A method to study protein biomarkers in saliva using an automated capillary nano-immunoassay platform (Wes™)

Abstract

Traditional immunoprobing techniques like Western blot continue to play a crucial role in the discovery and validation of biomarkers. This technique suffers from several limitations that affect reproducibility and feasibility for large-scale studies. Modern immunoprobing techniques have addressed several of these limitations. Here we contrast the use of Western blot and an automated capillary nano-immunoassay (CNIA), Wes™. We provide evidence highlighting the methodological advantages of Wes™ over Western blot in the validation of a novel biomarker, Calcium-binding protein and spermatid-associated 1 (hCABS1). While Wes™ offers a faster, more consistent approach with lower requirements for sample and antibody volumes, variations in expected molecular weights and computational algorithms used to analyze the data must receive careful consideration and assessment. Our data suggests that CNIA approaches are likely to positively impact biomarker discovery and validation.

Keywords: Biomarker(s); CABS1; Capillary nano-immunoassay; Immunoprobing; Saliva; Stress; Western blot; Wes™.

Fig. 1.. In Wes, hCABSl overexpression cell lysates render a different immunoreactive pattern than human saliva when using antibodies to hCABSl raised against different parts of the protein.

Both antibodies, H1.0 and H2.0, detect hCABSl moieties at 94 and 65 kDa in a recombinant hCABSl overexpression cell lysate (rhCABSl OEL) (A, n = 4; B, n = 4) and no hCABSl moieties negative control cell lysate (NCL) (C, n = 4; D, n = 4). In rhCABSl OEL and NCL, both antibodies interact with a protein used in Wes as an internal marker peak. In a reference saliva sample (Refsal) used to optimize Wes running parameters, H1.0 detects an hCABSl moiety at 60 kDa (E, n = 19), and H2.0 detects two hCABSl moieties at 58 and 34 kDa (F, n = 19).

Fig. 2.. Increasing stacking matrix and sample quantity in the capillary, as well as primary antibody incubation time, enhances signal of protein-of-interest.

(A) Electrophoretogram of TSST cohort human saliva screened with hCABSl antibody H2.0 generated by running default Wes protocol. No signal of hCABSl is observable (B) Electrophoretogram of the same saliva sample screened with H2.0 generated by modifying Wes running protocol. hCABSl H2.0-expected 34 kDa moiety is observed, along with additional forms at 58 and 65 kDa. Moieties at 18 and 114 kDa were detected by our analysis software but fell below the 2,000 a.u. threshold.

Fig. 3.. CNIA analysis of saliva samples using antibodies H1.0 and H2.0 showed different trends of hCABSl levels.

During the Trier Social Stress Test, participants (n = 16) gave a saliva sample at 15 min pre-challenge, immediately before (−0) challenge, just after ( + 0), and at 15, 30, and 45 min post-challenge. In CNIA, all saliva samples gave a single peak of chemiluminescent signal. The area under each peak was normalized to that of a homolog peak in a reference saliva sample. (A) Antibody H2.0 detected a peak at 34 kDa. Differences were observed when comparing time points relative to measurement immediately after stress test ( + 0). The trend seems to indicate that hCABSl is associated to stress. (B) Antibody H1.0 detected a 60 kDa peak. Differences were observed when comparing time points relative to 15 min ( + 15) post-stress. Interestingly, this 60 kDa peak shows no association to stress. **P < 0.01