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Salivary Transferrin

Salivary Transferrin Quick Start Research Guide

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Biomarkers

1. How to Assay for Salivary Blood Contamination

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Order Code5300

Assay Protocol
Rev. 04.19.19
Specifications
Catalog#: 1-1302
Regulatory Status: RUO
Format: 96-well plate
Assay Time: ~ 1.5 hrs
Sample Volume/Test: 25 µL
Sensitivity: <0.007 ug/dL
Assay Range: 0.012-3.000 ug/dL
Storage Requirements: 2-8°C
Tests Per Kit
Singlet: 80
Target Analyte

Technical Documentation

Assay Kit Overview

Intended Use

The Salimetrics Blood Contamination Enzyme Immunoassay Kit is a competitive immunoassay specifically designed and validated for the quantitative measurement of the presence of blood in saliva samples. It is not intended for diagnostic use. It is intended only for research use in humans and some animals as an analytical tool to screen saliva samples that should be excluded from assays for other salivary analytes because of blood component leakage into the oral mucosa. Salimetrics has not validated this kit for serum or plasma samples.

Introduction

The quantitative measurement of analytes in saliva is invalid if the physiochemical and physical barrier between the general circulation and the oral mucosa is compromised such that there is a “leakage” of blood or plasma into saliva. This is especially true when levels of the analyte of interest in blood are substantially higher than levels observed in saliva (i.e., ng/mL in serum vs. pg/mL in saliva). Screening samples and excluding those contaminated by blood may be critical if valid conclusions are to be drawn from salivary data. A computerized literature search revealed that, with few exceptions, investigators working with salivary measurements rarely screen samples for this problem. This suggests that most researchers may be unaware that this phenomenon might be affecting their salivary data. Studies that report screening samples typically use a dipstick method designed for use with urine specimens. However, saliva contains peroxidases, which cause the same color change on the dipstick as does hemoglobin, leading to false positive results. Therefore, the dipstick method is not an accurate measurement of blood contamination in saliva. To solve this problem, Salimetrics designed a simple, efficient, and inexpensive immunoassay for researchers to use as an analytical tool to screen samples for blood presence in saliva. Each 96-well plate screens 80 samples with only 1 hour of total incubation time and uses only 20 µL of sample per test. We expect that the routine application of this screening tool will significantly improve the quality of the information learned in future studies. The Salimetrics Salivary Blood Contamination Enzyme Immunoassay quantitatively measures transferrin, a large protein (mol wt of 76,000) present in abundance in blood that is normally present in only trace amounts in saliva. Higher levels of transferrin measured in saliva by this assay indicate the presence of blood contamination and serve as a warning to investigators that samples should be excluded from subsequent quantitative assays for salivary analytes and statistical analyses. As a general guideline, saliva samples with transferrin values 0.5 mg/dL or higher should be candidates for exclusion when assaying salivary samples for testosterone. Values greater than 1 mg/dL should be considered as candidates for exclusion in other salivary assays. The criterion score adopted will vary depending on the analyte under investigation and the protocol used to measure it. We recommend computing a regression between transferrin levels and the levels of the analyte of interest and establishing a cut score based on statistical analysis of this associative relationship.

 

Salivary Transferrin/Blood Contamination Assay Principle

This is a competitive immunoassay kit. Transferrin in standards and samples compete with transferrin conjugated to horseradish peroxidase for the antibody binding sites on a microtitre plate. After incubation, unbound components are washed away. Bound Blood Contamination Enzyme Conjugate is measured by the reaction of the horseradish peroxidase enzyme to the substrate tetramethylbenzidine (TMB). This reaction produces a blue color. A yellow color is formed after stopping the reaction with an acidic solution. The optical density is read on a standard plate reader at 450 nm. The amount of Blood Contamination Enzyme Conjugate detected is inversely proportional to the amount of transferrin present in the sample.

 

Biomarkers

2. Technical Summary

Analyte Summary
Analyte: Transferrin
Aliases: Blood Contamination
Serum-Saliva Correlation: NA
Optimum Test Volume: 75 μL*
*Add 300 µl to the total volume of all tests for liquid handling
Assay Summary
Methodology: ELISA
Sensitivity: 0.08 mg/dL
Assay Range: 0.08 mg/dL - 6.6 mg/dL
Assay Type: Quantitative

Background

​In the bloodstream the majority of steroid hormones are bound either to non-specific proteins such as albumin or to specific proteins such as corticosteroid binding globulin (CBG) or sex hormone binding globulin (SHBG). These protein molecules are generally too large to diffuse from the blood stream into the saliva glands. Consequently, only the 1-15% of circulating hormone molecules that are found in an unbound state are free to diffuse from blood into saliva. Steroid hormone concentrations in saliva are therefore much lower than those in blood. If the barrier between the bloodstream and the oral mucosa is compromised by inflammation or microinjury such that there is leakage of blood or plasma into saliva, then there is a possibility that the higher levels of hormones found in the blood stream will contaminate saliva samples, causing falsely high readings. (1) Visual inspection of saliva samples is not adequate to detect blood contamination, since microinjuries to blood vessel membranes may allow hormone molecules and proteins to pass through while still retaining the much larger red blood cells. Dipstick tests for blood, which look for the presence of hemoglobin, are also not a reliable means of screening saliva samples, due to the presence of peroxidases in saliva, which can generate false positive results. (2,3) The Salimetrics Salivary Blood Contamination Enzyme Immunoassay quantitatively measures transferrin, a large protein (mol wt 76,000) present in abundance in blood that is normally present in only trace amounts in saliva. Higher levels of transferrin measured in saliva indicate the presence of blood contamination and serve as a warning to investigators that samples should be excluded from subsequent quantitative assays for salivary analytes and statistical analyses. (2,3,4) Saliva samples collected from populations that have little or no dental care, or known oral health problems, are especially appropriate for screening with the Blood Contamination Assay Kit.​

References & Salivary Transferrin Research

  1. Malamud, D., & Tabak, L. (Eds.). (1993).  Saliva as a diagnostic fluid. Ann N Y Acad Sci, (Vol. 694).  New York, NY.
  2. Kivlighan, K.T., Granger, D.A., Schwartz, E.B., et al. (2004). Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva.  Horm Behav, 46(1), 39-46.
  3. Schwartz, E., & Granger, D.A. (2004). Transferrin enzyme immunoassay for quantitative monitoring of blood contamination in saliva. Clin Chem, 50(3), 654-656.
  4. Granger, D.A., Cicchetti, D., Rogosch, F., et al. (2007).  Blood contamination in children’s saliva: Prevalence, stability, and impact on the measurement of salivary cortisol, testosterone, and dehydroepiandrosterone.  Psychoneuroendocrinology, 32(6), 724-33.

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