After obtaining approval from the local ethics board (CEIC 1868), 41 patients with pleural TB and 48 with non-TB effusions were recruited from a prospectively maintained database and biobank (IRBLleida Biobank B.0000682) at the University Hospital Arnau de Vilanova (Lleida, Spain). An elevation of pleural fluid ADA levels >35 U/L (the diagnostic cutoff for TB in our center) in at least 20% of the non-TB samples was established as a prerequisite. This criterion ensured an adequate number of participants with non-tuberculous effusions and elevated total ADA levels in pleural fluid, thereby accurately reflecting clinical reality.

A definitive diagnosis of TB effusion was made if auramin staining or cultures (either in solid or liquid media) of pleural fluid, sputum, or pleural biopsy specimens were positive or if the latter showed granulomas in the parietal pleura. TB was considered probable in patients with lymphocytic exudates from the first or subsequent thoracentesis, high pleural fluid ADA levels (>35 U/L), negative cytological examinations, and clearance of effusion with anti-TB therapy. If malignant cells were detected on cytological examination of the pleural fluid or biopsy samples, the effusion was categorized as malignant. Parapneumonic effusion was defined as any effusion associated with bacterial pneumonia that was either cured with antibiotics (uncomplicated) or required chest tube drainage (complicated). Other causes of pleural effusion were determined using well-established clinical criteria.

Pleural fluid samples obtained during thoracentesis were collected in 5 mL sterile heparinized tubes for immediate routine biochemical analysis, including ADA. Total ADA activity was determined using an automated spectrophotometric method (Roche Diagnostics, Barcelona, Spain).

A. Coating beads with ADA2 protein

Tosyl-activated M-280 Dynabeads (Invitrogen Life Technologies, Norway) were coated with ADA2 protein, according to the manufacturer’s instructions. Specifically, the coating process involved incubating the beads overnight at 37°C with 60 µg of ADA2 protein and 80 µL (2.4 mg) of beads in 200 µL of 75 mM borate buffer (pH 9.5) containing 75 mM ammonium sulfate. The coated beads were subsequently blocked using PBS (pH 7.4) containing 0.5% (w/v) BSA for 1 h at 37°C, followed by a single wash with PBS (pH 7.4) containing 0.1% (w/v) BSA. Finally, the beads were dissolved in wash buffer to achieve a concentration of 20 mg/ml.

B. Selection of ADA2-specific scFv antibodies by phage display

Antibodies against ADA2 were enriched from a synthetic human antibody library using phage display technique. For selection, two libraries, scFvP (size 1 × 10¹⁰) and scFvM (size 6 × 10⁹), were mixed (19). Both libraries were in the phagemid vector pEB32x (19), which uses a truncated p3 coat protein to display scFv on the surface of the filamentous phage M13. In the first selection round, 2.4x10¹² library phages (mixture of scFvP and scFvM libraries) in 2.5 mL of 50 mM Tris-PBS pH 7.0, 150 mM NaCl, 1% (w/v) BSA, 0.05% (v/v) Tween20 were mixed with 50 µL (1 mg) of ADA2 coated beads and incubated for 2h, at room temperature in rotation. Beads were then collected with a magnet and washed twice with 1 mL of 50 mM Tris-PBS pH 7.0, 1% (w/v) BSA, 0.1% (v/v) Tween20, followed by one wash with 1 mL of 50 mM Tris-PBS pH 7.0, 0.1% (v/v) Tween20. To elute the bound phages, the beads were resuspended in 100 µL TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and 100 µg/ml trypsin (Sigma, USA) and incubated for 30 min at room temperature. Next, 100 µL of 100 µg/ml soybean trypsin inhibitor (Sigma, USA) in TBS was added. E. coli XL1-Blue cells were infected with the eluted phages, and the phages were amplified using the VCS M13 helper phage (Stratagene, USA) as described previously (20). The subsequent two selection rounds were carried out in a similar way, but fewer phages (10¹¹ and 10¹⁰ phages in 1 mL, respectively) were used, and the binding time was 1 h.

E. coli XL1-Blue cells were infected with the 3^rd selection round phages. Gradual dilutions of the infected cells were grown at 37°C on LA plates (tryptone 10 g/L, yeast extract, 5 g/L, NaCl 5 g/L, and agar 15 g/L) with 25 µg/mL chloramphenicol, 12,5 µg/mL tetracycline, and 0.5% glucose to obtain separated colonies. Each well of a 96-well V-bottom-shaped plate containing SB medium, 10 µg/mL tetracycline, 25 µg/mL chloramphenicol, and 0.05% glucose was inoculated with an individual colony. Phage particles were produced using the VCS M13 helper phage (Stratagene, USA). Phages were screened for desired binding activity using Maxisorp plates (Nunc, Denmark) coated with ADA2. The coating was carried out overnight at room temperature with 1 µg/mL ADA2 in 50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN₃ at 100 µL/well. The coated plates were blocked with 1% bovine serum albumin (BSA) in the same buffer solution. For the detection of phage particles in the screening assay, Eu-N1-labelled anti-phage Mab was used, as previously described (20).

The genes encoding the most promising scFv clones were digested from the phagemid vector with SfiI and cloned into the pAK600H vector (21), followed by transformation into E. coli XL1-Blue cells. Five milliliters of SB/Amp-Tet-Glu medium (Tryptone 30 g/L, yeast extract 20 g/L, MOPS 10 g/L, 100 µg/ml Ampicillin, 12,5 µg/mL tetracycline, and 0.5% glucose) were inoculated with individual colonies and grown at 37°C. The following day, the preculture was diluted 100-200 times with fresh SB/Amp-Tet medium (without glucose) and grown until the OD600 reached 0.6. scFv production was induced using 200 µg/L IPTG. The induced cultures were grown overnight at 20°C. The next day, the bacterial cells were centrifuged, resuspended in IMAC30 (20 mM phosphate buffer, 150 mM NaCl, 30 mM imidazole, pH 7.4), and sonicated. Purification was performed using Nickel Sepharose HiTrap columns (GE Healthcare). IMAC30 was used as the binding/wash buffer, and IMAC300 (20 mM phosphate buffer, 150 mM NaCl, 300 mM imidazole, pH 7.4) was used to elute the bound proteins. The buffer was changed to TSA using GE desalting columns (Sigma Aldrich).

Maxisorp plates (Nunc, Denmark) were coated with 5 µg/mL anti-hADA2 rabbit polyclonal antibodies (22) in PBS at +4°C overnight. The next day, the plates were washed three times with PBS and 0.05% Tween 20 and blocked with 2% BSA in PBS at room temperature for 2 h. After aspiration of the blocking buffer, the plates were stored at +4°C for three months. Next, 100 µL of the samples and hADA2 dilutions in 0.5% BSA and PBS were added to the wells and incubated for 2 h at room temperature. The plates were then washed three times with PBS and 0.05% Tween 20 and incubated for 1 h with 100 µL of 1 µg/mL H11 scFv-AP at room temperature. The plates were then washed four times with PBS containing 0.05% Tween 20. Then, 100 µL of 5 mM para-nitrophenyl phosphate (Thermo Fisher) in 50 mM Tris (pH 9), 200 mM NaCl, and 1 mM MgCl₂ was added to each well and incubated at 37°C for 5 h. Optical density at 405 nm was determined using a Synergy H4 microplate reader (BioTek).

Continuous and categorical variables are expressed as medians (25 and 75 percentiles) and percentages, respectively. For between-group comparisons, either the Kruskal-Wallis or Fisher’s exact test was used, as appropriate. Correlations were assessed using Spearman’s correlation coefficient. Receiver operating characteristic (ROC) curves were constructed to illustrate the diagnostic accuracy of ADA and its isoenzymes. The measures of test efficacy included sensitivity, specificity, and likelihood ratios. The level of significance was set at p<0.05 (two-tailed). Data were analyzed using a statistical software package (SPSS version 18.0; SPSS Inc., Chicago, IL, USA).

ADA2 specific antibodies were enriched from synthetic single-chain antibody fragment phage libraries (19, 20) using phage display. The selections were performed against human ADA2 produced in S2 insect cells (22) covalently bound to magnetic beads. After each panning round, the phage libraries were tested for their ability to bind to ADA2 in a phage immunoassay, where phage binding to ADA2 coated 96-well plates was detected via time-resolved fluorescence spectrocopy using anti-phage Eu-labeled antibodies. After three selection rounds, when a significant increase in the immunoreactivity of the phage was observed, 95 bacterial colonies were used to inoculate cultures on a 96-well plate to produce individual scFv-phage clones. When tested for their ability to bind to ADA2 using an immunoassay, 15 phage clones were found to selectively bind to the enzyme. The anti-ADA2 single-chain antibody encoding genes were cloned from the phagemid into the pAK600H vector for the expression of scFv as a fusion with bacterial alkaline phosphatase (scFv-AP). Recombinant anti-ADA2 scFv-AP-fused proteins were overexpressed in E. coli and purified using Ni-NTA Chelating Sepharose. The antibodies were tested for their ability to bind to ADA2 using biolayer interferometry. The anti-ADA2 scFv-AP fusion proteins ( Figure 1 ) with the highest apparent dissociation constant (Kd<10 nM) were selected for testing in the ADA2 ELISA.

For the ELISA, 96-well high-binding plates were coated with anti-ADA2 rabbit polyclonal antibodies (23) ( Figure 2A , step 1). The second step involved standard dilutions of recombinant ADA2 and clinical samples with unknown ADA2 concentrations. After incubation and washing of the unbound proteins, anti-ADA2 scFv-AP was added to the plates (Step 3). The concentration of anti-ADA2 scFv-AP bound to ADA2 was determined using the alkaline phosphatase substrate p-nitrophenyl phosphate (pNPP). Following incubation, the increase in absorbance at 405 nm was measured using a microplate reader. A typical standard curve is presented in Figure 2B . The detection limit for ADA2 concentration in clinical samples was 1 ng/ml. Anti-ADA2 scFv-AP was remarkably stable, providing reliable and reproducible results after 5 years of storage ( Supplementary Figure S1 ).

To validate the ADA2 ELISA with anti-ADA2 scFv-AP ( Figure 1A ), we analyzed pleural effusions from 41 patients with pleural TB and 47 with non-TB effusions. The characteristics of the study population are presented in Table 1 . Patients with TB were considerably younger (median age 32 years) and uniformly exhibited total ADA values above the diagnostic threshold of 35 U/L, in contrast to the older age and variable total ADA levels observed in the non-TB groups. As expected, the total ADA and ADA2 concentrations were significantly higher in pleural fluid from patients with TB, both confirmed and probable cases ( Table 2 ) (9). This finding suggests a robust association between TB and increased ADA2 secretion. The distribution of the ADA measurements is shown in Figure 3 . Panel A shows that ADA2 concentrations segregate TB from non-TB effusions, whereas Panel B shows a broader overlap in the total ADA activity. This visual distinction underscores the enhanced discriminatory capability of ADA2. Additionally, Figure 4 demonstrates a strong positive correlation between ADA2 and total ADA (Pearson’s r = 0.727; Spearman’s r = 0.788), confirming that ADA2 constitutes a significant component of total ADA but provides enhanced diagnostic specificity when measured selectively. The receiver operating characteristic (ROC) analysis in Figure 5 further supports the utility of the ADA2 assay, as its ROC curve shows a higher area under the curve than that of the total ADA assay, indicating improved accuracy in distinguishing between tuberculous and non-tuberculous effusions. In terms of assay specificity, Table 3 shows that several non-TB specimens (e.g., 4 of 14 UPPE and 2 of 8 CPPE cases) exhibited false-positive total ADA values, while the corresponding ADA2 levels remained low, except for one CPPE. This reduction in false positives is critical for achieving diagnostic precision. Complementing these findings, Table 4 presents the diagnostic performance of the ADA2 assay: using a cutoff of 300 ng/mL, the assay achieved a sensitivity of 98% and specificity of 91%, with a positive likelihood ratio (LR+) of 11.5 and a negative likelihood ratio (LR–) of 0.03. These metrics indicate that a positive ADA2 result increases the pre-test probability of TB by approximately 50%, whereas a negative result virtually rules out the disease. Finally, Table 5 focuses on malignant pleural effusions and shows that, although most malignancies exhibit low ADA2 concentrations, lymphoma cases have significantly elevated ADA2 levels (median, 300 ng/mL), a finding that warrants cautious interpretation when malignancy is considered.