Source: UNIV OF MINNESOTA submitted to NRP
DEVELOPING MULTIPLEX GIANT MAGNETORESISTANCE (GMR) BIOSENSORS FOR THE DETECTION OF SWINE RESPIRATORY PATHOGENS.
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
1014656
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 17, 2017
Project End Date
Mar 30, 2020
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Population Medicine
Non Technical Summary
The pork industry has an annual gross production output of ~$20 billion (US Census of Agriculture, 2012) and respiratory diseases affect up to ~50% of the herd at any given stage of production have a significant economic impact on production. Yet, advancements in enhancing diagnostic capacity for porcine respiratory disease complex (PRDC) remains stagnant. Laboratory based-tests, which require specialized skills, costly equipment and infrastructure, and time, continue to be the mainstay. The need for a diagnostic solution, where the tests can be performed rapidly and with the accuracy on the farm, is un-deniable. Rapid identification of pathogen(s) involved at the farm could facilitate efficient implementation of control and prevention strategies that curtail disease outbreaks in swine production systems. In addition, simultaneous detection of multiple pathogens in a single test would further influence time, effort, and cost of production for producers dealing with these diseases on a regular basis. To this end, we propose to develop a Giant Magnetoresistance (GMR)-based biosensor chip for detecting three PRDC pathogens using functionalized magnetic nanoparticles (MNP), utilizing the property of the biosensor to detect minute changes in magnetic fields. Ultimately, our long term objective is to extend this capacity of GMR sensors to detect multiple swine respiratory pathogens, in clinical sample matrices commonly collected on a farm using a portable device.The project will develop a GMR sensor array that can detect multiple PRDC pathogens simultaneously and then use the array to detect thes pathogens in clinicla sample matrices, like nasal swabs, oral fluids, laryngeal swabs and tracheobronchial lavage fluid. The proposed research project will help bring the power of pathogen diagnostics to the pen-side, providing a means for early detection of PRDC. Ultimately, this technology could be translated into new approaches for rapid infectious disease detection and novel strategies for surveillance and control.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3114030110160%
3114010110040%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria; 4030 - Viruses;

Field Of Science
1101 - Virology; 1100 - Bacteriology;
Goals / Objectives
We propose to develop a Giant Magnetoresistance (GMR)-based biosensor chip for detecting three Porcine respiratory disease pathogens using functionalized magnetic nanoparticles (MNP), utilizing the property of the biosensor to detect minute changes in magnetic fields,. Our research team, including faculty from the School of Engineering and the College of Veterinary Medicine, have demonstrated this proof of concept using a GMR system with MNPs functionalized to detect influenza virus with high sensitivity and specificity in the laboratory. Our objective is to extend this capacity of GMR sensors to detect multiple swine respiratory pathogens, in sample matrices commonly collected on a farm using a portable device.Aim 1: To develop multiplex GMR biosensor array for detection of multiple PRDC pathogens with functionalized magnetic nanoparticles (MNP). In this specific aim, we propose to develop multiplex GMR biosensor based assay for simultaneous detection of SIV, PRRSV, and Mycoplasma hyopneumoniae.Aim 2: To evaluate the GMR biosensor array for use with respiratory clinical sample matrices. It is known that sample matrices could interfere with the sensitivity of immune-based detection assays. To determine if GMR biosensor sensitivity is altered with various respiratory samples, we propose to test nasal swabs, oral fluids, laryngeal swabs and tracheobronchial lavage fluid in the GMR system for the detection of SIV, PRRSV, and M hyopneumoniae.
Project Methods
Rabbit polyclonal and mouse monoclonal antibodies (MAb) to M. hyopneumoniae will be developed using a deoxycholate (DOC) extract of whole cell lysate from the reference strain 232 as immunogen. The antigen will be injected using standard immunization protocols for rabbits and mice to obtain hyper-immune serum and antigen specific B cells (for production of MAbs), respectively. Mouse splenocytes (a source of B cells) will be fused with myeloma cells to generate hybridomas that produce antibodies to Mycoplasma. Hybridomas, screened and selected based on antibody titers and specificity using ELISA, will be cloned by limit dilution to generate single cell clones producing MAbs. ELISA and western blotting techniques will be used to evaluate specificity of antibodies by comparing binding affinities to the reference strain (232) and to related Mycoplasma sp. such as M. flocculare, M. hyosynoviae, and M. hyorhinis. Cross-reactivity to other respiratory pathogens including Streptococci, Pasteurella, Actinobacillus, PRRSV and SIV will be evaluated prior to selecting antibodies for GMR sensors. MAbs and rabbit polyclonal antibodies with desired specificity will be purified and used to develop capture and detection reagents, respectively, for the GMR biosensor based assay.Multiplex GMR sensor array development. APTES-Glu modified GMR sensors will be robotically printed with capture antibody using the sci-FLEXARRAYER S5. The 29-sensor array will be divided into 5 regions. Five sensors each will be spotted with capture antibodies specific to Influenza, PRRSV, and M. hyopneumonia. Mouse IgG (non-specific) and anti-mouse IgG will be similarly printed onto GMR sensors to serves as negative (-) and positive (+) controls, respectively. Printed chips will be incubated at 4oC for 12 h. Optimal concentration of each detection antibody will be determined for a fixed concentration of capture antibody and a saturating concentration of antigen using serial dilutions of antigen and detection antibody in a two-way design matrix. The assay will be proportionally scaled down for the GMR assay to be perfomred on a hand held devise. Standardization of the GMR assay will be performed using cultured organisms or purified proteins as targets.To evaluate the GMR biosensor array for use with respiratory clinical sample matrices clinical sample matrices including washings from nasal and laryngeal swabs, oral fluids, and tracheobronchial lavage fluid, collected from healthy pigs will be spiked with multiple pre-determined concentrations of SIV, PRRSV, and M. hyopneumoniae, both individually and in multiple permutations. The dynamic range of the GMR biosensor for each pathogen tested in the array will be determined. In addition, the analytical sensitivity of GMR biosensors will be determined by comparing biosensor results with the gold standard assay.

Progress 10/17/17 to 03/30/20

Outputs
Target Audience:The data was presented to researcher groups by way of publications in lay-articles and peer-reviewed journals. Research data was also used in grant applications to the USDA and pork commodity groups for additional funding for this work. We have also presented the data obtained from this project at national and local swine conferences. An oral presentation was given at the Leman Swine health conference in St. Paul describingthe GMR no-wash system for IAV in 2018 and the Mycoplasma assay development work in 2019. Presentation to students and the swine researchers at the College have been made as well, describing the work done on this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project served as a platform for training of DMV students in the Summer Scholar program and High school students, from the Wayzata High school Honorsmentor program,interested in research experiences as a career. How have the results been disseminated to communities of interest?Several posters were presented at the CVM Research day and Leman Swine Conference. An oral presentation was given at the Leman Swine Conference in 2019 describing the results obtained from the Mycoplasma mAb data. Data obtained from this grant was also used in three USDA grant applications, including one for development of new technology for increased sensitivity in pen-side assays What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? AIM 1: To develop a multiplexed GMR sensor array to detect multiple PRDC pathogens in swine. Our long-term goal is to develop a portable diagnostic immunoassay platform that can perform pen-side testing for detection of Mycoplasma hyopneumoniae, influenza A virus (IAV), and porcine reproductive and respiratory syndrome virus (PRRSV) directly from swine respiratory samples in a single test with minimum requirements for sample handling and laboratory skills. In order to develop these tests, there is a need for pathogen-specific antibodies, which detect M. hyopneumoniae but do not cross-react with other commensal or pathogenic species of swine mycoplasmas. In this current project year, we developed both rabbit polyclonal and mouse monoclonal antibodies (MAb) to M. hyopneumoniae using a tween 20 extract (membrane fraction) of whole cell lysate from the reference strain 232. Polyclonal antibodies were developed in two rabbits and the titer of both polyclonal antibodies was >640,000 as determined by ELISA. Polyclonal antibodies were purified by protein A affinity chromatography and used as capture-antibody in sandwich ELISA. Mouse monoclonal antibodies were developed by hybridoma technique by fusing splenocytes from 3 immunized mice with Sp2/O myeloma cells. Screening of hybridoma clones for the secretion of M. hyopneumoniae specific antibodies by direct ELISA revealed 23 clones reactive to M. hyopneumoniae with absorbance ranging from 0.5 to 4.00. Of these, four clones (MAb 2, MAb 4, MAb 7, and MAb 8) were selected to study cross-reactivity to M. hyorhinis and M. flocculare antigens. We identified 3 clones that were specific to M. hyopneumoniae, which showed no cross-reactivity to M. hyorhinis and M. flocculare. Two of the hybridoma clones (MAb 2 and MAb 4) with an absorbance of >1.00 by ELISA were further characterized by Western blotting and found that they are reactive to two different proteins of M. hyopneumoniae. These two clones were sub-cloned by limiting dilution and used to optimize sandwich ELISA. These clonesare being sequenced and will be tested in GMR based assays when additional funding is procured. Furthermore, we developed an ELISA-based assay to detect M. hyopneumoniae directly from the sample. Using this assay, we tested ten M. hyopneumoniae field isolates collected from different swine farms in addition to reference strain and found that MAb 2 reacts with all M. hyopneumoniae isolates tested, whereas MAb 4 detects only reference strain. Aim 2: To evaluate the GMR biosensor array for use with respiratory clinical sample matrices. In addition, we simplified our previously developed Giant magnetoresistance (GMR) based sensing platform to use in the field, as a pen-side assay. We developed a wash-free magnetic bioassay for the detection of IAV in swine respiratory samples from swine using this assay and portable handheld detection platform. This sensing platform for IAV showed an analytical sensitivity of 250 TCID50/ml in virus spiked nasal swab samples. This test also detected IAV in nasal swab samples from known IAV infected pigs. Diagnostic sensitivity and specificity analysis are planned for future field studies.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Su D, Wu K, Krishna VD, Klein T, Liu J, Feng Y, Perez, A, Cheeran MC-J, and Wang JP. 2019; Detection of Influenza A virus in Swine Nasal Swab Samples with A Wash-Free Magnetic Bioassay and A Handheld Giant Magnetoresistance Sensing System. Front Microbiol. 2019 May 21; 10:1077. doi: 10.3389/fmicb.2019.01077.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Krishna VD, Wu K, Su D, Cheeran MC-J, Wang JP, Perez, A. 2018; Nanotechnology: Review of concepts and Potential application of sensing platforms in food safety. Food Microbiology. doi: 10.1016/j.fm.2018.01.025
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Wu K, Liu J, Saha R, Su D, Krishna VD, Cheeran MC-J, and Wang JP. Detection of influenza A virus nucleoprotein through the self-assembly of nanoparticles in magnetic particle spectroscopy-based bioassays: A method for rapid, sensitive, and wash-free magnetic immunoassays. 2019. Submitted to Biosensors and Bioelectronics
  • Type: Journal Articles Status: Other Year Published: 2020 Citation: Krishna VD, Pieters M, and Cheeran MC-J. Development and characterization of monoclonal antibodies specific to Mycoplasma huopneumonia and its application in detection of M. hyopneumoniae from swine respiratory clinical samples. In preparation
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Krishna VD, Pieters M, Nair MS, Munhoz dos Anjos Leal F, Wu K, Wang JP, and Cheeran MC-J. Development of monoclonal antibodies specific to Mycoplasma hyopneumoniae. CVM Research Day, 2019
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Krishna VD, Pieters M, Nair MS, Munhoz dos Anjos Leal F, Wang JP, and Cheeran MC-J. Development of monoclonal antibodies specific to Mycoplasma huopneumoniae. Allen D. Leman swine conference, 2019 (oral presentation)


Progress 10/17/17 to 09/30/18

Outputs
Target Audience:A portable handheld platform using a GMR-biosensor to detect veterinary pathogen(s) in clinical samples was developed. The GMR-basedimmunoassay was standardized as a wash-free single step assay that integrates seamlessly into a handheld GMR biosensor platform. This one-step immunoassay approach makes it possible to perform the test at the pen-side on any farm. Six nasal swab samples (five IAV qRT-PCR positive and one negative) were tested using the one step GMR assay and compared with standard lab-based ELISA. The results from this preliminary trial demonstrated complete congruence of the wash-free GMR assay with ELISA for IAV. In addition, the wash-freeIAV GMR testwas found to detectpurified recombinant IAV nucleocapsid as low as 30 ng/ml. In order to multiplex the GMR platform, a PRRSV and Mycoplasma hyopneumoniae detection system using the same method (as IAV) is being developed. Antibodies specific to ahighly conserved region of PRRSV nucleocapsid protein have been procured. Rabbit polyclonal hyperimmune serum specific to M. hyopneumoniae was generated with titers >640,000 by ELISA. Mouse monoclonal antibodies (MAb) to Mycoplasmahyopneumoniaeis currently being generated. Once the single-analyte GMR bio-assays are optimized, GMR sensors will be printed to enable detection of all three PRDC pathogens simultaneously. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided development opportunities for a post-doctoral fellow and a graduate student from the College of Science Engineering. They have learned laboratory techniques and conceptsin diagnostic assay development for veterinary medicine. How have the results been disseminated to communities of interest?Data generated have been presented at local and national meetings and conferences in the respective scientificdisciplines. One manuscriptis under reviewfor publication in Frontiers in Microbiology. What do you plan to do during the next reporting period to accomplish the goals?Several IAV RT-PCR positive and negative nasal swab samples are currently being assessed to determine the sensitivity and specificity of this newly developed assay. PRRSV immunodiagnosticis being currentlybeingdeveloped for the GMR platform. Monoclonal antibodies are being generated againstM. hyopneumoniae and will be typed and used for the development of the GMR immunodiagnostic for the pathogen.Once a wash free GMR assay for PRRSV andM. hyopneumoniae are standardizedindividually, multiplex GMR biosensor-based assay for simultaneous detection of all three pathogens will be developed and tested. Optimization of GMR assay for detection of all three pathogens;IAV, PRRSV, and M. hyopneumoniae, in swine respiratory samples including nasal swab, oral fluids, laryngeal swabs, and tracheobronchial lavage fluid will be performed in the next funding period. Known amounts of pathogenswill be added to negative samples from healthy pigs to determine if they can be detected simultaneously and separately. After standardization, the multiplex GMR assay will be evaluated by using respiratory samples collected from the field from infected pigs and assay sensitivity and specificity will be determined.

Impacts
What was accomplished under these goals? AIM 1:A portable handheld platform using a GMR-biosensor immunoassay was standardized as a wash-free single step assay. This one-step immunoassay approach makes it possible to perform the test at the pen-side on a farm. The assay does not require any washing steps, which effectively reduces assay runtime and simplifies the detection process considerably. In order to multiplex the GMR platform, a PRRSV detection system using the same method is being developed. Antibodies specific to a highly conserved region of PRRSV nucleocapsid protein have been procured and the diagnostic assay is being optimized for the GMR platform. In addition to PRRSV, the GMR sensor is will also be functionalized to detect Mycoplasma hyopneumoniae. Since reagents for Mycoplasma are notcommercially available,rabbit polyclonal hyperimmune serum specific to M. hyopneumoniae was generated. The titer of the serum was determined to >640,000 by ELISA. Mouse monoclonal antibodies (MAb) to Mycoplasmahyopneumoniae are also beingdeveloped for the diagnostic test. Mice were immunized with a Tween 20 extract of whole cell lysate from the reference strain 232 and tested for antibody responsesto M hyopneumoniae by ELISA. With the development of these antibodies, a wash free GMR assay for M. hyopneumoniae will be standardized. After the IAV, PRRSV, and M. hyopneumoniaeassays are standardized individually, multiplex GMR biosensor-basedassay for simultaneous detection of all three pathogens will be developed and tested. Aim 2: Using this one-step GMR assay, influenza A virus (IAV) detection from nasal swab samples obtained from infected pigs was successfully tested.Six nasal swab samples (five IAV qRT-PCR positive and one negative) were tested using the one step GMR assay and compared with standard lab-based ELISA. Samples were incubated with a mixture of detection antibody and magnetic nanoparticles for 1 hour and added to the functionalized GMR sensor.Real-time signals were collected within 4 minutes of sample addition.The results from this preliminary trial demonstrated complete congruence of the wash-free GMR assay with ELISA for IAV. Several RT-PCR positive and negative nasal swab samples are currently being assessed to determine the sensitivity and specificity of this newly developed assay.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: 1. Su, D., Wu, K., Krishna, V. D., Klein, T., Zhao, Z., Zhang, X., Feng, Y., Perez, A. M., Cheeran, M & Wang, J. P. Wash-free Magnetic Bioassays Based on Handheld Platform: Potential for Future Easy-to-use, Rapid, and On-site Diagnosis. Frontiers in Microbiology.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Krishna, V. D., Wu, K., Klein, T., Su, D., Perez, A. M., Wang, J. P., & Cheeran, M. Influenza A Virus Detection Using a Giant Magnetoresistance (GMR) Biosensing Portable Handheld Device. Leman Swine Conference Poster, 2017.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Krishna, V. D., Wu, K., Klein, T., Su, D., Perez, A. M., Wang, J. P., & Cheeran, M. Influenza A Virus Detection Using a Giant Magnetoresistance (GMR) Biosensing Portable Handheld Device. CVM research day Poster, 2017 (Best Poster Award).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: 4. Su, D., Wu, K., Krishna, V.D., Wang, J.P., Giant Magnetoresistance Based Handheld Platform for Rapid Detection of Influenza A Virus . 62nd Annual Conference on Magnetism and Magnetic Materials, November 6, 2017, Pittsburgh, PA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Su, D., Wu, K., Krishna, V.D., Klein, T., Perez, A.M., and Wang, J.P., A GMR Handheld Platform for One-Step and Rapid Biomarker Detection. Annual Conference and Retreat for the Institute for Engineering in Medicine, November 6th, 2017, Minneapolis, U.S. (Best Poster Award).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Su, D., Wu, K., Krishna, V.D. and Wang, J.P., Detection of Influenza A Virus in Nasal Swab Samples With A Wash-Free Magnetic Bioassay and A Handheld Giant Magnetoresistance Sensing System. Annual Conference and Retreat for the Institute for Engineering in Medicine, September 24th, 2018, Minneapolis, U.S.