Depolymerization and conversion of lignin to value-added bioproducts by microbial and enzymatic catalysis

Chondrex, Inc

Abstract

Chondrex, a major secretory protein of human chondrocytes and synovial fibroblasts, is increased in the serum of patients with cartilage and joint disease. We have developed a sandwich ELISA to quantify chondrex in serum. The CVs between trials were 2.8% to 3.7% and the average CVs within the series and overall were 3.6% and 5.4%, respectively. The limit of detectability by linear dilution was 20 µg / L, recovery after dilution was 102% ± 5%, and analytical recovery (of the added analyte) was 98% ± 11%.

The reference interval (central 90% interval) for chondrex in healthy adults was 25 to 95 μg / L. Chondrex values ​​for patients with active rheumatoid arthritis or osteoarthritis were significantly higher than in healthy adults, rheumatoid arthritis patients. inactive and patients with diabetes (P <0.05). In patients treated with disease-modifying antirheumatic drug therapy, the decrease in chondrites values ​​reflected the clinical improvement observed in the responders, while the values ​​were maintained or increased in the non-responders. In conclusion, chondrex may be a useful marker in arthritis clinical research.

Materials and Methods

1. Monoclonal antibodies

Monoclonal antibodies were generated by immunizing A / J mice with purified chondrex. After an initial intraperitoneal boost of 50 µg of chondrites incomplete Freund’s adjuvant, the mice were allowed to rest for 8 weeks. They were then boosted intraperitoneally at 2-week intervals; first with 25 μg of chondrex in incomplete Freund’s adjuvant; then with 10 µg of condrex in 10 mmol / L of phosphate, 150 mmol / L of sodium chloride, pH 7.2; and finally with 5 μg of condrex in 10 mmol / L of sodium phosphate, 150 mmol / L of sodium chloride, pH 7.2, intravenously.

The spleens of two mice were then fused according to standard procedures. The supernatants were screened with a solution-phase ELISA in which the chondrex bound to a well of the microplate was incubated with the supernatant to be analyzed. Sequential incubations with anti-mouse IgG-horseradish peroxidase conjugate and then substrate allowed the detection of anti-chondrex antibodies. Four IgG antibodies were subcloned, isotyped and further examined in a sandwich assay format. A pairwise search included combinations of all monoclonal antibodies as well as polyclonal material.

The final clone (116) was chosen due to its superior sensitivity in a monoclonal: polyclonal sandwich ELISA format, as well as its growth characteristics in cell culture. Monoclonal antibody 116 was purified from hybridomas grown in HB Pro serum-free medium (Irvine Scientific). Terminal culture supernatants were harvested by centrifugation when cell viability dropped to <10-20%.

The supernatants were purified on fast-flow protein A-sepharose (Pharmacia) after 0.2 μm filtration (pore size) and the addition of saturated sodium borate and sodium chloride to final concentrations of 100 g / L and 3 mol / L, respectively. Bound antibody was eluted with 0.1 mol / L glycine, pH 3.0, and the fractions were neutralized by the addition of 10% (by volume) 1.2 mol / L Tris, pH 8.5. Fractions containing antibodies were pooled and dialyzed in 10 mmol / L phosphate, 150 mmol / L sodium chloride, pH 7.2.

2. Biotin-Fab conjugate

Monoclonal antibodies were digested into Fab fragments by incubation with immobilized papain (Pierce Chemical Co.) according to the manufacturer’s instructions. Fab fragments were purified on fast-flow protein A-Sepharose with the use of 10% saturated sodium borate and 3 mol / L sodium chloride. The purified Fab in unbound material was concentrated and dialyzed at 50 mmol / L of sodium carbonate, pH 8.5. Biotinylation was performed with a 15-fold molar excess of D-biosignal-ε-aminocaproic acid N-hydroxysuccinimide ester (Boehringer Mannheim) according to the manufacturer’s instructions.

3. Polyclonal antibody

New Zealand white rabbits were immunized every 3 weeks for 90 days with 50 µg of heparin-purified chondrex. Primary immunization was with chondrex incomplete Freund’s adjuvant, the first boost was in incomplete Freund’s adjuvant, and all subsequent boosters were in 10 mmol / L sodium phosphate, 150 mmol / L sodium chloride, pH 7.2. Rabbits were bled 10 days after immunization. The antisera were purified by protein A-sepharose chromatography (Pierce). Bound antibody was eluted with 0.1 mol / L glycine, pH 3.0, and neutralized by the addition of 10% (by volume) 1.2 mol / L Tris, pH 8.5. Fractions containing antibodies were pooled and dialyzed in 50 mmol / L sodium phosphate, pH 7.5.

4. Calibrators

Calibrators were prepared by dilution of heparin-purified condrex at 0, 20, 50, 100, 200 and 300 μg / L in a calibrator base (10 mmol / L sodium phosphate, 100 g / L bovine serum albumin , 1 g / L sodium azide, pH 7.0).

5. Streptavidin plates

Streptavidin-coated MaxiSorp ™ Nunc-Immuno ™ microtiter plates (from VWR) were prepared by incubating 150 μL (per well) of 10 mg / L streptavidin (Scripps Labs) in 50 mmol / L sodium phosphate, pH 7.2, for 16-24 h at room temperature. The liquid was then aspirated from the wells and the remaining binding sites were blocked by incubation for 2 hours at room temperature with Tris 20 mmol / L, sodium chloride 150 mmol / L, bovine serum albumin 15 g / L, 0, 1 mL / L Tween, 1 g / L sodium azide, pH 7.5.

After washing 3 times with wash buffer (Tris 20 mmol / L, sodium chloride 150 mmol / L, Tween-20 1 mL / L, pH 7.5), the plates were coated overnight (16-24 h) at room temperature with 200 μL / well of 150 g / L sucrose in 10 mmol / L sodium phosphate, 150 mmol / L sodium chloride, pH 7.2. The liquid was aspirated and the plates were dried overnight in low humidity at 25 ° C. Plates were stored in aluminium foil bags with a desiccant at 4 ° C until needed.

Other reagents

Triglyceride solution (Pentex, Triglyceride Superstrate, # 96-052) was purchased from Bayer. Haemoglobin was prepared from a washed erythrocyte lysate. The hemoglobin concentration was determined spectrophotometrically at 578 nm ([heme, mg / L] = A578 nm / 0.0002278). Hydroxychloroquine was obtained from Geneva Pharmaceuticals.

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