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Examples of Successful Proposals

Salivary BioMarkers for Studies of Children and Youth in Families

Identifying Information

Lead Investigator:

Douglas A. Granger, Ph.D.
Assistant Professor Department of Biobehavioral Health
Director, Penn State University Behavioral Endocrinology Laboratory
(p) 863-8402
(f) 863-7525,
DAG11@psu.edu

Collaborating Investigators:

Leann Birch, Human Development and Family Studies
Alan Booth, Sociology
Karen Carver, Sociology
Mary Clements, Psychology
Cathy Cohan, Human Development and Family Studies
Nan Crouter, Human Development and Family Studies
Pamela Cole, Psychology
Susan McHale, Human Development and Family Studies
Liz Susman, Biobehavioral Health
Lynne Vernon-Feagans, Human Development and Family Studies
Karen Quigley, Psychology

Abstract

Technical advances that make possible the non-invasive measurement of biological processes in saliva have paved the way for new discovery regarding how biological, behavioral, and social environmental variables interact to influence human development and health. The Penn State Behavioral Endocrinology Laboratory has specialized expertise in the design, development, and application of salivary biomarkers in behavioral research. This project will develop new salivary immunoassays for biomarkers (Leptin, Oxytocin, and Estradiol) for use by Penn State faculty. Assay protocols will be designed and rigorously evaluated for internal and external validity. Guidelines for their use in the field will be developed. A testing service will be offered for researchers, and a hands-on lab practicum will train investigators interested in using these cutting edge research tools.

Specific Aims and Objectives

I. Basic research will be conducted to assess the feasibility of developing immunoassays for the quantification of Leptin, Estradiol, and Oxytocin in human saliva.

II. We will construct enzyme- or radio- immunoassay platforms and then rigorously evaluate internal and external validity of these new assays. Linearity, accuracy, precision, specificity, sensitivity, and serum-saliva comparisons will be established.

III.Conditions of saliva sample collection, handling, and storage will be extensively researched.

IV. The assays developed will be made available to PSU researchers via the BEL testing service, consultation will be provided on the implementation of sample collection, and the protocols will be made public via the BEL Lab Practicum and publications.

The overarching objective is to provide Penn State researchers with the tools to lead the cutting edge of the integration of biological measures into studies on children and youth in families.

Background and Rationale, and Description of Methods

Nature of the Problem

Early behavioral scientists assumed that neurobiological processes were critical components of the behavioral and psychological phenomenon they were studying (e.g., William James, 1842-1910). However, the influence of those assumptions on scientific thinking in subsequent decades was limited by a lack of knowledge regarding how to observe or quantify biological processes in humans let alone in social context. Consequentially, as the subdisciplines of the social and behavioral sciences emerged over the 20^th century, research on children, youth, and families focused primarily on the interface between the social environment and individual behavior. The integration of biological processes in this endeavor remained a core conceptual component, but studies explored their influence only indirectly using comparative methods.

In this decade, technical and conceptual advances in the neuroscience have made possible non-invasive measurement of some of the processes believed by early social and behavioral scientists to underlie behavior. As a result, there is renewed interest in the integration of biological, behavioral, and social environmental variables. This renewed empirical focus has contributed to new agendas at several National Institutes (including Mental Health, and Child Health and Development) and the National Science Foundation that prioritize the advance of fundamental knowledge about interactions between biological and behavioral processes as they relate to human development, health, and behavior.

In behavioral studies focused on children, youth, and families, the incorporation of salivary biomarkers has paved the way for new discovery. Several Penn State investigative teams have emerged as conceptual and methodological leaders at this cutting edge. One of the reasons for Penn State's excellence is that its researchers are supported by a unique interdisciplinary facility, the Behavioral Endocrinology Laboratory (BEL). During the previous 5 years, the BEL has developed a national and international reputation for assisting researchers integrate salivary biomarkers into their studies by (1) developing new highly sensitive immunoassays, (2) consulting on study design and sampling issues, (3) providing a testing service for researchers without access to laboratory facilities, and (4) teaching a lab practicum.

Penn State researchers working with the BEL rely on the highest quality method development to support their grant proposals and implement their projects. The majority of these investigators do not have their own wet laboratories; they depend on the BEL to work out the details of analytical methods, sample collection procedures, and testing protocols. Given that most of these researchers have behaviorally rather than biologically-oriented training backgrounds, the success of their grant applications is often dependent on BEL research and development efforts to provide extensive preliminary data and technical support. Interestingly, while there is considerable interest at federal institutes and private foundations for the integration of these measures into behavioral science, there have been no funds earmarked by the major agencies to support method development. It is also important to note that, based on our experience, PSU faculty are rarely able to support assay development with their existing funding.

Why Saliva?

Monitoring biomarkers in saliva has distinct advantages over doing so in other biological fluids (i.e., urine, serum or plasma). This is especially true when children and youth are involved as participants. Sampling saliva represents a less-invasive method for long-term or repeated sampling schedules; enables collection of samples in special populations (e.g., infants, and children), and in many circumstances in which blood or urine sampling is not viable. Salivary levels of biomarkers accurately reflect the unbound, biologically active, fraction of most serological markers in the general circulation. Also, unless visibly contaminated with blood, human saliva is not considered a class II biohazard (CDC) affording researchers administrative and safety benefits.

Problems and Pitfalls of Existing Salivary Immunoassays

Surprisingly, the availability of immunoassays for the measurement of biomarkers in saliva is very restricted. Despite, widespread use for research purposes, the FDA has yet to approve salivary assays for clinical-diagnostics. Most companies are just beginning to think about marketing immunoassay systems for saliva. Until recently, companies have assumed that with specialized sample collection techniques and simple modifications to serum-based immunoassay protocols that saliva biomarkers can be adequately measured. For several reasons that approach has created problems related to assay measurement validity and reliability.

First, with few exceptions the majority of available immunoassays for saliva are modifications of protocols developed for use with serum/plasma. The calibrators used in those assay kits are suspended in a human serum matrix. The composition of serum is markedly different from saliva (there is substantially less protein in saliva for instance), and so these simple modifications are likely to produce results that are influenced by matrix differences. To ensure accurate results, immunoassays for saliva biomarkers must be designed with matrices that match saliva.

Second, the levels of most biomarkers in saliva are considerably lower than levels in the general circulation (10 to 100 fold lower). The use of standard curves developed to capture the range of values expected in serum/plasma samples is often not sensitive enough to capture the complete range of individual differences in the level expected in saliva. To overcome problems associated with assay sensitivity serum assay modifications typically involve increasing the sample volume tested in the assay perhaps as much as ten-fold. For instance, a common saliva sample test volume is 200 m L (10x the 20 ul required for serum) and samples are typically assayed in duplicate. In this example, approximately 400 m l of sample would be devoted for each test of this particular marker (800 m l if repeated testing is implicated). Assays need to be designed that improve sensitivity and do not require large saliva test volumes. Collecting enough sample from subjects for test volumes now required can be a daunting task, especially when participants are children and youth.

Third, substances that we put in our mouths very easily lower or raise the pH of saliva. Performance of immunoassays becomes compromised as the pH of samples to be tested drops below 4. The result is artificially inflated results. Salivary assay systems need to be designed so as to be very robust to the effects of interference caused by collection techniques that affect pH (and viscosity).

Which Biomarkers?

We have informally surveyed a group of Penn State faculty and determined three biomarkers with promise to advance Penn State's behavioral research on children, youth, and families. Once these assays are developed there is commitment from the faculty to conduct pilot work which will most certainly lead to external funding. The markers are (1) Leptin, (2) Estradiol, and (3) Oxytocin. A brief description of each follows:

Leptin is an important regulator of food intake. It is produced in direct proportion to adiposity in humans, is increased in the blood by food intake, and acts as a satiety substance. Once produced, it is free to circulate in the blood stream and acts via a hypothalamic site to regulate ingestive behavior. Leptin is known to affect neuroendocrine regulation, food intake, energy expenditure, metabolism, and inhibit the cortisol and ACTH response to stress. It may be involved in the timing of puberty, and behavioral and physiological processes which are related to the facilitation of learning and memory. Its levels are decreased by both acute and chronic stress. The clear majority of studies on the reciprocal relationships between Leptin and behavior have been conducted with animals. We are not aware of non-invasive means to measure Leptin. Our preliminary literature searching suggests it is feasible.

Oxytocin is a neurohypophyseal peptide hormone that functions as a neuropeptide in several brain areas in addition to its role as a posterior pituitary hormone. It affects a variety of cognitive, grooming, affiliative, sexual, maternal, and reproductive behaviors. The neuropeptide participates in important reproductive functions, such as parturition and lactation, and homeostatic responses including modulation of the HPA axis. Recent evidence also implicates Oxytocin in social aspects of reproductive behaviors including the regulation of maternal-infant interactions, and both social contact and selective social interactions associated with social attachment and monogamous pair bonding. If a salivary measure of Oxytocin could be designed it would have wide spread impact on many research programs at PSU. Levels of Oxytocin are exceedingly low (less than a trillionth of a gram) in the general circulation. Thus, it will be challenging to design a salivary immunoassay to be ultra-sensitive.

Estradiol is the primary female reproductive hormone. It is implicated in the onset of puberty, menstruation, sexual and reproductive capacity, pregnancy, and menopause. It is associated with maternal behavior, but may be important to general social affiliative behavior. Its ratio to testosterone may be very important in understanding gendered behavior. It is also implicated in breast cancer, cognition, and osteoporosis. The BEL has conducted preliminary research and is able to measure Estradiol in the saliva of normally cycling adult women. We seek to improve this assay's sensitivity so that levels may be measured in prepubertal boys and girls as well as in adult men. To do so may require that we change formats from radioimmunoassay to an enzyme-based approach or use an extraction or separation.

Assay Development Plan

Step 1. Feasibility

1. Conduct literature search to characterize the markers source, size, half-life, and distribution in the body.
2. Search literature for assay methods reported in the research literature and characterize their levels of sensitivity, sample volumes used, and basic structure of assay protocols.
3. Search immunodiagnostic vendors for source of polyclonal antibodies, HRP-linked markers, and calibrators.
4. Design basic enzyme-immunoassay platform or radioimmunoassay platform for use with each marker.

Step 2. Internal and External Validation

1. Determine the theoretical minimum detection limit, defined as the minimal concentration of each marker that can be distinguished from zero at the 95% confidence limit, by interpolating the mean minus 2 SD for 10 replicates of the zero calibrator.
2. Determine the reproducibility of the standard curve across 10 replicate runs. Compute averages, SDs, and CVs for slope, correlation coefficient, ED80, ED50, and ED20.
3. Precision. Intra-assay variation (CV) computed for tests of 10 replicate tests of low, medium, and high concentrations. Inter-assay variation will be computed for the mean of average duplicates for 10 separate runs for low, medium, and high concentration samples across 3 independent runs.
4. Accuracy. Method accuracy will be determined from known amounts of unlabelled marker added to saliva samples containing various endogenous concentrations.
5. Linearity of dilution. Parallelism will be evaluated by measuring marker in high, medium, and low concentration samples, which will be serially diluted incrementally to the low end of the assay's range (ED95).
6. Drift. We will determine whether there is any position effect due to delays in addition of reagents, pairs of samples will be spread throughout for low, medium, and high level samples in the beginning, middle, and end of each of 10 runs.
7. Serum-saliva relationship. It will be determined whether salivary measurements correlate with serum-levels, 25 matched saliva and serum samples from children, adolescents, and adults will be assayed and compared.
8. Sensitivity of assay range. We will evaluate whether the range of sensitivity of the assay is sufficient to capture the full range of developmental, individual, gender, and diurnal differences.

Step 3. Sample collection, handling, and storage

1. Identify collection materials needed, vial sizes needed,
2. Determine effects on assay quality control parameters of conditions related sample handling and storage.
3. Based on data generated thus far write recommendations.

Relevance to the CYFC

The overarching objective is to provide Penn State researchers with the tools to lead the cutting edge of the integration of biological measures into studies on children and youth in families. This objective is consistent with CYFC's interest in increasing knowledge about the developmental trajectories associated with health risks and problem behaviors.

Anticipated Outcomes

1. Assays will be made available to PSU researchers via the BEL testing service
2. Assay protocols will be incorporated into curriculum of BEL Lab Practicum on Salivary Biomarkers
3. Make BEL Lab Practicum available to CYFC researchers at a limited cost (must check with Continuing and Distance Education about this)
4. Present findings at National and International Meetings
5. Write articles for publication, which specify the protocols, detail assay performance characteristics, serum-saliva comparisons, and references ranges for developmental and individual differences.
6. Provide consultation service to PSU faculty regarding implementation of sample collection and research design for their PHS proposals.

Budget and Personnel

The project will have access to BEL equipment but will need to support supplies, reagents, and person hours. Total budget requested from CYFC is $10,000. Dollar estimates are based on a 6-month project period.

Personnel

PI - will devote the equivalent of 5% time to this project over 12-months and will direct all aspects of the research activities. This salary will be contributed by the BEL.

Project Associate- is the senior/lead biotechnician in the BEL. Funds are requested to devote 50% time over 6 months to this project.

Administrative Assistant is responsible for managing budgets, standing orders, and ordering supplies for the BEL. The salary will be contributed by the BEL.

Lab Supplies

Several immunodiagnostic companies (DSL, Webster TX; ICN, Costa Mesa, CA; DPC, Santa Monica, CA) provide complimentary reagents and materials to the BEL. The estimated total budget for supplies, reagents, and materials is $8,000. However, we anticipate contributions from our industry sponsors. Thus, we request only $2,000 (~30% of the total supply costs) for consumable laboratory supplies and reagents.

Human Subject Fees

Sixteen children, 16 adolescents, and 16 adults will donate saliva (1-6 mLs) and/or serum samples to complete the analysis of assay external validity. As outlined in PSU IRB under protocol (#950054-02, -03, -04) each subject receives $15 compensation ($720). We expect the samples will be collected at the GCRC at no charge to the project. We request $720 to support costs of obtaining these critical biological specimens.

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