The new assay, described in Frontiers in Allergy by researchers from EPS Holdings, EPS Innovative Medicine, Intelligence and Technology Lab, and Wakayama Medical University, focuses on a molecule called secretory immunoglobulin A, or SIgA. Unlike the antibodies circulating in blood, SIgA is the workhorse of the body’s mucosal defenses, guarding the wet surfaces of the mouth, nose, and gut. Structurally, SIgA is polymeric—mainly dimeric—and is complexed with a secretory component that shields its enzymatically vulnerable regions. This molecular armor makes SIgA remarkably stable in harsh environments where other antibodies would be degraded. Because SIgA plays a central role in immune tolerance toward commensal bacteria and dietary antigens, allergen-specific SIgA in saliva has long been suspected to be a promising biomarker for judging whether sublingual immunotherapy is taking hold.

There has, however, been a stubborn technical obstacle standing in the way. Saliva is a notoriously difficult fluid for immunoassays. It teems with bacteria and other microorganisms that are physiologically absent from blood, and it contains abundant lytic enzymes, including matrix metalloproteases and lysozymes, that can degrade immunoglobulins and even attack the reagents of the assay itself. These components can alter the conformation of proteins or generate neo-antigens, producing non-specific binding and a high background noise that drowns out the true signal. In previous studies measuring antigen-specific SIgA in saliva, samples had to be assayed at low dilutions—typically up to 1:160, and between 1:2 and 1:160 in studies of salivary antibodies against SARS-CoV-2. Low dilutions mean more interfering material and therefore more background, which limits sensitivity and makes small changes in antibody levels nearly impossible to detect.

The research team’s solution combines two clever engineering choices. First, they adopted a signal amplification chemistry known as catalyzed reporter deposition, originally reported by Bobrow and colleagues, which uses biotinyl-tyramide, or BT-Tyr. In this scheme, horseradish peroxidase serves a double role, acting both as a reporter enzyme and as the catalyst for amplification. After the peroxidase-labeled reagent is immobilized in the ELISA well, BT-Tyr is added together with a trace amount of hydrogen peroxide. The enzyme converts the biotinylated phenolic compound into reactive free radicals that deposit covalently onto electron-rich moieties—tyrosine and tryptophan residues—of the blocking protein coating the well. A second dose of streptavidin-conjugated peroxidase then binds the deposited biotin, creating a clump of reporter signal. Crucially, the degree of amplification correlates with the amount of analyte originally present, so quantitativeness is preserved rather than sacrificed.

The second choice addressed specificity. Instead of detecting SIgA with a labeled anti-IgA antibody, the researchers biotinylated the allergen itself and used it as the reporter. The target allergen was Sugi basic protein, the principal antigen complex of Japanese cedar pollen, comprising the major antigens Cry j1 and Cry j2. In an absorption test designed to prove specificity, pooled saliva diluted 1:400 was preincubated with free SBP before assay. When biotinylated SBP served as the reporter in antibody-coated wells, preincubation with SBP at just 0.8 micrograms per milliliter was enough to absorb the specific SIgA and abolish the signal. In contrast, when a biotinylated anti-IgA antibody was used as the reporter in antigen-coated wells, the blocking effect never exceeded 80 percent even at 4 micrograms per milliliter, because non-specific binding of saliva components interfered. The biotinylated-antigen reporter approach was clearly superior.

Sensitivity figures were equally striking. Compared with a non-amplified version of the assay, the BT-Tyr-amplified method delivered more than 200-fold signal amplification. The limit of detection—defined as the blank mean plus three standard deviations—was 0.104 absorbance units for the amplified assay versus 0.039 for the non-amplified version, and raw readings from a saliva sample diluted 12,000-fold still rose above the detection threshold. Blocking chemistry mattered here too: sodium casein outperformed bovine serum albumin as a blocking agent, yielding blank absorbance of roughly 0.041 compared with 0.143 for BSA, thanks to the heterogeneous, small-sized casein proteins that blanket the plate surface efficiently without obscuring the immobilized capture antibodies.

With the assay validated, the team turned to the clinic. Twenty-two patients with Japanese cedar pollinosis receiving sublingual immunotherapy with CEDARCURE tablets—2,000 JAU daily for the first week, then 5,000 JAU daily—along with five untreated patients, provided saliva samples at three-month intervals over twelve months. Each sample was diluted just 1:450, a dilution far higher than conventional assays could tolerate. The results revealed clear individual differences. Defining a responder as a patient whose salivary SBP-specific SIgA exceeded an absorbance of 0.2 at any time point, eleven of the sixteen analyzed immunotherapy patients—69 percent—qualified as mucosal immune responders, while five, or 31 percent, never crossed the threshold and were classified as non-responders. None of the untreated patients showed any increase; their absorbance values never exceeded 0.2 throughout the study.

The timing of the immune response was perhaps the most clinically meaningful finding. Among responders, salivary SBP-specific SIgA levels rose significantly by nine months after starting treatment at the latest, and the differences were statistically robust: at the nine-month point, responders differed from non-responders with p = 0.002 and from untreated patients with p = 0.036; at twelve months, the corresponding values were p = 0.0004 and p = 0.0014. In practical terms, this means that within roughly three-quarters of a year—a fraction of the typical multi-year immunotherapy course—patients and physicians could, in principle, obtain an objective reading of whether the mucosal immune system is mounting the desired response, rather than waiting for pollen seasons to pass and symptoms to (or not to) fade.

The researchers are careful about the limits of their preliminary study. The sample size was small, and larger cohorts will be needed for definitive statistical evaluation. They also note that a standardized arbitrary unit, built from a standard curve generated by serially diluting a high-titer saliva sample, will be required to minimize variation between assays, and that future work must test whether rising SIgA levels actually correlate with symptom improvement. Yet the practical advantages of the platform are considerable. All reagents are commercially available, and readout requires only a conventional chromogenic plate reader measuring absorbance at 450 nanometers—far cheaper and more widely accessible than fluorescence or chemiluminescence instruments. And because most allergens are proteins that can be biotinylated with standard labeling kits, the same amplified format could be adapted to monitor immunity against virtually any allergen, giving clinicians a versatile new window onto the mucosal immune system and, potentially, an early warning system for patients who are not responding to treatment.

Subject of Research: A sensitive amplified ELISA for measuring allergen-specific salivary secretory IgA to monitor sublingual immunotherapy efficacy in Japanese cedar pollinosis

Article Title: A sensitive colorimetric ELISA to measure allergen-specific salivary SIgA for monitoring sublingual immunity efficiency in pollinosis SLIT

Article References: Yamamoto, T., Mitsunaga, F., Kotani, A., Tajima, K., Hotomi, M., & Nakamura, S. (2026). A sensitive colorimetric ELISA to measure allergen-specific salivary SIgA for monitoring sublingual immunity efficiency in pollinosis SLIT. Frontiers in Allergy, 7, Article 1857343. https://doi.org/10.3389/falgy.2026.1857343

Image Credits: AI Generated

DOI: 10.3389/falgy.2026.1857343

Keywords: sublingual immunotherapy, ELISA, secretory IgA, saliva, Japanese cedar pollinosis, biotinyl-tyramide, signal amplification, allergen-specific antibodies, Sugi basic protein, mucosal immunity, allergy biomarker, Frontiers in Allergy