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Differentiation of the Can F 1 Allergen in Hypoallergenic Dog Saliva Compared to Shedding Dog Saliva

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Differentiation of the Can F 1 Allergen in Hypoallergenic Dog Saliva Compared to Shedding Dog Saliva Murray State's Digital Commons Honors College Theses Honors College Spring 4-9-2021 Differentiation of the Can f 1 allergen in hypoallergenic dog saliva compared to shedding dog saliva Rose Miller Follow this and additional works at: https://digitalcommons.murraystate.edu/honorstheses Part of the Small or Companion Animal Medicine Commons, Veterinary Microbiology and Immunobiology Commons, and the Veterinary Preventive Medicine, Epidemiology, and Public Health Commons Recommended Citation Miller, Rose, "Differentiation of the Can f 1 allergen in hypoallergenic dog saliva compared to shedding dog saliva" (2021). Honors College Theses. 85. https://digitalcommons.murraystate.edu/honorstheses/85 This Thesis is brought to you for free and open access by the Honors College at Murray State's Digital Commons. It has been accepted for inclusion in Honors College Theses by an authorized administrator of Murray State's Digital Commons. For more information, please contact [email protected]. Murray State University Honors College Honors Thesis Certificate of Approval Differentiation of the Can f 1 allergen in hypoallergenic dog saliva compared to shedding dog saliva Rose Miller April 9, 2021 Approval to fulfill the requirements of HON 437 Dr. Kevin Miller, Professor Chemistry ______________________________ Dr. Laura Ken Hoffman, Professor Pre-Veterinary Medicine Approval to fulfill the Honors Thesis requirement Dr. Warren Edminster, Executive Director of the Murray State Honors Honors College Diploma Author: Rose Miller Project Title: Differentiation of the Can f 1 allergen in hypoallergenic dog saliva compared to shedding dog saliva Department: Veterinary Technology/Pre-Veterinary Medicine Date of Defense: April 9, 2021 Approval by Examining Committee ______________________________ ____________ (Dr. Kevin Miller, Co-Advisor) (Date) ______________________________ ____________ (Dr. Laura Ken Hoffman, Co-Advisor) (Date) ______________________________ ____________ (Dr. Chris Trzepacz, Committee Member) (Date) ______________________________ ____________ (Dr. Bill DeWees, Committee Member) (Date) Differentiation of the Can f 1 allergen in hypoallergenic dog saliva compared to shedding dog saliva Submitted in partial fulfillment Of the requirements For the Murray State University Honors Diploma Rose Miller April 9, 2021 Table of Contents Table of Contents…………………………………………………...……………………………...i List of Tables……………………………………………………………………………………..iii List of Figures……………………………………………………………………………………..v Acknowledgements……………………………………………………………………………....vii Abstract…………………………………………………………………………………………...ix I. Introduction……………………………………………………………………………1 A. Sub-problems……………………………………………………………………...7 B. Hypothesis…………………………………………………………………………7 C. Assumptions……………………………………………………………………….7 D. Limitations………………………………………………………………………...8 E. Definition of Terms……………………………………………………………......8 II. Methodology…………………………………………………………………………10 A. Purpose…………………………………………………………………………...10 B. Population and Sample…………………………………………………………...11 C. Pre-Experimental Survey………………………………………………………...12 D. Design of Research………………………………………………………….........13 E. Data Collection…………………………………………………………………...13 F. Data Analysis…………………………………………………………………….18 G. Statistical Analysis……………………………………………………………….19 i III. Results and Discussion..……………………………………………………………...20 A. Overview…………………………………………………………………………20 B. Survey Results…………………………………………………………………....20 C. Hair Follicle SDS-PAGE/Western Blot Results………………………………….21 D. Skin SDS-PAGE/Western Blot Results…………………………………………..22 E. Saliva SDS-PAGE/Western Blot Results………………………………………...23 F. Saliva ELISA Results…………………………………………………………….23 G. Discussion…………………………………………………………………..........35 IV. Conclusion…………………………………………………………………………...38 References………………………………………………………………………………………..39 Appendix A: IACUC Approval Form…………………………………………………………….44 Appendix B: IACUC Application..………………………………………………………………46 Appendix C: SDS-PAGE/Western Blot Results………………………………………………….58 Appendix D: Scatterplot of Linear Range from Can f 1 Control Sample…………………………61 Appendix E: Additional Tables of ELISA Analysis Results……………………………………...63 ii List of Tables Table 1 Individual Subject ELISA Data of Hypoallergenic Dog Group ………………….25 Table 2 Individual Subject ELISA Data of Shedding Dog Group……………………….....26 Table 3 Final ELISA Analysis of Can f 1 Concentration in Saliva by Breed of Hypoallergenic Dog Group……………………………………………………….27 Table 4 Final ELISA Analysis of Can f 1 Concentration in Saliva by Breed of Shedding Dog Group……………………………………………………………………………...27 Table 5 Plate 1 ELISA Raw Data for Can f 1 allergen control sample, S4 (Shedding), Archie (Shedding), Oliver (Hypoallergenic)………………………………………64 Table 6 Plate 2 ELISA Raw Data for Can f 1 allergen control sample, Amber (Shedding), Kash (Hypoallergenic), Louie (Hypoallergenic)…………………………………..68 Table 7 Plate 3 ELISA Raw Data for Can f 1 allergen control sample, Buttercup (Shedding), S2 (Shedding), Frasier (Hypoallergenic)………………………………………….72 Table 8 Plate 4 ELISA Raw Data for Can f 1 allergen control sample, S5 (Shedding), Willie (Shedding), Lynnie (Hypoallergenic)……………………………………...76 Table 9 Plate 5 ELISA Raw Data for Can f 1 allergen control sample, Harper (Shedding), Bentley (Hypoallergenic), Rue (Hypoallergenic)…………………………………80 Table 10 Plate 6 ELISA Raw Data for Can f 1 allergen control sample, Louie (Shedding), Gracie (Hypoallergenic), Max (Hypoallergenic)………………………………….84 iii Table 11 Plate 7 ELISA Raw Data for Can f 1 allergen control sample, S1 (Shedding), Dolly (Shedding), TJ (Hypoallergenic)……………………………………………88 Table 12 Plate 8 ELISA Raw Data for Can f 1 allergen control sample, S9 (Shedding), Gibson (Hypoallergenic)………………………………………………………….92 Table 13 Plate 9 ELISA Raw Data for Can f 1 allergen control sample, S3 (Shedding), H2 (Hypoallergenic), Buddy (Hypoallergenic)……………………………………96 Table 14 Plate 10 ELISA Raw Data for Can f 1 allergen control sample, Jenny (Shedding), H3 (Hypoallergenic), H4 (Hypoallergenic)……………………………………….99 Table 15 Plate 11 ELISA Raw Data for Can f 1 allergen control sample, S10 (Shedding), S7 (Shedding), H7 (Hypoallergenic)……………………………………………..103 Table 16 Plate 12 ELISA Raw Data for Can f 1 allergen control sample, S6 (Shedding), S2 (Shedding), H6 (Hypoallergenic)……………………………………………..107 Table 17 Plate 13 ELISA Raw Data for Can f 1 allergen control sample, S11 (Shedding), H1 (Hypoallergenic), H5 (Hypoallergenic)……………………………………...111 Table 18 Plate 14 ELISA Raw Data for Can f 1 allergen control sample, Juliet (Shedding), Joy (Hypoallergenic), Honey (Hypoallergenic)………………………………….115 Table 19 Plate 15 ELISA Raw Data for Can f 1 allergen control sample, Joe (Shedding), Smiley (Shedding), Stanley (Hypoallergenic)…………………………………...119 iv List of Figures Figure 1 The crystal structure of the Canis familiaris 1 lipocalin allergen ………………...1 Figure 2 Equation representing the pooled standard deviation of two data sets……………19 Figure 3 Equation representing the tcalculated value used to compare to the tcritical ………………...19 Figure 4 Plate 1 ELISA Analyzed Data for S4 (Shedding), Archie (Shedding), Oliver (Hypoallergenic) …………………………………………………………28 Figure 5 Plate 2 ELISA Analyzed Data for Amber (Shedding), Kash (Hypoallergenic), Louie (Hypoallergenic) ………………………………………………………….28 Figure 6 Plate 3 ELISA Analyzed Data for Buttercup (Shedding), S12 (Shedding), Frasier (Hypoallergenic) …………………………………………………………29 Figure 7 Plate 4 ELISA Analyzed Data for S5 (Shedding), Willie (Shedding), Lynnie (Hypoallergenic) ………………………………………………………...29 Figure 8 Plate 5 ELISA Analyzed Data for Harper (Shedding), Bentley (Hypoallergenic), Rue (Hypoallergenic) ……………………………………………………………30 Figure 9 Plate 6 ELISA Analyzed Data for Louie (Shedding), Gracie (Hypoallergenic), Max (Hypoallergenic) …………………………………………………………...30 Figure 10 Plate 7 ELISA Analyzed Data for S1 (Shedding), Dolly (Shedding), TJ (Hypoallergenic) ……………………………………………………………...31 Figure 11 Plate 8 ELISA Analyzed Data for S9 (Shedding), Gibson (Hypoallergenic) ……31 v Figure 12 Plate 9 ELISA Analyzed Data for S3 (Shedding), H2 (Hypoallergenic), Buddy (Hypoallergenic) …………………………………………………………32 Figure 13 Plate 10 ELISA Analyzed Data for Jenny (Shedding), H3 (Hypoallergenic), H4 (Hypoallergenic) ……………………………………………………………..32 Figure 14 Plate 11 ELISA Analyzed Data for S10 (Shedding), S7 (Shedding), H7 (Hypoallergenic) ……………………………………………………………..33 Figure 15 Plate 12 ELISA Analyzed Data for S6 (Shedding), S2 (Shedding), H6 (Hypoallergenic) ……………………………………………………………..33 Figure 16 Plate 13 ELISA Analyzed Data for S11 (Shedding), H1 (Hypoallergenic), H5 (Hypoallergenic) ……………………………………………………………..34 Figure 17 Plate 14 ELISA Analyzed Data for Juliet (Shedding), Joy (Hypoallergenic), Honey (Hypoallergenic) …………………………………………………………34 Figure 18 Plate 15 ELISA Analyzed Data for Joe (Shedding), Smiley (Shedding), Stanley (Hypoallergenic) ………………………………………………………...35 Figure 19 Can f 1 Allergen Control Sample Western Blot Results…………………………59 Figure 20 Hair Follicle Sample Western Blot Results from Buttercup Hoffman (Shedding); includes Can f 1 control sample…………………………………………………..59 Figure 21 Skin Excision Sample Western Blot Results from unknown euthanized patient at Westside Veterinary Clinic (Shedding); includes Can f 1 control sample………...60 Figure 22 Saliva Sample Western Blot Results from Louie Kamrud (Shedding); includes Can f 1 control sample…………………………………………………..60 Figure 23 ELISA Control Sample Curve Fitting for Linear Range Calculation; Plate 15 Example…………………………………………………………………62 vi Acknowledgements
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