(Invited) Salivary Lipopolysaccharide (LPS) Detection Using Lateral Flow Sandwich-Based Immunoassay Point-of-Care Devices

Abstract

Endotoxins, also known as lipopolysaccharides (LPS), are important macromolecules existing in the outmost part of Gram-negative bacteria, which are generated when the bacteria are in growing and stationary phases. Large numbers of LPS molecules are released when the bacteria die. For example, a single E. coli bacterium can release ~ 2 million LPS molecules. Once LPS is released, the lipid A part is exposed to the host’s immune system, eliciting strong immune response, including various inflammations, potentially life-threatening sepsis, and septic shock. Gingivitis is a sign of disturbance in the oral microbiome by overgrowing Gram-negative bacteria. More than 50% of global population has some level of gingivitis, which can worsen to periodontitis if it is not properly treated. Periodontitis is a severe gum infection disease that eventually leads to tooth loss and other serious health complications. [1]While endotoxins induce a strong immune response in hosts, they can also serve as important biomarkers to diagnose various bacterial diseases. P. gingivalis, one of > 500 bacteria species found in subgingival plaque is a major Gram-negative pathogenic bacterium that is responsible for chronic periodontitis. LPS from P. gingivalis (PG LPS) induces significant host responses in gingival tissue by increasing the production of inflammatory biomarkers. Furthermore, because of the close relationship between P. gingivalis and other important systemic diseases, such as cardiovascular diseases and neurodegenerative (Alzheimer’s) disease that has emerged in recent years, quantitative evaluation of PG LPS has become a key measure in oral health, as well as in the whole body health system.[2]Considering the increasing significance of P. gingivalis LPS, high demand for a novel point-of-care device has emerged for both early diagnosis and frequent monitoring of PG LPS levels. Traditional qualitative detection of bacterial biomarkers is carried out mostly utilizing the well-known PCR method, which requires trained personnel and a relatively longer time span in a laboratory setting. Lateral flow assay is one of the most promising POC platforms with many benefits, such as low cost, simple fabrication process, user-friendly colorimetric interface, rapid turnaround time, and no need for external instruments.[3]We have developed a point-of-care lateral flow assay (LFA) device to detect and quantify PG LPS concentrations in human saliva using the sandwich assay approach with two different antibodies. Fig. 1a shows the basic concept of our sandwich immunoassay on the LFA device. LPS is a large biomacromolecule with multiple binding sites, which is very beneficial for utilizing the more sensitive sandwich-type approach versus competitive immunoassay approach. Two different antibodies are utilized in the device. A monoclonal antibody (mAb) is conjugated to the surface of gold nanoparticles (AuNP) to capture PG LPS molecules in the sample solution. A polyclonal antibody (pAb) is printed and immobilized on the nitrocellulose membrane of LFA device in order to form a test line for capturing the AuNP conjugates bound to PG LPS from the sample solution. When LPS is present in the sample solution, the mAb-conjugated AuNP particles (mAb-AuNP) bind to LPS. Then the combined LPS-mAb-AuNP conjugate particles flow through the porous nitrocellulose membrane and are captured by the immobilized pAb in the test line stripe on the LFA. Polyclonal antibodies can bind to other available binding sites on the LPS membrane that were not utilized by the monoclonal antibody. When sufficient LPS-mAb-AuNP particles are captured a reddish test line is observed on the nitrocellulose membrane. The control line on the LFA is printed on the LFA using protein G, which captures the monoclonal antibody on the AuNP’s in order to validate the test by confirming the conjugation status between mAb and AuNP. Fig. 1b shows the test line intensity at multiple LPS concentrations. The test line intensity increases linearly with LPS concentration, with the effective linear dynamic range up to ~2 µg/mL concentration and a limit of detection of ~ 22 ng/mL. Also, while the assay presented the strongest test line intensity for P. gingivalis, neither a-mucin, which is most abundant protein in saliva, nor E. coli LPS produced a test line signal. P. pallens LPS, which is related to Gingivitis disease, resulted in a much weaker test line formation. Park, B.S. and J.-O. Lee, Recognition of lipopolysaccharide pattern by TLR4 complexes. Experimental & Molecular Medicine, 2013. 45(12): p. e66-e66.He, W., et al., Point-of-Care Periodontitis Testing: Biomarkers, Current Technologies, and Perspectives. Trends in Biotechnology, 2018. 36(11): p. 1127-1144.Dalirirad, S., D. Han, and A.J. Steckl, Aptamer-Based Lateral Flow Biosensor for Rapid Detection of Salivary Cortisol. ACS Omega, 2020, 51: p. 32890–32898.doi.org/10.1021/acsomega.0c03223 Figure 1