Duoset elisa ancillary reagent kit

We measured serum protein concentrations of tPA, BDNF, proBDNF, p75NTR and TrkB using enzyme linked immunosorbent assay (ELISA). Serum concentrations of tPA, BDNF and p75NTR were measured by ELISA kit (tPA (DTPA00; R&D Systems, Minneapolis, MN, USA); BDNF (DBD00, R&D Systems); and p75NTR (ab155436, Abcam, Cambridge, UK)) following the manufacturer's instructions. ProBDNF and TrkB concentrations were estimated using Duoset human ELISA Kit (ProBDNF: DY3175 and TrkB: DYC397; R&D Systems) combined with DuoSet ELISA Ancillary Reagent Kit (DY008; R&D Systems) following the manufacturer's instructions. All the experiments were performed in duplicate. The details of the procedure are shown in the Supplementary Data.

Human (R&D Systems, USA) and mouse IL-1α (R&D Systems, USA) DuoSet ELISA kits, as well as human (R&D Systems, USA) and mouse IL-1β (R&D Systems, USA) DuoSet ELISA kits, were used in combination with DuoSet ELISA Ancillary Reagent kit (R&D Systems, USA). The assays were performed following the manufacturer’s instructions.

All the subjects underwent a peripheral venous blood draw between 08:00 and 10:00 am. The samples were collected in anticoagulant-free tubes and immediately centrifuged for 20 min at a speed of 3000 r/min to obtain patients’ plasma. Plasma samples were then separated and stored at −80 °C until future analysis. ELISA kits were used to measure plasma levels. The details of all the ELISA kits were as follows: mBDNF (DBD00—R&D Systems, Minneapolis, MN, USA); tPA (DTPA00—R&D Systems, Minneapolis, MN, USA); and IL-1β (ab214025—Abcam, Cambridge, MA, USA). Plasma proBDNF, TrkB, TNF- $\mathsf{\alpha }$ , and IL-6 levels were estimated using a DuoSet human ELISA kit (proBDNF: DY3175; TrkB: DYC397; TNF- $\mathsf{\alpha }$ : HSTA00E; IL-6: HS60DC—R&D Systems, Minneapolis, MN, USA) combined with a DuoSet ELISA Ancillary Reagent kit (DY008—R&D Systems, Minneapolis, MN, USA). Based on the manufacturers’ instructions, we determined the concentration of factors in each sample. The results are exhibited in pg/mL. All the experiments were performed in duplicate.

To quantify secreted lubricin, cell culture medium was collected from the TGFβ1 dose response on day 16 and from the 3D bioprinted disc constructs on days 1, 10, and 22. Cell culture medium was frozen at −20 °C until use. Enzyme-Linked Immunosorbent Assay (ELISA) kit (DuoSet ELISA Ancillary Reagent Kit and Human Lubricin/PRG4 kit, R&D systems) was used following the manufacturer’s protocol. Lubricin content was calculated based on the standard curve.

Anti-HPV16-L1 antibody was detected by an enzyme-linked immunosorbent assay (ELISA). The DuoSet ELISA Ancillary Reagent Kit (DY008, USA) was purchased from R&D company. The clear 96-well microplate was coated with 100 μL of purify HPV16-L1 protein at a concentration of 10 μg/mL. Seal the plate and incubate overnight at 4 °C. Then, the plate was washed with the wash buffer (0.05% Tween-20 in PBS, PBST). After blocking the plates with 1% BSA diluting in PBST, 50 μL of diluted serum (10 μL of each sample was diluted in 40 μL blocking buffer) was added to each well. The plates were incubated for 45 min at 37 °C. After washing four times, 50 μL of biotin-labeled goat anti-human IgG (V7830, Promega, China) was added for 30 min at 37 °C. After washing four times, 50 μL of the working dilution of streptavidin-HRP was added to each well for 30 min at 37 °C. After washing 4 times, each well was added with color reagent A and color reagent B. After 15 min of incubation in the dark at 37 °C, the reaction was stopped by the addition of 50 μL of stop solution to each well. The absorbance at 450 nm were detected with a Microplate Reader (BioTke, China). IL-2 and IFN-γ in serum and genital secretions were detected by ELISA kits according to the manufacturer’s instructions (M2000 for IL-2, MIF00 for IFN-γ, R&D, USA).

Cord blood exosomal CNTN2 and BDNF levels were quantified by ELISA. CNTN2 levels were determined using DuoSet Human CNTN2/TAG1 ELISA Kit (DY1714-05) and DuoSet ELISA Ancillary Reagent Kit (DY008) using the protocol provided (R & D Systems). BDNF levels were determined using Quantikine Total BDNF ELISA Kit (DBNT00) using the protocol provided (R & D Systems). CNTN2 and BDNF protein concentrations were determined using standard curves (CNTN2 R² = 0.96−0.99, BDNF R² = 0.99) created using the optical density of known concentrations of a standard solution per the manufacturer’s protocols, and a best fit line was generated using linear regression analysis. The flowchart of experimental study and the validation of exosomal isolation are shown in Figure 1. The levels of exosomal CNTN2 and BDNF in 50 µL of cord blood samples were calculated using the following formula: