A Rapid, Sensitive, Specific Assay for the Detection of Chlamydia trachomatis

1. cut-off 5 min 5 min A Rapid, Sensitive, Specific Assay for the Detection of Chlamydia trachomatis Benahmed F, Simpson J, Chakraborty N, Mielzynska I, Modarress K, Hazel T and Shyamala V. Innovative Biosensors Inc., Rockville, MD RESULTS Analytical sensitivity in Assay Buffer and Donor urine Two parameter analysis: Signal:Noise ratio & Reaction kinetics ABSTRACT Background of the CANARY® Technology Analytical sensitivity in assay buffer (AB) (Panels prepared and quantitated at University of Washington) A rapid, simple, sensitive and specific method for the detection of Chlamydia trachomatis (CT) is highly desirable for use in hospital laboratories, public health facilities, and point-of-care settings. In these environments such a test would allow for real-time diagnosis of infection; enabling results to be delivered to the client as they wait and leading to improved efficacy of treatment. CANARY® is a cell-based technology that enables rapid detection of low levels of analytes based on the emission of luminescent signal. This signal is triggered upon interaction of analytes with target-specific antibodies that are engineered for expression in CANARY® B cell lines. The potential of this technology has been demonstrated by its application to the detection of >30 different viral and bacterial pathogens of importance to biodefense, food testing, and health care targets (References 1-4). CT infection is associated with ectopic pregnancy, pelvic inflammatory disease and infertility in women. There have been approximately 15 CT serovars identified to date in the literature. The focus of this application of the CANARY® technology is the detection of CT elementary bodies (EBs). The monoclonal antibody used for CT detection is highly specific with picomolar affinity. Formalin fixed (inactivated) and non-fixed CT EBs were serially diluted in assay buffer and tested directly in the CANARY® system. Under these conditions, the limit of detection for all eight serovars was in the range of 5-250 EB per assay. No difference in detection was observed between fixed and non-fixed EBs. To further evaluate the performance of CANARY® in a relevant matrix, CT EBs were spiked into urine for testing. The detection of EBs in urine matrices required filtration-centrifugation followed by a wash step. The CANARY® CT assay detected 25-500 CT EBs/ mL urine using a procedure that required < 30 minutes. Current activities are directed at optimizing the assay for use with clinical samples and data derived from these studies supports the application of this assay in settings where rapid, sensitive detection is critical. In summary the CANARY® technology for CT detection requires very basic instrumentation including a bench-top centrifuge, luminometer, and a laptop computer only. It is particularily suited for the detection of as few as 25-500 CT EB/mL urine using a procedure that requires < 30 minutes. The CANARY® technology developed at MIT Lincoln labs is relatively simple and requires only that a monoclonal antibody be available for the target of interest (Rider et al., 2003). Genes encoding the light and heavy chains of the antibody are cloned into vectors that control expression in B cells. These vectors are transfected into a parental B cell line (biosensor) expressing a bioluminescent protein. Translation and assembly of the light and heavy chains results in the production of functional antibodies that are expressed and localized to the cell surface. Exposure of the biosensor cell line to its corresponding pathogen triggers release of calcium from internal stores, thus activating the luminescent properties of the biosensor. Using the two parameter analysis, non-fixed EB could be detected in the range of 5-100 in Assay Buffer and between 25-500EB/ml in urine Serovar I is detected at 25 EB/assay (for non-fixed) and 50 EB/assay (for fixed) according to Signal:Noise ratio of 3. SUMMARY AND CONCLUSIONS Analytical sensitivity in Donor urine • The CANARY® technology provides a valuable tool in the detection and identification of pathogenic agents. By taking advantage of the signal amplification pathways intrinsic to mammalian B cells CANARY® enables detection of low levels of target organism(s) in a number of matrices, including urine, blood, nasal swabs and environmental samples. • Advantages of the CANARY® assay include: • (1) Rapid detection time of less then 30 minutes • (2) High sensitivity with ability to detect low levels of organisms and proteins • (3) High specificity through antigen-antibody interaction • (4) Requires very basic instrumentation • The results obtained in this study show a limit of detection (LoD) between 2.5-50 EB/assay in AB and between 25-500 EB/ml in urine for serovars D, E, F, G, H, I, J and K, all of which demonstrates the high sensitivity of the assay. The speed and simplicity with which CANARY® can be performed is a significant advantage over alternative, often time-consuming and complicated technologies with similar detection limits such as PCR. OBJECTIVES Determine the analytical sensitivity of eight serovars spiked into assay buffer (AB) and in human urine using the CANARY® assay MATERIALS AND METHODS Sample Preparation Using a CANARY® B cell line engineered to detect CT EBs, Innovative Biosenors, Inc., (IBI) has been able to design a simple sample preparation method that facilitates sensitive detection of this pathogen in human urine. After a filtration step to remove inhibitors, centrifugation at 13,500 x g is used to concentrate the target followed by two washes with buffer, this method enables reproducible detection of as few as 25 – 500 EB/ml of urine in less than 30 minutes Serovar D is detected at 10 EB/ml of urine in both fixed and non-fixed according to preset Signal:Noise ratio of 3 INTRODUCTION CT Serovars Analytical Sensitivity: Reaction kinetics Chlamydia trachomatis (CT) is a sexually transmitted pathogen, with high rates of infection among females. Women with lower genital tract CT infections often have no symptoms, putting them at risk of spreading the infections to sexual contacts. Without diagnosis and treatment, they are at risk for serious complications of pelvic inflammatory disease, ectopic pregnancy, or infertility. There are many nucleic acid-based assays for CT detection, but they are laborious and time consuming assays. Therefore there is a need for the development of rapid and sensitive methods for the detection of CT at the point of care. Although about 15 serovars have been identified for CT, serovars D-K are common in the urogenital tract and they are sexually transmitted. Here we describe the application of the CANARY® technology to the detection of CT elementary bodies (EB) in human urine. Results indicate that CANARY® is capable of detecting as few as 25 to 500 EB per ml of donor urine spiked with 8 serovars D-K using a simple procedure that requires less than 30 minutes. REFERENCES 1. Rider T.H., Petrovick M.S., Nargi F.E., Harper J.D., Schwoebel E.D., Mathews R.H., Blanchard D.J., Bortolin L.T., Young A.M., Chen J., Hollis M.A. 2003. A B cell-based sensor for rapid identification of pathogens. Science 301:213-215. 2. Hazel, T.J., I. Mielzynska, Q-X. Zheng, L. Li, and K. J. Modarress. 2007. Rapid Detection of Food Pathogens on Stainless Steel Surfaces Using a B Cell-Based Biosensor. Amer. Soc. Microbiol. 107th conference June 5-9th Atlanta, GA, Page 448. 3. Hazel, T.J., S. Cui, I. Mielzynska and J. Meng. 2005. Application of a Biosensor for Rapid Detection of E. coli O157:H7 Contamination in Ground Beef. Amer. Soc. Microbiol. 105th conference May 21-25th Orlando, FL, Page 514. 4. Mielzynska, I., and T. J. Hazel, T.J. 2007. Rapid detection of Chlamydia trachomatis in human urine using a B cell-based biosensor. 39th Annual Amer. Assn. of Clin. Chem. Oakridge conference Apr.19-20, St.Louis.Abstract #52 In addition to Signal:Noise ratio, algorithm for a second parameter evaluating signal kinetics at a specific period of detection is used to score for positivity. This ensures two levels of ascertainment for positives. The algorithm is still being refined