Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real-Time Quaking-Induced Conversion (RT-QuIC) Assay

<div><p>Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP^d) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease <i>ante mortem</i>. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay’s sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an <i>in vitro</i>-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.</p></div

The effect of batch variability and seed genotype on RT-QuIC.

<p>RT-QuIC was performed using two different batches, Batch #1 (A) and Batch #2 (B), of hamster rPrP. Both assays employed hamster rPrP at a concentration of 60 µg/well as the substrate and sCJD M/V brain homogenate (BH) at the indicated dilutions as the seed. (C) RT-QuIC was performed using hamster rPrP Batch #1 at a concentration of 60 µg/well as the substrate and sCJD M/M brain homogenate (BH) at indicated dilutions to seed conversion. Reactions contained minimal salt (5.5 mM NaCl).</p

The effect of substrate concentration on RT-QuIC.

<p>RT-QuIC was performed using hamster rPrP substrate at concentrations of 20, 35, 45, and 60 µg/well. Reactions contained minimal salt (5.5 mM NaCl) and employed sCJD M/V brain homogenate (BH) diluted 10⁻⁴ to seed conversion.</p

MS-H: A Novel Proteomic Approach to Isolate and Type the <em>E. coli</em> H Antigen Using Membrane Filtration and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

<div><p>Serotyping is the long-standing gold standard method to determine <i>E. coli</i> H antigens; however, this method requires a panel of H-antigen specific antibodies and often culture-based induction of the H-antigen flagellar motility. In this study, a rapid and accurate method to isolate and identify the <i>Escherichia coli</i> (<i>E. coli</i>) H flagellar antigen was developed using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Flagella were isolated from pure culture, digested with trypsin, and then subjected to LC-MS/MS using one of two systems (Agilent-nano-LC-QSTAR XL or Proxeon-nano-LC-LTQ-Orbitrap XL). The resulting peptide sequence data were searched against a custom <i>E. coli</i> flagella/H antigen database. This approach was evaluated using flagella isolated from reference <i>E. coli</i> strains representing all 53 known H antigen types and 41 clinical <i>E. coli</i> strains. The resulting LC-MS/MS classifications of H antigen types (MS-H) were concordant with the known H serogroup for all 53 reference types, and of 41 clinical isolates tested, 38 (92.7%) were concordant with the known H serogroup. MS-H clearly also identified two clinical isolates (4.9%) that were untypeable by serotyping. Notably, successful detection and classification of flagellar antigens with MS-H did not generally require induction of motility, establishing this proteomic approach as more rapid and cost-effective than traditional methods, while providing equitable specificity for typing <i>E. coli</i> H antigens.</p> </div

Diagnostic specificity, sample stability and run-to-run repeatability test results for MS-H with the Orbitrap platform^a.

<p>NM, non-motile; N/A, not attainable.</p>a<p>The same flagella digest was tested by LC-MS/MS three times within a one-week period; ^ba two-year old residual digest was re-used.</p

Comparison of H serotyping and MS-H of <i>E. coli</i>.

<p>Comparison of H serotyping and MS-H of <i>E. coli</i>.</p

H7 identification of reference strains by MS-H with the QSTAR platform^a.

a<p>11 known H7 positive <i>E. coli</i> strains and one non-motile strain (E32511) were twice tested for MS-H, and the sequence coverage were obtained by Mascot database search.</p

Side-by-side comparison of H serotyping and MS-H typing of <i>E. coli</i> H types with the Orbitrap platform^a.

<p>-, serotyping titration or MS identification was not reached.</p>a<p>Serotyping and MS-H were performed concurrently from subcultures of single bacterial colonies. MS-H was repeated on two consecutive days; <b>^b</b>motility induction was performed for these inconsistent strains after initial MS-H; ^cuntypeable by serotyping although previous MS and PCR-based sequencing showed type H21; ^dusing designated antisera by MS-H.</p

MS-H identification of all references strains with the QSTAR platform^a.

<p>UI, unidentified strain.</p>a<p>Known reference strains encompassing all 53 H types were tested by MS-H. If a primary strain could not be identified after three consecutive MS-H analyses, an alternate strain was selected for MS-H typing.</p