Detection of Salivary Endotoxins Using an Antibody-Based Combined Mono/Polyclonal Lateral Flow Immunoassay Device Summary We report a point-of- care/use lateral flow assay (LFA) for the detection of P. gingivalis endotoxin , an important saliva biomarker for oral health. Two different sandwich LFA design approaches using the combination of mono- and polyclonal antibodies or polyclonal antibody alone have been evaluated to detect P. gingivalis endotoxins, with a detection limit of ~22 ng mL -1 and 46.5 ng mL -1 for water-based and saliva-based samples, respectively. LFA also shows good selectivity to P. gingivalis endotoxin versus other endotoxins and proteins. Saliva pretreatment combining syringe filtration and potato starch successfully inhibits α-amylase activity and provides better results in LFA devices. |
Biological fluids, such as blood, urine, saliva and mucus, are perhaps the most important source of information about our medical condition. Historically, and remains the case today, blood testing is the most common diagnostic tool. Conventional detection of molecular markers found in biofluid samples is mainly carried out using the well-known polymerase chain reaction (PCR) method, which requires trained personnel and a relatively longer period of time in a laboratory. However, with the increasing use of point-of-care (POC) diagnostics, other biofluids are gaining importance. Rapid and low-cost analysis of biomarkers present in various biofluids can provide an early indication of various medical conditions, thereby improving treatment outcomes and reducing medical costs.
As demonstrated during the coronavirus disease (COVID) pandemic, the lateral flow assay (LFA) is a versatile POC platform with many benefits, such as fast analysis time, low cost, easy-to-use colorimetric interface, manufacturing process simple and without the need for external control. Because COVID LFA home test kits were widely used during the pandemic, there is now global familiarity with lateral flow assay (LFA) test kits. Furthermore, the use of smartphones for LFA applications has been developed not only to improve the analysis of test results but also to improve the sensitivity of LFA.
Endotoxins induce a strong immune response in hosts, but also serve as important biomarkers useful for diagnosing various bacterial diseases . Porphyromonas gingivalis (P. gingivalis), one of more than 700 bacterial species found in the oral cavity and one of 500 species present in subgingival plaque, is an important gram-negative pathogenic bacteria responsible for chronic periodontitis . Periodontal disease presents chronic inflammation caused by a bacterial infection, causing gum disease and even tooth loss.
The salivary presence of this bacteria in high concentration suggests that the patient has chronic periodontitis disease. Furthermore, due to the close relationship between P. gingivalis and other important systemic diseases, such as cardiovascular diseases, rheumatoid arthritis and neurodegenerative diseases (Alzheimer’s) as has emerged in recent years, quantitative evaluation of PG LPS has become an key measure in oral health, as well as in the entire body health system.
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Engineers at the University of Cincinnati have developed a new device that can warn consumers about the early risks of cavities due to diseases such as gingivitis and periodontitis.
Gingivitis, the earliest form of gum disease, is caused by bacteria. But not just any bacteria. The problem for researchers was getting a device to identify the particular type responsible for the disease, said Andrew Steckl, an eminent Ohio scholar and distinguished research professor in the UC College of Engineering and Applied Sciences.
"It’s been a challenge to get to the point where we can detect this toxin created by the bacteria responsible for gingivitis," he said. Steckl and UC senior research associate Daewoo Han collaborated with Sancai Xie, a senior scientist at Procter & Gamble Co., and described their results in a paper published in the journal Sensors and Diagnostics of the Royal Society of Chemistry .
Steckl’s research team has been exploring biosensing for various applications. They studied stress hormones in sweat in collaboration with the Air Force Research Laboratory at Wright-Patterson Air Force Base. Now they are studying saliva.
"There are good reasons to use saliva," he said. "It is relatively abundant and easy to obtain through non-invasive methods. And saliva has many important elements that can act as indicators of health."
Gingivitis bacteria can travel through the bloodstream, causing cardiovascular disease and other serious health problems, Steckl said. But saliva is a complicated biofluid, said Han, the study’s lead author.
The researchers pre-treated the sample with potato starch to eliminate a protein called amylase that could interfere with the test results. Their test uses antibodies that react to endotoxins found in bacteria.
"Daewoo worked very hard through many dead ends before achieving success," Steckl said. "I tell my students that research is searching, searching and researching until you find the answer."
Home health tests have been available for generations in specific uses such as pregnancy detection. But the COVID-19 pandemic introduced a wide audience of consumers to the concept of monitoring their health with new technology.
The at-home testing industry is expected to generate $45 billion annually by 2031, according to Allied Market Research.
Steckl said he sees many opportunities for new consumer products. "Our results are definitely promising," Steckl said. "Sometimes it’s easy. Most of the time you have to persevere."
Summary conclusion We have demonstrated an antibody-based sandwich lateral flow assay (LFA) to detect P. gingivalis LPS, an important biomarker of oral health. The LFA device has an LOD of approximately 22 ng ml -1 for aqueous samples and excellent selectivity against other LPS and salivary proteins. Detection in human saliva was evaluated in combination with potato starch and syringe filtration to reduce interference from biomolecules in saliva, especially α-amylase enzymes. LFA immunoassay reactions with saliva samples gave comparable results (LOD ~46.5 ng mL -1) to those with water-based samples. Further development will be made to improve the sensitivity of the assay using saliva samples. The ability to detect multiple disease-related LPS molecules will also be explored to make more accurate diagnoses of patients’ health. Finally, because the sensitivity of current antibody-based detection is significantly affected by the performance of the conjugated antibody, the development of aptamer-based sandwich LFA will be investigated to improve flexibility and performance. The pretreatment for saliva samples discussed above can also be used with aptamer-based sandwich LFA devices. |
