorable results, yielding a linear calibration curve from 0.1 μg/ml to 0.1 ng/ml, Figures 3 and 4. I think it could be possible to push the limits of detection lower, with additional steps to suppress the NTC problem. Since a linear range was established and the ELISA conditions confirmed from Neil, we then began the preliminary immuno-PCR assays. The first assay gave favorable results, yielding a linear curve from 0.1 ug/ml –0.1 ng/ml (Figures 3 & 4). These two graphs represent two different sets of buffers used in the ELISA portion. Figure 3 was done using immuno-PCR buffers, while Figure4 was done using the original ELISA buffers. The difference in slopes indicated that the immuno-PCR buffers allowed for a steeper slope, suggesting a more accurate wash and capture of antibodies.

The second aspect studied was the Mg2+ ion concentration. Since the concentration in the SYBR® green Master Mix was not known, separate reagents including, dNTP’s, Hot start polymerase and PCR buffer were used. The Qiagen Hot start Polymerase Kit included a 10X PCR buffer with 15 mM Mg2+. In each reaction the 10XPCR buffer was diluted to 1X by using 10 uL in a 50 uL reaction. Now each reaction was 1.5 mM in Mg2+ concentration. A 25 mM Mg2+ concentration was also included in the kit; with adding 2 uL of this solution the total Mg2+ ion concentration was raised in 1 mM increments. A series of samples from 1.5 mM to 5.5 mM were assayed at three different oligonucleotide concentrations. The first experiments showed a favorable response at 3.5 mM Mg2+ ion. When the assay was repeated with increasing the SYBR® green I dye amount, little to no correlation was found. In fact there were samples that fluoresced less than the blank indicating insignificant amounts of amplification occurring. To try and increase the small signals, we decided to add an additional 50 uL of SYBR® Green to each sample and read the plate again. In this case the extra dye increased fluorescence in each well to approximately 3 times what it was before. It was realized after this trial that the SYBR® Green I dye being used was to be dissolved in 1X Tris-HCl EDTA (TE) buffer. This buffer will chelate any free Mg2+ ions in solution that are necessary for the PCR reaction to run. It will be worthwhile in accurately optimizing the Mg2+ ion concentration to run the assay free of SYBR® Green I dye in the Eppendorf Gradient Thermocycler. Then, after transferring the solutions to the 96 well plate, add an additional 25-50 uL of SYBR® green I Dye. This way the TE buffer will not effect the PCR amplification.

Findings

We obtainined numerous sources and samples of PrPc and Abs against PrPc/PrPres. Purification, characterization, and quantitation of PrPc protein and its Abs was conducted. CZE, cIEF and ACE analysis of anti-PrP Abs to demonstrate purity, activity of the various isoforms, and determination of binding constants was performed. Biotinylation of prion Abs to form B-Ab was concluded. Identification of optimum Ab pairs in conventional ELISA to produce the highest S/N ratio. Determination of LODs and calibration curves for conventional ELISA assays for PrPc. The development and optimization of PCR small, single-stranded DNA fragment, using various templates and primer pairs, to generate optimum S/N ratios. Results showed elimination of most/all contamination when doing RT-PCR. Interfacing of ELISA with PCR to generate iPCR, optimization of overall conditions, minimization of nonspecific Ab binding, minimization of DNA contamination, generation of iPCR plots of cycle numbers vs. FL response for low levels of PrPc protein vs various controls. We came to the realization of LOD of <1 ppt for direct PCR and about 1-10 ppt for iPCR, though not yet confirmed and reproduced. Need to reduce nonspecific binding and contamination in theiPCR approach (January, 2004). And finally, the initiation of ExIA studies, obtaining needed DNA plasmid and cell line, initial efforts to express the plasmid thru conventional methods for production of the needed gene/DNA.

Implications

We have now almost optimized iPCR so that it can detect, at first, healthy PrPc in simple buffers, at the 1 parts-per-trillion (ppt) level, and below when interfaced with newer iPCR approaches (e.g., competitive DNA). Further method optimization and validation studies are in progress, aimed at applying iPCR to actual biofluid samples from chronic wasting disease (CWD) deer and/or elk, using simple biofluids rather than brain matter. We have also studied the binding constants for various prion antibodies being used today in all commercial and research ELISAs, in order to determine which are the best pairs for iPCR and related immunoassays. Finally, we have initiated studies into using expression immunoassay, a form of ELISA, which has significantly lower limits of detection (LOD) than conventional ELISAs. These will be eventually applied as ante-mortem assays for the presence of CWD in animal biofluids.