Development of a multiplex microsphere immunoassay for the quantitation of salivary antibody responses to selected waterborne pathogens

Oak Ridge Institute for Science and Education, Oak Ridge, TN, USA.
Journal of immunological methods (Impact Factor: 1.82). 02/2011; 364(1-2):83-93. DOI: 10.1016/j.jim.2010.11.005
Source: PubMed


Saliva has an important advantage over serum as a medium for antibody detection due to non-invasive sampling, which is critical for community-based epidemiological surveys. The development of a Luminex multiplex immunoassay for measurement of salivary IgG and IgA responses to potentially waterborne pathogens, Helicobacter pylori, Toxoplasma gondii, Cryptosporidium, and four noroviruses, involved selection of antigens and optimization of antigen coupling to Luminex microspheres. Coupling confirmation was conducted using antigen specific antibody or control sera at serial dilutions. Dose-response curves corresponding to different coupling conditions were compared using statistical tests. Control proteins in the specific antibody assay and a separate duplex assay for total immunoglobulins G and A were employed to assess antibody cross-reactivity and variability in saliva composition. 200 saliva samples prospectively collected from 20 adult volunteers and 10 paired sera from a subset of these volunteers were used to test this method. For chronic infections, H. pylori and T. gondii, individuals who tested IgG seropositive using commercial diagnostic ELISA also had the strongest salivary antibody responses in salivary antibody tests. A steep increase in anti-norovirus salivary antibody response (immunoconversion) was observed after an episode of acute diarrhea and vomiting in a volunteer. The Luminex assay also detected seroconversions to Cryptosporidium using control sera from infected children. Ongoing efforts involve further verification of salivary antibody tests and their application in larger pilot community studies.

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    • "Finally, after washing off unbound Streptavidin-PE, the beads were suspended in Bio-Rad assay buffer and analyzed on a Bio-Rad 96-well plate reader using the Bio-Plex 200 Array System (Bio-Rad Laboratories, Inc.). The median fluorescence intensity (MFI) was calculated from a standard curve using Bio-Plex Manager software (Bio-Rad Laboratories, Inc.) [33] and considered to be proportional to analyte concentration. The protein content in the lysates was determined by Bio-Rad DC protein assay (Bio-Rad Laboratories, Inc.). "
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    BMC Cancer 11/2013; 13(1):525. DOI:10.1186/1471-2407-13-525 · 3.36 Impact Factor
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    • "Luminex-based methods have been used to assess murine immune responses to Hp infection (Taylor et al., 2008) and vaccination (Taylor et al., 2007) in splenocyte culture supernatant and recently to quantify gastric cytokine concentrations in Hp-infected mice (Schumacher et al., 2012). A method to measure Hp-specific IgG in human saliva samples has also been developed, using Luminex beads conjugated with antigens including Hp whole cell sonicate and recombinant urease (Griffin et al., 2011). However, to our knowledge, Luminex assays have not been optimised for human gastrointestinal mucosal tissue samples, though were recently used to quantify interleukin-1β, interleukin-1 receptor antagonist, interleukin-6 and tumour necrosis factor-α in gastric tissue samples (Serelli-Lee et al., 2012). "
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    Journal of immunological methods 05/2013; 394(1-2). DOI:10.1016/j.jim.2013.04.009 · 1.82 Impact Factor
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    Journal of clinical microbiology 07/2011; 49(9):3184-90. DOI:10.1128/JCM.00557-11 · 3.99 Impact Factor
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