Elisa

To determine cortisol and testosterone concentration, the available immunoenzymatic commercial assay dedicated and validated for equine species was used (cortisol cat. no.: ELK8554; testosterone cat. no.: ELK8556; ELISA, ELK Biotechnology Co., Ltd., Wuhan, China). Intra-assay precision (precision within an assay): CV% < 8%; inter-assay precision (precision between assays): CV% < 10%.
SAA concentrations were measured using a multispecies but validated for equine species immunoenzymatic commercial assay (cat. no. TP-802; PHASE SAA Assay, Tridelta Ltd., Maynooth, Ireland). Sample dilution was 1:1000 instead of 1:2000 recommended by the manufacturer’s protocol, and the results were appropriately recalculated. The assay, including the dilution, was previously validated for the determination of SAA concentrations in horses (16 (link)). The used enzyme immunoassay has an intra-assay precision CV% < 5% and an inter-assay precision CV < 11. The absorbance was measured by Multiscan Reader (Labsystem, Helsinki, Finland) using a Genesis V 3.00 software program.

Serum samples were diluted twice, and the level of transforming growth factor beta1 (TGF-β1) was measured with commercially available ELISA (Cusabio, Wuhan, China). The value was read at 450 nm, and the detection range was 0.78 ng/mL-50 ng/mL. The interleukin-6 (IL-6) and collagen type-1 C-terminal telopeptide (CITP) levels were measured with a sandwich enzyme immunoassay with a commercially available ELISA (ELK Biotechnology), following the instructions. The intra- and interassay precision of all assays were < 8% and < 10%, respectively.

After co-transfection with the adalimumab HC and LC constructs, cells were selected in the presence of G418 as described above to establish stable CHO cell colonies. Then, the cells were suspended in a serum-free medium (ProteinEasy Biological Products Co., Ltd., Xinxiang, China) without antibiotic at a concentration of 5 × 10⁶ cells/mL with a working volume of 30 ml in 125-ml Corning shake flasks. After 7 days, supernatants were collected, and adalimumab expression was analyzed by Western blotting and enzyme-linked immunosorbent assay (ELISA) as previously described (Wang et al., 2018 (link)). For Western blot analysis, 20 μl aliquots of the supernatant containing adalimumab were electrophoresed on a 10% SDS-PAGE gel, after which proteins were transferred to polyvinylidene fluoride membranes and incubated with 1:5,000 dilutions of goat anti-human antibody (EarthOx, San Francisco, CA, USA) for 2 h at room temperature. After washing with PBST (PBS with 0.1% Tween20), the protein bands were visualized using an enhanced chemiluminescence substrate kit (Beyotime Biotech Co., Ltd., Shanghai, China). At the same time, adalimumab expression was quantified with ELISA (ELK Biotechnology Co., Ltd., Wuhan, China) according to the provided protocol. Cell concentration was determined with a Countstar cell counter, and adalimumab specific productivity (pg/cell/day) was calculated.

The hepatic I/R injury-induced expression of inflammatory cytokines in skeletal tissues, specifically tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1-beta (IL-1β), was determined by ELISA (ELK Biotechnology, Wuhan City, China) according to the manufacturer’s instructions. In addition, qRT-PCR was used to detect the mRNA levels of inflammatory factors at the transcriptional level. According to the manufacturer’s protocol, total RNA was extracted from frozen skeletal muscle tissue using Trizol reagent (Invitrogen Life Technologies). First strand cDNA was synthesized using PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa Bio Inc.). Candidate gene expression levels were measured using an RT-PCR thermocycler (StepOne™ Life Technologies) with the following specific primers: IL-1β, forward: 5′-GTG​GCA​GCT​ACC​TAT​GTC​TTG​C-3′, reverse: 5′-CCA​CTT​GTT​GGC​TTA​TGT​TCT​GT-3′; IL-6, forward: 5′-GCC​AGA​GTC​ATT​CAG​AGC​AAT-3′, reverse: 5′-CTT​GGT​CCT​TAG​CCA​CTC​CT-3′; and TNF-α, forward: 5′-CAC​CAC​GCT​CTT​CTG​TCT​ACT​G-3′, reverse: 5′-GCT​ACG​GGC​TTG​TCA​CTC​G-3′. The mRNA level for each target gene was calculatedusing the Delta-Delta-CT method and normalized to the β-actin mRNA level from the same sample. The primers for β-actin were as follows: forward: 5′-CGT​TGA​CAT​CCG​TAA​AGA​CCT​C-3′, reverse: 5′-TAG​GAG​CCA​GGG​CAG​TAA​TCT-3′.

The concentrations of the following growth factors were evaluated by ELISA (ELK Biotechnology—Biolake, Donghu New & High Technology Development Zone, Wuhan, China): PDGF-AB (pg/mL, IGF-1 (ng/mL); EGF (pg/mL), FGF2 (pg/mL), TGF-ß1 (pg/mL). The assay was performed in triplicate on each of the 3 batches of PL, PLS, and FBS.
Additionally, the concentrations of the following cytokines and growth factors were evaluated by Luminex (MILLIPLEX Human Cytokine/Chemokine Magnetic Bead Panel—Immunology Multiplex Assay, Luminex xMAP technology, Merck, Darmstadt, Germany): interleukin 6 (IL-6), interleukin 10 (IL-10), the chemokine CCL5 or RANTES, platelet-derived growth factor isoform AA (PDGF-AA), vascular endothelial growth factor A (VEGF-A), tumor necrosis factor-alpha (TNF-α), receptor antagonist interleukin 1 (IL1RA), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-colony-stimulating factor (G-CSF). The evaluation was performed in duplicate on each of the 3 batches of PL, PLS, and FBS.

After reperfusion procedure, the blood samples were drawn from the postcaval vein and centrifuged (3,000 rpm, 15 min) at 4°C. The serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured by an automatic analyzer (Chemray 240 analyzer, Dulei Biotechnology, Shenzhen, China). The serum tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1-beta (IL-1β) were analyzed by ELISA (ELK Biotechnology) following the manufacturer's instructions.