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

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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

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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

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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

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

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

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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

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

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

Acknowledgements

First and foremost, I would like to send my thanks to my two mentors, Dr. Kevin Miller and Dr. Laura Hoffman, for walking with me through the entire process. Dr. Miller opened the door for me to learn from him, and I cannot express enough how thankful I am to have had such an incredible research mentor. His willingness to guide me through research gives me confidence that that will be felt for years to come. He has been so important to me as a mentor in and out of the lab, and I am so incredibly grateful that he has persevered with me through thick and thin. I am also especially thankful for his kiddos, Joshua and Adelyn, and the joy they brought in my final year at Murray State, and his wife, Christine, for her willingness to open her home to me.

Dr. Hoffman has been helping me through my journey in veterinary medicine since the beginning, and I could not have imagined the process without her positivity and support. Had she not been so optimistic about this research idea when I approached her my freshman year, I would not be writing this today. I always knew I could approach her when I needed encouragement, and she always brought a smile to my face while helping me through the rough patches of research. I truly could not have asked for a better pair of mentors to encourage and challenge me as a student.

Second, I would like to thank my remaining committee members, Dr. Chris Trzepacz and

Dr. Bill DeWees. Dr. Trzepacz was instrumental in the research process, as he allowed me to use his lab for a year and stuck with me through all the failed attempts and brainstormed new testing ideas with me after hours. I gained countless technical laboratory skills during my time in Dr.

Trzepacz’s lab, which is all due to his sacrifice to guide me through various testing processes.

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Dr. DeWees was not only my professor, but became my family away from home, and I cannot thank him and his wife, Luane, enough for being there for me through the rough patches of the past four years, and for celebrating with me when I had exciting news. Even though he may not have offered any technical help in the research process, his support was priceless. Dr. DeWees and

Luane will always be family to me.

I would be remiss not to mention the people who allowed me to collect samples from their pups. I would not have learned as much as I did from this project had it not been for them.

I would also like to thank my dear friends and peers who stuck with me through this process and listened to me when I needed to talk things through. Thanks to my best friend, Kathryn

Kamrud, who was so patient with me during the times when all I could talk about was the poor results when the project first started. Thanks to Cristen Shaw, who offered me guidance from her first-hand experience as an undergraduate researcher, as well as her loving support from day one.

Thanks to Katey Lindenmeyer, who walked alongside me as we both went through the ups and downs of research. I could not have asked for better friends throughout this experience.

Thank you to my mom, who read countless rough drafts of abstracts, application essays, letters, etc. Her support, as well as my dad’s, mean the world to me, and I am so incredibly thankful to have such loving parents. Day after day, they continuously teach me more about the Lord and push me to be more like Christ. Their support and encouragement were instrumental in helping me see God’s plan for my future, and I am so grateful to Him for using them as vessels to teach me and train me to love and encourage those around me. All the glory to Him!

Finally, I would also like to extend my appreciation to my dear friend, Kristin Anderson, for this research idea in the first place. Never would I have thought that a Spanish Education major would have suggested a research idea that led to a future career.

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Abstract

Dog saliva contains a large variety of proteins, each with specific functions throughout the body. While some aid in digestion, others provide immune support for the body. In the case of human dog allergies, dog skin dander has been proven to cause allergic reactions to those who are sensitive to the allergens in the dogs. However, past studies have shown a difference in allergic reaction to specific breeds, breaking dog breeds into two main categories: hypoallergenic and non- hypoallergenic. Within these groups, there are different hypoallergenicity levels, but overall, the hypoallergenic dog group does not cause an allergic reaction, whereas non-hypoallergenic, or shedding dogs, do. Because all dogs in both groups shed dander from their skin, the allergic reaction must be catalyzed by a different physiological substance, one that can be potentially paired with the dogs’ shedding or lack thereof. The Can f 1 allergen, the most abundant allergen in canines, is suspected to initiate allergic reactions in humans. In this study, the allergens specific to the saliva in each dog group were compared via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot tests, and enzyme-linked immunosorbent assay

(ELISA). In preliminary testing using SDS-PAGE and Western blot technology, the saliva samples were homogenized in a buffer solution and, with the use of an electric current, run through a gel made to separate specific proteins. The Western blot test pins a specific protein or allergen needed with the use of the corresponding allergen’s antibody. To quantify the concentration of the Can f

1 allergen in each sample, the Can f 1 allergen was isolated from dogs in each group and quantified to indicate the presence of the allergen in the saliva via ELISA. In these tests, each saliva sample was placed in the microtiter plate in individual wells and serially diluted. After the addition of a

ix primary and secondary antibody, the amount of the Can f 1 allergen in each sample was quantified and comparatively analyzed to saliva samples in both dog groups. The testing process was hypothesized to yield results of a higher Can f 1 allergen in the saliva of dogs in the shedding group, and lower Can f 1 allergen in the saliva of dogs in the hypoallergenic group. The results displayed inconclusive evidence regarding the correlation between the Can f 1 allergen concentration in dog saliva to human allergies due to the small sample pool and high variability in dog breeds. With these results, further studies can be conducted to neaten the testing process and advance the current treatment for human allergies or prevent reactions via combating the source.

I. Introduction

Saliva not only plays an important role in digestion in most animals, but also carries significance in numerous systems throughout the body. Particularly in canines, there are over 2,000 proteins present in saliva, some of which aid in immune protection and others in antimicrobial mechanisms. Saliva as a whole has a primary responsibility of protecting the oral cavity, and in turn the rest of the body, against infection. [1] Along with endogenous peptides, each protein present in saliva has a specific role to play in the body. When focusing on the Canis familiaris 1

(Can f 1) allergen, that role is still unknown.

Figure 1: The crystal structure of the Canis familiaris 1 lipocalin allergen. [2]

The Can f 1 allergen, a ligand-binding protein produced by tongue epithelial cells, is most present in saliva but can be found in other areas throughout a dog’s body, such as the dander and hair follicles. [3] It has been shown that households with residential dogs have high levels of the

Can f 1 allergen in the environment, which is most often the cause of canine-specific allergic

1 reactions in humans. [4] Allergens present in the saliva, dander, hair follicles, and other areas of the body are released into the environment, which is then agitated by movement and causes an allergic reaction to humans with sensitivities to these allergens. A study by Ramadour showed that the Can f 1 allergen found in the hair shaft of dog subjects varies not by breed, hormonal status, and hair length, but by gender and skin type. Dogs within the same breed showed great variability of the Can f 1 allergen concentration and had similar averages when compared to other breeds.

However, when comparing different breeds, there was no significant variability. Similarly, when comparing male versus female results, subjects in the same breed showed no difference, but when comparing overall average male concentration to female concentration, males had more of the allergen found in their hair shafts. Hormone levels measured by the castration or intact status of subjects suggested there was no effect on the Can f 1 concentration. In the interest of dogs with seborrheic, or dry, flaky skin, the Can f 1 allergen increased compared to those without seborrheic skin. [5] Although this study attempted to correlate the Can f 1 allergen with specific breeds, along with the previously mentioned variables, it did not focus on the allergen at the source, which is the saliva.

The hair follicle is the base of every hair that grows in a mammal’s dermal and epidermal layers of the skin. Each follicle goes through a process of regeneration and growth as the hair grows. In each follicle, there are ten layers of differing cells that each have a different purpose in protecting the follicle, aiding growth, and preventing decay. However, every hair follicle has one main ingredient: keratinocytes. During the growth phase of each follicle (anagen), keratinocytes differentiate between the layers of the follicle. In one specific zone in the hair follicle, known as the hair matrix zone, rapid proliferation occurs in the keratinocytes and hair growth is induced.

Through this, an area referred to as the dermal papilla is surrounded by keratinocytes, which is an

2 essential stimulus for both hair growth and hair follicle induction. In this extensive process of follicle formation, growth, and hair growth, proteins are heavily involved. In the hair growth itself, proteins interact with keratin to form a matrix from which hair grows. The cuticle cells that surround this matrix are flattened, and the hair is rooted in the follicle and grows upward through the epidermal and dermal layer of the skin. [6]

The integumentary system, consisting of skin, hair, nails, and exocrine glands, is a highly protective organ of the body. The skin contains three layers: epidermis, dermis, and hypodermis.

The epidermis, the thickest layer of skin, incorporates several types of cells, including keratinocytes, melanocytes, Langerhans’ cells, and Merkel’s cells. Keratinocytes are responsible for the secretion of lipids, which forms the water barrier in the skin, and the production of keratin.

Melanocytes are responsible primarily for the pigmentation of the skin, caused by the production of melanin. Langerhans’ Cells, also known as dendritic cells, are vital for the role of antigen presentation, and are responsible in defending the body against intruders. Merkel Cells, bound with keratinocytes, are mainly found in areas of the body responsible for the sense of touch, such as the fingertips and oral area. The dermis, separated into the papillary layer and the reticular layer, contains hair follicles, along with several other areas incorporated in the skin. The hypodermis, also called the subcutaneous fascia, may also contain hair follicles. [7] Pet dander is commonly found in dust samples in homes with pets and has been known to provoke an allergic reaction in humans. [8,9]

The canine species has countless derivative breeds that differ significantly. These breeds may differ in size, color, behavior, and shedding. The latter seems to be the catalyst for many humans’ allergic reactions to canines. Dogs will shed their old or damaged hair and replace them with newer hair. [10] However, there is a universal grouping of dogs that have not provoked an

3 allergic reaction and they are referred to as hypoallergenic. These dogs have a significantly lower rate of shedding compared to those considered non-hypoallergenic. Many research studies have proven that human allergic reaction is caused by the dander, or dead skin, that enters the environment surrounding the dog. One particular study isolated specific protein (lipocalin) allergens and combined each allergen to form a multi-allergen, which was then used to diagnose and vaccinate for dog allergies. This breakthrough opens the door for further diagnoses and vaccinations for human patients experiencing dog allergies, or other animal-specific allergies. [11]

However, the issue with this study is that they imply that, since all dogs contain some sort of lipocalin allergen, they all can cause human allergies. Based on previous medical cases, dog allergies in humans are highly breed specific.

A second study took this evidence and attempted to measure the levels of lipocalin allergens in different dog breeds for the purpose of understanding why humans with dog allergies only react to specific breeds. After extracting allergens from both the hypoallergenic and non- hypoallergenic dog group, a conclusion was drawn that the two dog groups had no differing types of proteins present that could account for the difference in breed-specific allergic reactions in humans. [12] Thus, if the allergens in both hypoallergenic and non-hypoallergenic dogs do not differ, then dog-allergic individuals are at a loss to understand why their allergies are breed- specific.

The unknown that is still present is the lack of understanding why some breeds cause an allergic reaction in humans and some do not. Dog breeding groups associate the hypoallergenic dog group with dogs that do not shed or shed minimally, and the non-hypoallergenic dog group with dogs that shed a typical amount with the changing seasons. However, it has not been proven that the presence of shedding in a dog is the primary cause for an allergic reaction in humans.

Thus, a study must be conducted to understand if these two dog groups are appropriately described as hypoallergenic and non-hypoallergenic due to their appropriate shedding rates. In this study, it is hypothesized that dogs that typically do not provoke an allergic reaction, grouped together as hypoallergenic, have lower levels of the Can f 1 allergen present in its saliva compared to non- hypoallergenic, or shedding dogs. As dogs groom their bodies, they spread the Can f 1 allergen from their saliva in their mouths to the rest of their body. Dogs that typically shed will spread the allergen with their hair in their surrounding environment, subsequently causing a higher likelihood of humans experiencing an allergic reaction.

There are many different ways to go about testing via gel electrophoresis; this study will focus on the proteins, which narrows the testing down to three different types: SDS-PAGE, IEF, and 2-D PAGE. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) will be utilized in this project because, unlike the other two routes of testing, SDS-PAGE measures the molecular weights of the different proteins run through the gel. Although it will not specify the exact types of proteins present in the sample, proteins will be grouped into bands in the gel according to their relative molecular weight. This allows for other samples to be compared according to the two groups’ differing proteins, or lack of proteins, coherent with the weights of the protein groups. [13] Utilizing SDS-PAGE will allow for confirmation of the presence of the

Can f 1 allergen in each saliva sample. The molecular weight of the natural Can f 1 allergen was found to be 20 kDa (kilodaltons) under SDS-PAGE reducing conditions. [14] As a baseline level of the allergen cannot be used, quantification is not possible using gel electrophoresis.

It has been found that the allergen(s) that often cause an allergic reaction have a low molecular weight. The Can d 1 and Can f 1 allergens are the two most common allergens to provoke a reaction in human patients. However, much like the previously mentioned study, another

5 study found that these two allergens were equally present in homes with hypoallergenic and shedding dogs. [15] However, these allergens initially came from the dander from the skin, which seems to have no correlation with the rate of shedding in which each breed differs.

To allow for a more quantitative result, a monoclonal enzyme-linked immunosorbent assay

(ELISA) will be conducted on each saliva sample. Each saliva sample, along with a pure Can f 1 allergen sample, will be loaded onto the ELISA plate and immobilized, allowing for a primary and secondary antigen to be added to each well. Through this process of Can f 1-specific antigens flagging the allergen in each sample, quantification of the Can f 1 allergen in each sample can be calculated from the optical density and compared to samples in the contrasting group.

This research project, although not curative or preventative for allergies, will lead to further studies that show the reasoning behind breed-specific allergic reactions in humans. The hypothesis aims to answer this specific problem, rather than provide human patients with a solution to their allergic reactions. However, with the conclusion from this project, doctors will be able to further understand what initially causes a reaction in their patients, and thus how to go about treatment.

The hypothesis of this study is this: hypoallergenic dogs will have lower levels of the Can f 1 allergen present in their saliva, whereas shedding dogs will have higher levels of the Can f 1 allergen. This can be expounded upon in that, because hypoallergenic dogs cause little or no reaction to those with a sensitivity to canine allergens, and because the Can f 1 allergen has been proven to be a primary cause of canine-specific allergies in humans, dog breeds that typically cause an allergic reaction in humans will have more allergen present to provoke the reaction.

Sub-problems

1. The issue of potential difference in the concentration of the Can f 1 allergen in

different dog breeds leads to the conclusion of determining if there is such a thing

as a hypoallergenic dog breed. This can be quantified from an ELISA test.

2. Variance of the Can f 1 allergen concentration within dog groups may lead to a

conclusion of if the Can f 1 allergen is truly the cause of a majority of allergic

reactions in humans.

3. The correlation between the frequency of allergic reactions in humans and dog

breeds may further support the connection between the Can f 1 allergen

concentration in saliva between different dog breeds.

Hypothesis

0 (Null): There is no significant correlation between breed of dog and the concentration of the Can f 1 allergen in saliva.

1 (Research hypothesis): Dog breeds defined as hypoallergenic by the American

Kennel Club have significantly less Can f 1 allergen in their saliva compared to non- hypoallergenic dogs.

Assumptions

1. There is little significant variance of the concentration of the Can f 1 allergen in

subjects’ saliva throughout the day.

2. Subject size does not affect the Can f 1 allergen concentration in saliva due to the

increased saliva production to compensate for increased body surface area.

3. Diet has little to no effect on Can f 1 allergen concentration in saliva.

4. Owners were truthful in the breed history of their dogs; thus the classification of

the dog subjects was accurate and appropriate.

Limitations

1. This study will not attempt to define what dog breeds explicitly cause human

allergic reactions and which breeds do not.

2. This study will not attempt to explicitly define what constitutes a hypoallergenic

dog breed.

3. This study will not separate the Can f 1 allergen from any other allergen that may

or may not cause allergic reactions in humans.

Definition of Terms

1. Can f 1 allergen: refers to the primary allergen that is known to “induce histamine

release from blood basophils” in dog-allergic human patients. The Can f 1 allergen

is produced in the tongue epithelial tissue of canines. [16]

2. Hair follicle: follicles go through a life cycle including anagen, catagen, and

telogen. These three stages are hypothesized to promote different amounts of

protein expression. [17]

3. Skin excision: skin, a part of the integumentary system, contains three layers, the

epidermis, the dermis, and the hypodermis. The hypodermis is the layer closest to

the body and contains hair follicles, along with sensory neurons and blood vessels.

[7] When excising a sample of skin, for surgical reasons or other, all three layers

are removed to provide wide margins of the sample obtained. [18]

4. Saliva: saliva contains proteins and endogenous peptides that aid in immune

support and digestion and serve as antimicrobial protection. [1] Dog saliva is a

primary source involved in allergic immune responses and has “IgE-binding protein

profiles” that vary in each dog. [19]

5. SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis “provides

an easy way to estimate the number of polypeptides in a sample and thus assess the

complexity of the sample or the purity of a preparation”, as well as “allows samples

from different sources to be compared for protein content”. [13]

6. Western blot: these tests “identify specific proteins from a complex mixture of

proteins extracted from cells”, [20] and more recently, have the ability of

“quantitative interpretation of western blot data in terms of fold changes in protein

expression between samples”. [21]

7. ELISA: enzyme-linked immunosorbent assays focus on “specific antigen-antibody

interactions” which are “recognized by assaying an enzyme label conjugated to one

reactant, usually antibody”. [22]

II. Methodology

A. Purpose

The topic of species-specific allergies in humans has been of interest in the medical field for decades. Dog and cat allergies affect 10-20% of people worldwide, and as this statistic steadily increases, more research is being conducted to find different ways to prevent or treat these allergic reactions. [23] In the United States alone, the cost in prevention in households with people who suffer from acute asthma from dog allergies is estimated to add up to $500 million to $1 billion.

[24] There is evidence that suggests that dogs who are known to not cause an allergic response in dog-sensitive individuals have a genetic mutation, but it is unknown how this mutation affects the production of the Can f 1 allergen; or, more specifically, if the structure of the protein itself is altered, or if the amount of the protein is merely less in dogs that are considered hypoallergenic.

[15] The purpose of this study was to test the hypothesis that the saliva of hypoallergenic dogs, or dogs that are known to cause less of an allergic reaction in humans, contains a higher concentration of the Can f 1 allergen compared to shedding dogs, or dogs that do cause more of an allergic reaction in humans. The objectives of this study were:

1. To first confirm the presence, or lack thereof, of the Can f 1 allergen in different

samples, including hair follicles, skin, and saliva via sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot testing.

2. To quantify the levels of the Can f 1 allergen in dog hair follicles, skin, and saliva

and compare between hypoallergenic and shedding dogs via SDS-PAGE, Western

blot, and enzyme-linked immunosorbent assay (ELISA).

3. To determine if there is a statistically significant difference in the levels of the Can

f 1 allergen in the saliva of hypoallergenic dogs compared to shedding dogs.

B. Population and Sample

A total of forty-eight privately-owned dogs were used in this study: two for the hair follicle testing, two for the skin testing, and forty-four for the saliva ELISA testing. Subjects were recruited through Murray State University’s Veterinary Technology/Pre-Veterinary Program and Club, private practice veterinary clinics in Murray, KY and St. Louis, MO, and personal connections.

All subjects were up to date on their rabies vaccine as required by the state, and had no chief complaints pertaining to their physical health. All methods were carried out in accordance with the approval from the Institutional Animal Care and Use Committee (IACUC), and all efforts were made to minimize suffering.

During the hair follicle collection procedure, a hemostat was used to grasp roughly 10-15 hairs at the base and the follicle was extracted. This procedure was repeated three more times to increase the chance that viable hair follicle samples were collected. Samples were placed in a labeled container and immediately placed on dry ice and held at roughly -78°C for transport.

Subjects were monitored for 10 minutes after sample collection for any irritation or discomfort.

All samples were placed in a freezer held at -80°C until the testing process began.

Two privately-owned dogs were utilized for skin excision samples. For reasons other than this study, the subjects were euthanized humanely. Owners of the subjects previously voiced their

11 desire for the veterinary clinic to dispose of their pet as they saw fit. A licensed veterinarian performed a procedure of removing a 50 cm2 sample from the caudal dorsal area toward the base of the tail. Both samples included all three layers of the skin, as alluded to in the introduction.

The Salimetrics SalivaBio’s Children’s Swab (SCS) System has features capable of collecting animal saliva. The SCS System features a 125 mm swab, 8 mm in diameter, which is able to be held in the subjects’ mouths with no risk of choking. Each subject was first allowed to smell a treat to provoke salivation, then one end of the swab was held in their mouth for 15-30 seconds. The swab was flipped, and the collection process was repeated. The volume of saliva recovered ranged from 200-1000 µL. After sample collection was complete, the swab was placed in its corresponding container and equipped with a center container allowing for isolation of the product through centrifugation. Sample tubes were placed in the LW Scientific Combo-V24

Centrifuge and spun at 2000 rpm for 10 minutes, then placed directly on dry ice and held at roughly

-78°C for transport and storage until the testing process began.

C. Pre-Experimental Survey

Fifty-eight individuals with self-identified canine-specific allergies were asked to take an optional survey regarding their allergy severity and specific dog breeds to which they find themselves allergic. Individuals involved in the survey had no relation to the dog subjects, and surveyors were recruited through social media advertisement and personal connections. The survey was distributed through email which included a Google Drive link.

D. Design of Research

Sample collection of hair follicles, skin, and salvia was first conducted and analyzed via

SDS-PAGE and Western blot tests. Categorization of each dog subject was based on the breed of each subject as understood by the owner, as well as secondary variables such as shedding rate and personal experience with allergic reactions, or lack thereof, from each subject. Saliva samples were then analyzed via an ELISA test to quantify the concentration of the Can f 1 allergen in each sample and compared to subjects of the opposing dog group to determine if there is a statistically significant difference in concentration of the Can f 1 allergen in the saliva of hypoallergenic dogs versus shedding dogs.

E. Data Collection

1. Control Can f 1 SDS-PAGE Sample Preparation

In preparation of the positive control sample, a 1X Laemmli Sample Buffer was first made by diluting 4X Sample Buffer (TruPAGE LDS Sample Buffer; 40% w/v Glycerol, 4% w/v Lithium

Dodecyl Sulfate (LDS), 4% Ficoll 400, 0.025% Phenol Red, 0.025% Brilliant Blue G250, 2 mM

EDTA) (1.25 µL) with DI H₂O (5 µL) in a microcentrifuge tube, then a recombinant dog Can f 1 sample (RayBiotech Recombinant Dog Can f 1; 0.3 mg/mL) (0.75 µg) was added. The solution was mixed until fully combined, then stored on ice until use.

2. Hair Follicle SDS-PAGE Sample Preparation

Before running the SDS-PAGE, hair samples were examined for complete removal of the hair follicle. The hair shafts were cut from roughly 2-5 mm at the base to isolate the hair follicles.

Forty hair follicles were homogenized in 1X Sample Buffer (200 µL) then shaken at 4 ℃ for 1 hour. Remaining hair strands were then removed from sample tubes before centrifuging and the

13 microcentrifuge tubes were centrifuged at 10,000 rpm, 4 ℃ for an additional 1 hour. The supernatant was then decanted into a separate container and the samples were heated to 100℃ for

90 seconds to denature the protein, then chilled on ice until use.

3. Skin SDS-PAGE Sample Preparation

Skin samples were stored at -20 ℃ until use. Before thawing, the hair shafts were shaved at the base of the hair. The hypodermis, or the loose connective tissue, was removed along with the lipid layer in the sample. A final yield of 220 mg of skin was obtained, and measurements of the follicle depth (1 mm) and dermis depth (3 mm) were taken. The skin sample was cut into pieces

(approximately 3 mm3) and placed in a microcentrifuge tube along with 1X Sample Buffer 1100

µL). The sample tube was heated to 100 ℃ for 20 minutes to denature the proteins, and intermittently vortexed to break up skin samples within the buffer. After the denaturing process was complete, the sample tube was centrifuged at 10,000 rpm for 20 minutes to allow for the skin proteins to form a pellet and the fat to float in the supernatant. While the sample was cooling to room temperature, 1X Sample Buffer (20 µL) was added to 10 empty microcentrifuge tubes. The cooled supernatant was decanted, and the pellet containing denatured skin proteins was weighed

(2000 µg). To form an accurately diluted sample with a known concentration (100 µg/µL), 1X

Sample Buffer (20 µL) was added and combined with the pellet. 1X Sample Buffer (20 µL) was added to the tube to dilute the sample in half. This diluent (20 µL) was added to the next sample tube containing Sample Buffer to dilute this solution by half. Serial dilutions were done to the four remaining microcentrifuge tubes. Each diluted sample (10 µL) was added to their respective wells in the gel.

4. Saliva SDS-PAGE Sample Preparation

Each saliva sample (15 µL) was diluted with 1X Sample Buffer (15 µL) in microcentrifuge tubes and serially diluted seven times. The saliva sample tubes were heated to 100 ℃ for 10 minutes to denature the proteins, then chilled on ice until use.

5. SDS-PAGE Procedure

1X Running Buffer was prepared by diluting 20X Running Buffer (TruPAGE TEA-Tricine

SDS Running Buffer; 1.2 M Triethanolamine, 0.8 M Tricine, 2.0% w/v Sodium Dodecyl Sulfate

(SDS)) (10 mL) with DI H₂O (190 mL). A pre-poured gel (Sigma-Aldrich TruPAGE Precast Gels

10%) was placed into the apparatus and submerged in 1X Running Buffer. The samples were then loaded into the gel in wells 3-8 and 10-15 in the Mighty Small II SE 260 Mini-Vertical Gel

Electrophoresis Unit. A protein ladder (ThermoFisher Spectra Multicolor Broad Range Protein

Ladder; Amersham ECL Rainbow Marker Full Range) (15 µL) was loaded into wells 2, 9, and 16.

Blank samples of 1X Sample Buffer were loaded into wells 1 and 17 and the gel was run at 100 V

(35 MA) for 1 hour.

6. Western Blot Procedure

The gradient gel was removed from the apparatus and blotted to a nitrocellulose membrane

(Novex by Life Technologies Nitrocellulose/Filter Paper 0.2 µm). 1X Transfer Buffer was prepared by diluting 20X Transfer Buffer (TruPAGE Transfer Buffer; 0.25M Trizma Base, 1.92

M Glycine) (10 mL) with DI H₂O (190 mL). The Western blot sponges were hydrated with 1X

Transfer Buffer and placed in the sandwich along with the Western blot membrane and SDS-

PAGE gel, then fit into the transfer apparatus (Life Technologies Mini Gel Tank). The apparatus was filled with 1X Transfer Buffer and ran at 25 V for 1 hour. A 1X Tris-Buffered Saline 0.1%

Tween 20 (TBS-T) solution was made by diluting 10X TBS (200 mM Tris Base, 1500 mM NaCl)

(100 mL) in DI H2O (900 mL) and adding Tween (Sigma Aldrich Tween 20) (1 mL). In a small beaker, nonfat instant dry milk (2.5 g) was dissolved in the 1X TBS-T solution (50 mL) to make a

5% nonfat dry milk blocking solution. The blocking solution (5 mL) was placed in a centrifuge tube along with the western blot membrane with the transfer side facing inward. The tube was rolled for 30 minutes. A primary polyclonal rabbit anti-Can f 1 antibody (RayBiotech Rabbit anti-

Can f 1 antibody) (100 µL) was added to the centrifuge tube to make a 0.2 µg/µL primary antibody solution in a 1:1000 solution. The tube was rolled for 1 hour. The membrane was removed from the tube and washed with TBS-T three times for 5 minutes each rinse. Then, the nonfat milk blocking solution (5 mL) was added to a centrifuge tube along with a horseradish peroxidase

(HRP) conjugated goat anti-rabbit antibody (Jackson Laboratories Peroxidase AffiniPure Goat

Anti-Rabbit IgG) (0.5 µL), creating a 1:10,000 solution. The membrane was placed in the tube and rolled for 1 hour, then from the tube and washed with TBS-T three times for 5 minutes each rinse.

Finally, the membrane was developed by adding 1:1 chemiluminescent detection reagents

(Amersham ECL start Western blotting Detection Reagent) and placed in a dark chamber

(Amersham Imager 600) with a 2 minute exposure.

7. ELISA Procedure

Polystyrene microtiter plates (NUNC Maxisorp flat-bottom 96 well plate) were coated with a monoclonal antibody 10D4 (INDOOR biotechnologies Can f 1 ELISA kit (10D4/CF1); 10

µL/mL) in a 50 mM carbonate-bicarbonate buffer (pH 9.6) (100 µL) and left overnight at 4 ℃. A

Dulbecco’s Phosphate Buffered Saline-0.05% Tween 20, pH 7.4 (PBS-T) solution was prepared by adding Tween (250 µL) to 1X PBS (130 mmol Sodium Chloride, 2.7 mmol Potassium Chloride,

10 mmol Sodium Phosphate, 1.8 mmol Potassium Phosphate) (500 mL). The wells were washed three times with PBS-T. A 1% Bovine Serum Albumin (BSA), PBS-T solution was prepared by

16 dissolving BSA (Fisher Bioreagents Bovine Serum Albumin Fraction V) (2.5 g) in PBS-T (50 mL). The plate was incubated for 30 minutes at room temperature with 1% BSA, PBS-T (100 µL per well), then washed three times with PBS-T (250 µL). A control curve was used, ranging from

250-0.5ng/mL Can f 1 using doubling dilutions of the Universal Allergen Standard (UAS)

(INDOOR biotechnologies Can f 1 ELISA kit 10D4/CF1). To prepare the standard, UAS (20 µL) was pipetted into 1% BSA, PBS-T (180 µL) and placed in wells A1 and B1 on the microtiter plate.

Nine serial dilutions were performed by transferring the UAS diluent (100 µL) across the plate into 1% BSA, PBS-T diluent (100 µL). Wells A11, B11, A12, and B12 contained only 1% BSA,

PBS-T (100 µL) as blanks.

To prepare the starting dilution of each saliva sample, a recently thawed saliva sample (5

µL) was pipetted into 1% BSA, PBS-T (495 µL) to create a 1:100 dilution. This saliva diluent (100

µL) was pipetted into 1% BSA, PBS-T (100 µL) in column A of the sample’s corresponding row to create a 1:200 dilution. Each sample dilution was duplicated to allow for accurate results; thus, the first sample was placed in rows C-D, the second sample in E-F, and the third sample in G-H.

Eleven serial dilutions were performed in each row by transferring the saliva diluent (100 µL) across the plate into 1% BSA, PBS-T (100 µL). The entire plate was incubated at room temperature for 1 hour. The wells were washed three times with PBS-T (250 µL), then each well was coated in a polyclonal rabbit anti-Can f 1 antibody (INDOOR biotechnologies Can f 1 ELISA kit 10D4/CF1;

10 µL/10 mL) in 1% BSA, PBS-T (100 µL) and incubated at room temperature for an additional hour. The wells were then washed three times with PBS-T (250 µL), and an HRP conjugated goat anti-rabbit antibody (Thermo Fisher Scientific Invitrogen Goat anti-Rabbit IgG; 10 µL/10 mL) in

1% BSA, PBS-T (100 µL) was added to each well. The plate was incubated at room temperature for another hour. The wells were washed three times with PBS-T (250 µL), then the assays were

17 developed by adding water- soluble peroxidase substrate developing solution (Thermo Fisher

Scientific 1-Step ABTS Substrate Solution) (150 µL). The plate was allowed to develop for 20 minutes. 1% SDS stop solution (Thermo Scientific Sodium Dodecyl Sulfate, Lauryl) (100 µL) was added to each well after developing. The plate was read on the Western Blot Imager (Molecular

Devices SpectraMax i3x) when the absorbance at 405 nm reached 2.0-2.4.

F. Data Analysis

All objective data was analyzed on SoftMax Pro 7.1, then comparatively analyzed on

Microsoft Excel using the three-variable exponential equation given by the initial analysis program. This equation (y=A+B*(1-e-x/C) was utilized to form a scatterplot, which allowed for the calculation of the linear range of each ELISA plate using the standard sample, as seen in Figure

21. Subjective data, including the categorization of each dog subject, was done by referencing the

American Kennel Club’s classification of each breed as hypoallergenic or non-hypoallergenic. For the subjects with an unknown breed, their categorization was based upon the owner’s given history of the dogs rather than the specific genetic history.

G. Statistical Analysis

Comparisons between the two dog groups’ values for the Can f 1 saliva concentration were

done using a dependent two tailed sample t-test. Microsoft Excel was used to calculate the tcalculated

value and comparatively analyzed with the tcritical value using a confidence interval of 95% and forty-

two degrees of freedom. The following equations were used to determine tcalculated:

Figure 2. Equation representing the pooled standard deviation of two data sets

Figure 3. Equation representing the tcalculated value used to compare to the tcritical

III. Results and Discussion

A. Overview

Forty-four canine subjects participated in this study, twenty-two of which were categorized as hypoallergenic and the other twenty-two as shedding. Of the hypoallergenic dogs, a variety of breeds were represented: Goldendoodle, Shih Tzu, Maltese, Poodle, Labradoodle, Havanese,

Miniature Schnauzer, Terrier Mix, and Yorkshire Terrier. Of the shedding dogs, a variety of breeds were represented: Terrier Mix, German Shepherd, Labrador Retriever, Pit Bull, Coonhound,

Beagle, Husky, Bullmastiff, Weimaraner, Great Pyrenees, and Newfoundland. Of all forty-four subjects, eleven were breeds that were listed as unknown due to the unknown breed history and ownership of the dog. These eleven subjects were patients at Westside Veterinary Service. Of all forty-four subjects, twenty were breeds with a genetic history that was not fully known by the owner, thus causing discrepancy with the categorization of the subjects’ respective groups. For these subjects, classification was done based on owner’s accounts of shedding rate and personal experience with allergies and their interaction with their dog.

B. Survey Results

With the purpose of correlating breed-specific allergies in humans with the concentration of the Can f 1 allergen in different dog breeds, a survey was initially conducted for individuals with a known canine allergy. When asked how severe, in their opinion, their allergies were gauged on a scale from 1 to 10 and the majority of participants answered 8. Out of 58 responses, 15 said

20 their allergies were minor (1-3), 18 said their allergies were moderate (4-6), and 25 said their allergies were severe (7-10). When asked what symptoms they commonly experience during an allergic reaction, the majority of participants answered itchy eyes. Out of 54 responses, 37 said they experience red eyes, 48 said they experience itchy eyes, 45 said they experience a runny nose,

47 said they experience sneezing, 8 said they experience their throat swelling, 11 said they experience an itchy tongue, 14 said they experience hives, 3 said they experience no symptoms, 2 said they experience coughing, 1 said they experience nausea, 1 said they experience eczema flare- ups, 1 said they experience red and itchy skin, 1 said they experience trouble breathing, 1 said they experience their eyes swelling shut, and 1 said they experience an itchy chest and discomfort in their lungs. When asked what other animals, if any, they also experience allergic reactions to, the majority of participants answered cats. Out of 58 responses, 39 said cats, 11 said rabbits, 7 said rodents, 19 said any animal with fur, 5 said horses, 2 said farm animals, 1 said birds and 1 said they did nott know. Nine participants said they did not have any other allergies. When asked to list specific dog breeds they know they experience allergic reactions to, the majority of participants answered Golden Retriever. Out of 53 responses, 43 answered Golden Retriever, 39 answered

Labrador Retriever, 20 answered Pit Bull, 36 answered Husky, 38 answered German Shepherd, 24 answered Hound, 21 answered Dachshund, 13 answered Labradoodle, 10 answered Poodle, 11 answered Bishon Frise, 19 answered Maltese, 14 answered Havanese, 15 answered Schnauzer, 14 answered Shih Tzu, 2 answered any dog breed that sheds, and 4 answered none.

C. Hair Follicle SDS-PAGE/Western Blot Results

In order to confirm the presence, or lack thereof, of the Can f 1 allergen in different samples, including hair follicles, skin, and saliva, an SDS-PAGE was performed. The use of a pure

Can f 1 allergen sample as a control in each gel allowed for confidence that the process was done correctly. Separation of the protein ladders also allowed for secondary confirmation of success, as the ladder included colored protein samples that were separated out at their respective molecular weights. The Can f 1 allergen was expected to separate at 20 kDa.

The Western blot transfer allowed for the samples in the gel to be visualized after the addition of a two-step chemiluminescent developer. The exposure time was increased to two minutes after an absence of bands in the expected area of sample. Figures 1-4 display the image of the Western blot membrane after exposure in the dark chamber. The control test, consisting of only pure Can f 1 allergen which was serially diluted, showed unknown separation in areas before the expected separation at 20 kDa, seen in Figure 1. However, there were strong bands at 20 kDa, thus the testing process of the hair follicle, skin, and saliva samples proceeded.

Based on previous literature showing the presence of the Can f 1 allergen on hair shafts, as mentioned in the Introduction, the Western blot that contained hair follicle samples had an expected result of bands at 20 kDa. However, after prolonged exposure in the dark chamber, no bands were seen in the areas where the hair follicle sample was placed. Minimal presence of the control sample was seen, as shown in Figure 2.

D. Skin SDS-PAGE/Western Blot Results

Similarly to the hair follicle samples, the skin samples also had an expected result of the presence of bands at 20 kDa. Literature which showed the presence of the Can f 1 allergen in the environment due to the presence of dander, as discussed in the Introduction, led to this hypothesized result. However, after prolonged exposure in the dark chamber, no bands were seen

22 in the areas where the skin sample was placed. There was a heavy band in the area where the control sample was placed, as shown in Figure 3.

E. Saliva SDS-PAGE/Western Blot Results

Due to literature indicating the presence of the Can f 1 allergen in saliva, as described in the Introduction, saliva samples were expected to be seen at 20 kDa. After a 2-minute exposure in the dark chamber, slight bands were seen in the expected area. Severe separation of the control sample was also seen, displaying nonspecific interactions, as shown in Figure 4. This may indicate a poor control sample purity or improper blocking of the gel itself. As a result of these issues, the

ELISA testing process was utilized instead to allow for accurate quantification of the Can f 1 allergen in saliva samples.

D. Saliva ELISA Results

The Can f 1 allergen was detected in 100% of samples. The concentrations of the Can f 1 allergen in hypoallergenic dogs are shown in Table 1. The concentrations of the Can f 1 allergen in shedding dogs are shown in Table 2. The weighted average concentration of the Can f 1 allergen in hypoallergenic dogs was 18.32 µg/µL, and the weighted average concentration of the Can f 1 allergen in shedding dogs was 16.08 µg/µL. The range of concentration in hypoallergenic dogs was 1.22-94.67 µg/µL, and the range of concentration in shedding dogs was 2.51-83.04 µg/µL.

When comparing dogs within the same breed, statistically significant variability was found, as seen in Tables 3 and 4. However, based on the data collected from the hypoallergenic dog group, the Mini Goldendoodle breed had the lowest average concentration of the Can f 1 allergen at 2.56

µg/µL, and the Maltese subject had the highest concentration at 94.67 µg/µL. Based on the data

23 collected from the shedding dog group, the Terrier Mix subject had the lowest concentration of the

Can f 1 allergen at 2.51 µg/µL, and the Unknown breed group had the highest average concentration at 83.04 µg/µL.

A dependent two sample t-test was performed to determine the significance in comparing

the tcritical and the tcalculated values. When comparing the hypoallergenic group average Can f 1 allergen concentration of 18.32 µg/µL to the shedding group average concentration of 16.08 µg/µL, a

degrees of freedom value of forty two and a confidence interval of 0.95 was used. A t critical value was

determined to be 2.018 and a tcalculated value was calculated to be 1.149. This result suggests that there is no statistically significant difference between the concentration of the two groups. Thus, the null hypothesis can be accepted, implying that there is no correlation between the categorization of canine subjects as hypoallergenic or shedding and the subject’s concentration of the Can f 1 allergen in saliva. However, further studies need to be conducted with canine subjects with a known genetic history to demonstrate that there is no correlation between dog categorization and allergen in saliva.

Table 1. Individual Subject ELISA Data of Hypoallergenic Dog Group

Name Breed Average Conc (µg/µL) Standard Confidence Deviation Interval

Gracie Goldendoodle 3.70 0.24 6.35%

Gibson Goldendoodle 10.81 1.69 15.65%

Stanley Goldendoodle 11.04 0.69 6.28%

TJ Goldendoodle 18.42 1.97 10.67%

Oliver Goldendoodle 19.7 0.55 2.79%

Buddy Havanese 11.16 1.30 11.62%

Frasier Labradoodle 4.9 0.12 2.49%

Kash Labradoodle 10.37 0.47 4.53%

Max Labradoodle 19.52 1.21 6.20%

H2 Maltese 94.67 8.41 8.88%

Joy Mini Goldendoodle 1.22 0.11 8.66%

Honey Mini Goldendoodle 5.38 0.85 15.76%

Bentley Shih Tzu/Maltese/Poodle 4.36 0.42 9.70%

H1 Shih Tzu/Poodle 5.35 0.02 0.33%

Rue Shih Tzu/Poodle 16.17 1.6 9.90%

Louie Shih Tzu/Yorkie 16.92 1.17 6.92%

Lynnie Terrier Mix 56.01 5.21 9.30%

H5 Unknown 3.42 0.33 9.53%

H7 Unknown 7.79 0.29 3.67%

H4 Unknown 9.12 0.99 10.84%

H6 Unknown 10.48 1.14 10.87%

H3 Yorkshire Terrier 62.49 1.41 2.25%

Table 2. Individual Subject ELISA Data of Shedding Dog Group

Name Breed Average Conc (µg/µL) Standard Confidence Deviation Interval

Willie Beagle 6.11 0.31 5.04%

Dolly Beagle 13.41 0.25 1.86%

S9 German Shepherd 4.57 0.29 6.43%

Amber Labrador Mix 3.97 0.58 14.58

S7 Labrador Mix 6.06 0.98 16.15%

S6 Labrador Mix 6.28 0.29 4.56%

Smiley Labrador/Husky 9.94 0.73 7.33%

Archie Labrador/German Shepherd 11.17 0.51 4.56%

S5 Labrador/Bullmastiff 11.86 0.42 3.52%

Joe Labrador Mix 21.00 2.20 10.30%

Jenny Labrador/Weimaraner 29.94 3.44 11.50%

Louie Pit Bull Mix 4.88 0.56 11.46%

Juliet Pit Bull/Coonhound 5.19 0.29 5.67%

Buttercup Pyrenees/Newfoundland 56.82 4.18 7.35%

Harper Terrier Mix 2.51 0.27 5.67%

S10 Unknown 5.30 0.35 6.64%

S12 Unknown 5.67 0.06 1.07%

S2 Unknown 7.61 0.39 5.15%

S1 Unknown 10.11 0.28 2.78%

S11 Unknown 13.21 0.22 1.65%

S3 Unknown 35.13 4.96 14.13%

S4 Unknown 83.04 6.80 8.19%

Table 3. Final ELISA Analysis of Can f 1 Concentration in Saliva

by Breed of Hypoallergenic Dog Group

Represented Breeds Number (n) Geometric mean Minimum Maximum

Goldendoodle 5 10.99 3.70 19.7

Havanese 1 11.16 - -

Labradoodle 3 9.97 4.90 19.52

Maltese 1 94.67 - -

Mini Goldendoodle 2 2.56 1.22 5.38

Shih Tzu Mix 4 8.94 4.36 16.92

Terrier Mix 1 56.01 - -

Unknown 4 7.10 3.42 10.48

Yorkshire Terrier 1 62.49 - -

Total 22 18.32 1.22 94.67

Table 4. Final ELISA Analysis of Can f 1 Concentration in Saliva

by Breed of Shedding Dog Group

Represented Breeds Number (n) Geometric mean Minimum Maximum

Beagle 2 9.05 6.11 13.41

German Shepherd 1 4.57 - -

Labrador Mix 8 10.28 3.97 29.94

Pit Bull Mix 2 5.03 4.88 5.19

Pyrenees/Newfoundland 1 56.82 - -

Terrier Mix 1 2.51 - -

Unknown 7 13.67 5.30 83.04

Total 22 16.08 2.51 83.04

Figure 4. Plate 1 ELISA Analyzed Data for S4 (Breed: Unknown),

Archie (Breed: Lab/German Shepherd Mix), Oliver (Breed: Goldendoodle).

Figure 5. Plate 2 ELISA Analyzed Data for Amber (Breed: Lab Mix),

Kash (Breed: Labradoodle), Louie (H) (Breed: Shih Tzu/Yorkie).

Figure 6. Plate 3 ELISA Analyzed Data for Buttercup (Breed: Pyrenees/Newfoundland),

S12 (Breed: Unknown), Frasier (Breed: Labradoodle).

Figure 7. Plate 4 ELISA Analyzed Data for S5 (Breed: Lab/Bullmastiff), Willie (Breed: Beagle),

Lynnie (Breed: Terrier Mix).

Figure 8. Plate 5 ELISA Analyzed Data for Harper (Breed: Terrier Mix),

Bentley (Breed: Shih Tzu/Maltese/Poodle), Rue (Breed: Shih Tzu/Poodle).

Figure 9. Plate 6 ELISA Analyzed Data for Louie (S) (Breed: Pit Bull Mix),

Gracie (Breed: Goldendoodle), Max (Breed: Labradoodle).

Figure 10. Plate 7 ELISA Analyzed Data for S1 (Breed: Unknown), Dolly (Breed: Beagle),

TJ (Breed: Goldendoodle).

Figure 11. Plate 8 ELISA Analyzed Data for S9 (Breed: German Shepherd),

Gibson (Breed: Goldendoodle).

Figure 12. Plate 9 ELISA Analyzed Data for S3 (Breed: Unknown), H2 (Breed: Maltese),

Buddy (Breed: Havanese).

Figure 13. Plate 10 ELISA Analyzed Data for Jenny (Breed: Lab/Weimaraner),

H3 (Breed: Yorkshire Terrier), H4 (Breed: Unknown).

Figure 14. Plate 11 ELISA Analyzed Data for S10 (Breed: Unknown), S7 (Breed: Lab Mix),

H7 (Breed: Unknown).

Figure 15. Plate 12 ELISA Analyzed Data for S6 (Breed: Lab Mix), S2 (Breed: Unknown),

H6 (Breed: Unknown).

Figure 16. Plate 13 ELISA Analyzed Data for S11 (Breed: Unknown),

H1 (Breed: Shih Tzu/Poodle), H5 (Breed: Unknown).

Figure 17. Plate 14 ELISA Analyzed Data for Juliet (Breed: Pit Bull/Coonhound Mix),

Joy (Breed: Mini Goldendoodle), Honey (Breed: Mini Goldendoodle).

Figure 18. Plate 15 ELISA Analyzed Data for Joe (Breed: Lab Mix),

Smiley (Breed: Lab/Husky Mix), Stanley (Breed: Goldendoodle).

E. Discussion

Out of all employed individuals in the United States, 55% experience allergic rhinitis, the most common symptom of allergen-specific allergies. Those who experience allergic rhinitis regularly spend, on average, 593 US dollars per year to help alleviate their symptoms. [25] Not only are these individuals left with recurring symptoms from allergies over which they have no control, but employers within the United States, and other countries across the world, are left with the decision of whether or not to cover the costs for their employees’ treatment. Individuals who experience pet allergies often seek treatment that attributes to 0.25 to 0.5 billion US dollars in annual healthcare costs. [23] For individuals without employment, the cost becomes even harder to cover. When considering canine-specific allergies, a study done by Smallwood concluded that prolonged exposure to the Can f 1 allergen, and other similar allergens present in dogs, during developmental years may improve immune development and decrease canine-specific allergy symptoms. However, there were various inconsistencies regarding how improved each

35 individual’s immunity would become and which symptoms would decrease in future years. [26]

Most studies that look at canine-specific allergies focus on the source of the Can f 1 allergen being in the home and fail to focus on the physiological mechanism behind the production of the Can f

1 allergen and how that may differ between breeds. Thus, this study was conducted to provide scientific evidence that explains why some dog breeds provoke a severe allergic response, while others do not. Through the process of collecting samples from three different hypothesized sources of the Can f 1 allergen in canine subjects, this study focused on offering quantitative evidence of where the primary source of the Can f 1 allergen lies in canines and how that might differ between breeds.

The three samples taken from canine subjects, hair follicle, skin, and saliva suggest that the source of the Can f 1 allergen is the oral cavity; or, to be specific, the tongue epithelial tissue, which carries the allergen into the saliva. Due to previous studies reaching conclusive results from hair shafts and dander, it is hypothesized that the Can f 1 allergen moves from the saliva to the body, thus the hair and dander, during the grooming process. [5, 8, 12, 19] However, based on the results of this study, it can be conclusively stated that the primary source of the Can f 1 allergen is the saliva.

When comparatively analyzing the ELISA results from each saliva sample, there is great variability of the Can f 1 allergen concentration within each breed in each dog group. Although several owners provided a detailed history over their pet’s breed lineage, many other owners were only able to state the absence of shedding in their pet and a lack of allergic response when individuals with allergies interacted with their pet. Therefore, these results may suggest that the level of the Can f 1 allergen cannot be simplified to two groups, but must be analyzed further from

36 a dog’s genetic history. Unfortunately, this does not account for the statements made by the owners of the dog subjects that their pet did or did not provoke an allergic response.

The results from the dependent two tailed sample t-test suggest there is no statistically significant difference in Can f 1 allergen concentration in saliva between the two dog groups.

However, when considering the high variability of concentration values within each group and the result of the sample t-test, a definitive statement regarding the correlation between breed and Can f 1 allergen in saliva cannot be made. Nevertheless, there seems to be no trend when comparing the two groups as a whole.

Moving forward, further sample analysis needs to take place that focuses on specific breeds with known genetic history. More specifically, saliva samples from two breeds in each dog group should be taken and analyzed via ELISA with the same process done in this study. By narrowing the subject pool to canine subjects with known genetic history and that clearly fit into their respective groups, such as poodles versus huskies, their shedding rates can be deliberately compared to the concentration of the Can f 1 allergen in their saliva. Further research can then be conducted to correlate breed to allergic response in terms of the Can f 1 allergen.

IV. Conclusion

Based on the results from the dependent two tailed sample t-test, there is no statistically significant difference of the concentration of the Can f 1 allergen in the saliva of the hypoallergenic dog group versus the shedding dog group. However, due to the high variability of the allergen in dogs within their respective groups, the conclusion that the Can f 1 allergen has no correlation with breed categorization can only be tentatively suggested. This can be correlated to the lack of knowledge of the breed history for many of the dog subjects. Thus, further studies should be conducted with subjects with known genetic history to confirm the suspicion that hypoallergenic dogs have more Can f 1 allergen present in their saliva than shedding dogs.

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Appendix A:

IACUC Approval Form

Appendix B:

IACUC Application

Appendix C:

SDS-PAGE/Western Blot Results

Figure 19. Can f 1 Allergen Control Sample Western Blot Results.

Figure 20. Hair Follicle Sample Western Blot Results from Buttercup Hoffman

(Breed: Pyrenees/Newfoundland); includes Can f 1 control sample.

Figure 21. Skin Excision Sample Western Blot Results from unknown euthanized patient

at Westside Veterinary Clinic (Breed: Lab/Pit Bull Mix); includes Can f 1 control sample.

Figure 22. Saliva Sample Western Blot Results from Louie Kamrud (Breed: Pit Bull Mix);

includes Can f 1 control sample.

Appendix D:

Scatterplot of Linear Range from Can f 1 Control Sample

Figure 23. ELISA Control Sample Curve Fitting for Linear Range Calculation; Plate 15 Example

Linear Range: 0.77-1.422

Appendix E:

Additional Tables of ELISA Raw Data

Table 5.a Plate 1 ELISA Raw Data for Can f 1 allergen control sample.

Table 5.b Plate 1 ELISA Raw Data for S4 (Breed: Unknown).

Table 5.c Plate 1 ELISA Raw Data for Archie (Breed: Lab/German Shepherd Mix).

Table 5.d Plate 1 ELISA Raw Data for Oliver (Breed: Miniature Schnauzer).

Table 6.a Plate 2 ELISA Raw Data for Can f 1 allergen control sample.

Table 6.b Plate 2 ELISA Raw Data for Amber (Breed: Lab Mix).

Table 6.c Plate 2 ELISA Raw Data for Kash (Breed: Labradoodle).

Table 6.d Plate 2 ELISA Raw Data for Louie (H) (Breed: Shih Tzu/Yorkie).

Table 7.a Plate 3 ELISA Raw Data for Can f 1 allergen control sample.

Table 7.b Plate 3 ELISA Raw Data for Buttercup (Breed: Pyrenees/Newfoundland).

Table 7.c Plate 3 ELISA Raw Data for S2 (Breed: Unknown).

Table 7.d Plate 3 ELISA Raw Data for Frasier (Breed: Labradoodle).

Table 8.a Plate 4 ELISA Raw Data for Can f 1 allergen control sample.

Table 8.b Plate 4 ELISA Raw Data for S5 (Breed: Lab/Bullmastiff).

Table 8.c Plate 4 ELISA Raw Data for Willie (Breed: Beagle).

Table 8.d Plate 4 ELISA Raw Data for Lynnie (Breed: Terrier Mix).

Table 9.a Plate 5 ELISA Raw Data for Can f 1 allergen control sample.

Table 9.b Plate 5 ELISA Raw Data for Harper (Breed: Terrier Mix).

Table 9.c Plate 5 ELISA Raw Data for Bentley (Breed: Shih Tzu/Maltese/Poodle).

Table 9.d Plate 5 ELISA Raw Data for Rue (Breed: Shih Tzu/Poodle).

Table 10.a Plate 6 ELISA Raw Data for Can f 1 allergen control sample.

Table 10.b Plate 6 ELISA Raw Data for Louie (S) (Breed: Pit Bull Mix).

Table 10.c Plate 6 ELISA Raw Data for Gracie (Breed: Goldendoodle).

Table 10.d Plate 6 ELISA Raw Data for Max (Breed: Labradoodle).

Table 11.a Plate 7 ELISA Raw Data for Can f 1 allergen control sample.

Table 11.b Plate 7 ELISA Raw Data for S1 (Breed: Unknown).

Table 11.c Plate 7 ELISA Raw Data for Dolly (Breed: Beagle).

Table 11.d Plate 7 ELISA Raw Data for TJ (Breed: Goldendoodle).

Table 12.a Plate 8 ELISA Raw Data for Can f 1 allergen control sample.

Table 12.b Plate 8 ELISA Raw Data for S9 (Breed: German Shepherd).

Table 12.c Plate 8 ELISA Raw Data for Gibson (Breed: Goldendoodle).

Table 13.a Plate 9 ELISA Raw Data for Can f 1 allergen control sample.

Table 13.b Plate 9 ELISA Raw Data for S3 (Breed: Unknown).

Table 13.c Plate 9 ELISA Raw Data for H2 (Breed: Maltese).

Table 13.d Plate 9 ELISA Raw Data for Buddy (Breed: Havanese).

Table 14.a Plate 10 ELISA Raw Data for Can f 1 allergen control sample.

Table 14.b Plate 10 ELISA Raw Data for Jenny (Breed: Lab/Weimaraner).

100

Table 14.c Plate 10 ELISA Raw Data for H3 (Breed: Yorkshire Terrier).

101

Table 14.d Plate 10 ELISA Raw Data for H4 (Breed: Unknown).

102

Table 15.a Plate 11 ELISA Raw Data for Can f 1 allergen control sample.

103

Table 15.b Plate 11 ELISA Raw Data for S10 (Breed: Unknown).

104

Table 15.c Plate 11 ELISA Raw Data for S7 (Breed: Lab Mix).

105

Table 15.d Plate 11 ELISA Raw Data for H7 (Breed: Unknown).

106

Table 16.a Plate 12 ELISA Raw Data for Can f 1 allergen control sample.

107

Table 16.b Plate 12 ELISA Raw Data for S6 (Breed: Lab Mix).

108

Table 16.c Plate 12 ELISA Raw Data for S2 (Breed: Unknown).

109

Table 16.d Plate 12 ELISA Raw Data for H6 (Breed: Unknown).

110

Table 17.a Plate 13 ELISA Raw Data for Can f 1 allergen control sample.

111

Table 17.b Plate 13 ELISA Raw Data for S11 (Breed: Unknown).

112

Table 17.c Plate 13 ELISA Raw Data for H1 (Breed: Shih Tzu/Poodle).

113

Table 17.d Plate 13 ELISA Raw Data for H5 (Breed: Unknown).

114

Table 18.a Plate 14 ELISA Raw Data for Can f 1 allergen control sample.

115

Table 18.b Plate 14 ELISA Raw Data for Juliet (Breed: Pit Bull/Coonhound Mix).

116

Table 18.c Plate 14 ELISA Raw Data for Joy (Breed: Mini Goldendoodle).

117

Table 18.d Plate 14 ELISA Raw Data for Honey (Breed: Mini Goldendoodle).

118

Table 19.a Plate 15 ELISA Raw Data for Can f 1 allergen control sample.

119

Table 19.b Plate 15 ELISA Raw Data for Joe (Breed: Lab Mix).

120

Table 19.c Plate 15 ELISA Raw Data for Smiley (Breed: Lab/Husky Mix).

121

Table 19.d Plate 15 ELISA Raw Data for Stanley (Breed: Goldendoodle).

122