The LAP ELISA measured Latent TGF-β1 without preceding acidification of human samples and did not cross-react with LAP2 or − 3. No cross-reactivity of the LAP ELISA with Latent TGF-β in bovine serum was found making the ELISA suitable also for human cell supernatants containing bovine serum. BALB/c mice were immunized subcutaneously with 10 μg recombinant human (rh) Latent TGF-β1 (R&D Systems, Abingdon, UK) in 50 μl phosphate-buffered saline (PBS) and 50 μl Freund’s complete

adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Mice were boosted using Freund’s incomplete adjuvant week 4 and 7 (Sigma). At week 27, 10 μg antigen in PBS was given intraperitoneally and 3 days later spleen cells were fused with Sp2/0 cells as described (elGhazali et al., 1993). Positive hybridomas Ibrutinib identified by indirect ELISA were subcloned E7080 mouse and cultured. MAbs were purified and biotinylated as described (Zuber et al., 2005). Mice were housed and handled at the Karolinska Institute, Solna, Sweden, according to the guidelines of the Swedish Ethical Committee for Animal Protection. Maxisorp 96-well plates (Nunc, Roskilde, Denmark) were

coated for 16 h at 4 °C with 2 μg/ml of rh LAP1, Latent TGF-β1 or TGF-β1 (R&D Systems) in 100 μl PBS. Other assay steps were at room temperature (RT), using 100 μl/well. Five washes using PBS with 0.1% Tween 20 were made between assay steps. After coating, wells were blocked for 1 h with incubation for buffer (PBS with 0.05% Tween 20 and 0.1% bovine serum albumin). Hybridoma supernatants were diluted in incubation buffer and incubated 2 h. Next, goat-anti-mouse IgG alkaline phosphatase conjugate (Mabtech, Nacka Strand, Sweden) was added and incubated for 1 h, followed by development with para-nitrophenyl phosphate (Sigma) and absorbance measurement (405 nm) by an ELISA reader (Labsystems, Helsinki, Finland). In the LAP ELISA, mAb MT593 (IgG1) was coated at 2 μg/ml and wells were blocked as above. Volumes and temperature for all assay steps and washes in between

were as above. Human plasma was diluted in an ELISA diluent (Mabtech) preventing potential interference by heterophilic antibodies in the samples (Bolstad et al., 2011). Also rh LAP1, Latent TGF-β1 and TGF-β1 and other samples were diluted in ELISA diluent. ELISA diluent was added to acidified samples after the acid treatment. After sample incubation for 2 h, mAb MT517-biotin (IgG1) at 1 μg/ml in ELISA diluent was incubated 1 h followed by streptavidin-horse radish peroxidase conjugate (SA-HRP; Mabtech) in incubation buffer for 1 h. The assay was developed with 3,3′,5,5′-tetramethylbenzidine substrate (Mabtech) and stopped with 1 M H2SO4 followed by absorbance measurement (450 nm). The same protocol was used for TGF-β1 ELISA (Human TGF-β1 DuoSet kit specific for TGF-β1; R&D Systems). Sample levels were determined against standards using rhLAP1 in the LAP ELISA and rhTGF-β1 in the TGF-β1 ELISA.