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. 2025 Aug 14:16:1646134.
doi: 10.3389/fimmu.2025.1646134. eCollection 2025.

Developing and validating anti-ADA2 single-chain antibodies coupled to alkaline phosphatase for diagnosing pleural tuberculosis

Maksym Skaldin  1   2   3 , José M Porcel  4 , Urpo Lamminmäki  5 , Silvia Bielsa  4 , Andrey V Zavialov  1   2   6
Affiliations

Developing and validating anti-ADA2 single-chain antibodies coupled to alkaline phosphatase for diagnosing pleural tuberculosis

Maksym Skaldin et al. Front Immunol. .

Abstract

Introduction: Adenosine deaminases ADA1 and ADA2 reduce adenosine concentrations, which regulate cellular immune responses to activation signals. It has been shown that ADA2 activity increases in the pleural fluid of patients with tuberculosis (TB).

Methods: We engineered recombinant scFv-AP antibodies using phage display technology to select high-affinity binders against ADA2. These were incorporated into a sandwich ELISA, allowing for the precise measurement of ADA2 levels in pleural fluid.

Results: The assay was tested on pleural samples from 41 patients with TB and 47 with non-TB effusions, including those with malignancies and parapneumonic effusions. Results showed that ADA2 concentrations were significantly higher in patients with TB than in other groups, and the ADA2-based assay exhibited improved diagnostic specificity (91%) compared with total ADA testing (76%). A cutoff of 300 ng/mL for ADA2 yielded a sensitivity of 98% and a negative likelihood ratio of 0.03, effectively ruling out TB when the result was negative.

Discussion: The new ADA2 assay offers a simple, reliable, and more specific alternative for diagnosing pleural TB, with potential applications in other ADA2-related disorders.

Keywords: ADA2 (Adenosin deaminase 2); ADA2 ELISA; pleural ADA; pleural tuberculosis; pleural tuberculosis diagnosis; single chain (scFv).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. The reviewer LD declared a past co-authorship with the author AZ to the handling editor.

Figures

Figure 1
Figure 1
Anti-ADA2 scFv-AP structure (A), gene composition, and amino acid sequence (B).
Figure 2
Figure 2
Anti-ADA2 scFv-AP ELISA (A) Schematic representation of the four major steps of the anti-ADA2 scFv-AP ELISA. Step 1: Coating a 96-well plate with polyclonal anti-ADA2 antibodies. Step 2: Specific binding of ADA2 to anti-ADA2 polyclonal antibodies. Step 3: Binding of anti-ADA2 scFv-AP to ADA2. Step 4: Detection of anti-ADA2 scFv-AP bound to ADA2 using an alkaline phosphatase (AP) substrate. (B) A typical standard curve showing a linear dependence between ADA2 concentration and absorbance at 405 nm after incubation with the alkaline phosphatase substrate p-nitrophenyl phosphate (pNPP).
Figure 3
Figure 3
Distribution of pleural fluid ADA2 isoenzyme concentration and total ADA activity in the different diagnostic groups. (A) ADA2 concentrations in pleural fluid from different diagnostic groups were obtained using ADA2 ELISA with anti-ADA2 scFv-AP. (B) Total ADA activity in various diagnostic groups was determined using a colorimetric assay. ADA, adenosine deaminase; CPPE, complicated parapneumonic effusions; MPE, malignant pleural effusion; TB, tuberculosis; UPPE, uncomplicated parapneumonic effusions.
Figure 4
Figure 4
Correlation between total ADA and ADA2 isoenzyme.
Figure 5
Figure 5
ROC curves for total ADA activity (A) and ADA2 concentrations (B).
See this image and copyright information in PMC

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