Construction of the pET43a-His6-c-myc-GST- human IL-8 expression plasmid
The synthetic fragment His6-c-myc-GST-human-IL-8 was cloned into the pET43a vector (Novagen) with the restriction enzymes NdeI and SalI. The chimeric protein was constituted by a six histidine tag, one additional tag derived from a terminal fragment of the proto oncogene c-myc (EQKLISEEDL), the glutation-S-transferase protein (GST) and the human cytokine interleukin-8 (Gb: NM_00584.4). The sequence of the recombinant plasmid was confirmed by restriction digestion and DNA sequencing.
Induction, expression and purification of protein His6-c-myc-GST- human IL-8
E. coli BL21 (DE3) cells carrying the recombinant plasmid pET43a-His6-c-myc-GST-human IL-8 were grown in 0.5 L Luria Bertani (LB) medium containing 100 μg/mL of ampicillin at 37 °C. When the bacterial growth reached optical density of 0.8 at 600 nm (OD600 nm), 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to induce expression of the protein. The culture was incubated for a further 4 h at 37 °C. Then cells were collected by centrifugation (5000 x g for 5 min), washed with PBS supplemented with 1 mM EDTA and resuspended in the same buffer. The cells were disrupted by sonication with a Bandelin Sonoplus disruptor set at 70% of power. The soluble fraction was clarified by centrifugation (4 °C, 14000 x g, 30 min) and was filtered through 0.22 μm filter. Then it was loaded on a 1 mL HisTrap HP column (GE Healthcare 17–5247-01) connected to an AKTA Prime plus (GE Healthcare). After washing with 20 mM sodium phosphate pH 7.4, proteins were eluted by applying the same buffer plus 500 mM imidazole. Protein concentration was determined by the Bradford assay (Bio-Rad) using bovine serum albumin (BSA) as standard. Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis with 12% polyacrylamide was carried out.
Comparative ELISA assays for the detection of bi-specific model c-myc-GST-human IL-8 protein using two coating antibodies with different affinity
Maxisorp plates (Thermo 442,404) were coated with the antibodies anti-human IL-8 clone MT8H6 or anti-c-myc purified from 9E10 hybridoma diluted to 2 μg/mL each other in Na2CO3/NaHCO3 pH 9,6 and incubated overnight at 4 °C. After blocking with PBS pH 7 containing 1% (w/v) BSA, the model protein GST-c-myc-human IL-8 was added in serially dilutions from 4 to 1000 pg/mL in the anti-human IL-8 plates and from 0.4 to 200 ng/mL on anti-cmyc coated plates. The next steps were as those described for ELISA as follow. Both ELISAs were developed using an anti-human IL-8 biotin clone MT8F19 antibody at 1 μg/mL. After 5 washing steps 100 μl per well of streptavidin-HRP diluted at 100 ng/mL and TMB (3,3′,5,5′- tetramethylbenzidine) were sequentially added to them. Enzyme reaction was stopped with 1 M HCl and color development was measured at 450 nm on a microplate reader (FLUOstar OPTIMA, BMG Labtech).
Chimeric antibody anti c-myc-ChBD
Cloning of anti-cmyc 9E10 antibody from hybridoma cells and fusion with ChBD
RNA was isolated from 106 cells 9E10 hybridoma using the PRImeZOL™ Reagent (Canvax AN1100) following the manufacture’s protocol, reverse-transcribed (RT) into cDNA and VH and VL genes was amplified by PCR with the high-fidelity DNA polymerase (Canvax P0032). Retro transcription of the fragment VK-CK was performed with the primer pCK-2rev (5’TATGCGGCCGCCTTTGTCTCTAACACTCATTCCTG 3′). The primers pVK-1fw (5’GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT 3′) and pCK-2rev were used for the amplification. On the other hand, VH fragment cDNA was obtained using the antisense primer pVH9E10-6rev (5’TTTTAGATCTAATTTTCTTGTCCACCTTGGTGC 3′) and the elongation was developed with pVH9E10-4fw (5’TTTAAGCTTCGCCACCATGAACTTCGGGCTCAG 3′) and pVH9E10-6rev. PCR products were cloned into pSPARK® vector (Canvax C0001). The pSPARK® vector used for this purpose encoded the LacZ α-peptide when no insert was present. This allowed a blue/white selection on IPTG and X-gal containing LB-agar plates. The funtional obtained sequences were then subcloned into mammalian expression vector pCDNA3.4 driven by cytomegalovirus promoter. The VH fragment cloned was fused to the murine FC IgG.
ChBD (chitin binding domain) of chitinase A1 from Bacillus circulans WL-12 was synthetized with the flank restriction enzymes 5′NheI/ 3′NotI. The fragment was ligated simultaneously with the PCR fragment performed with the primers pVH9E10-4fw and pIgG1m-25rev (5’GATAGCTAGCTTTACCAGGAGAGTGGGAGAG 3′) on the pcDNA3.4 vector coding the VH fragment digested by the restriction enzymes HindIII/ NheI. Both fragments were ligated at once into the expression vector opened with the restriction enzymes HindIII/ NotI.
Expression and purification of anti c-myc- ChBD antibody in mammalian cells
Both VH-IgG1 and VL-CK chain of the anti c-myc-ChBD expression vectors were co-transfected in CHO-S (Chinese hamster ovary) cells using the ExpiCHO Expression system kit (Thermo A29133). The antibody was expressed at 32 °C and 5% CO2 for 10 days attending to the manufacturer’s instructions. Cells were isolated by centrifugation and the supernatant was measured by mouse IgG sandwich ELISA and purified by affinity chromatography with protein G column (HiTrap Protein G, GE Healthcare 29–0485-81). Pure antibody concentration was measured by absorbance at 280 nm and purity grade was analyzed by SDS-PAGE.
Flow cytometry to detect chitin binding domain activity using chitin magnetic beads
For the activity detection of the chitin binding domain fused to the heavy chain of anti c-myc antibody, chitin magnetic beads were used (BioLabs E8036S), following the manufacture’s guidelines. The antibodies were diluted in binding buffer at 1 μg/mL (500 mM NaCl, 20 mM Tris HCl, 1 mM EDTA, 0.05% Triton-X100, pH 8) and incubated with 50 μl of beads at 4 °C for 1 h. After 3 washing steps with the same buffer, 1 μg/mL of anti-mouse FITC was added and incubated at 4 °C for 30 min. Beads were analyzed on a flow cytometer (Guava EasyCyte mini, Guava Technologies).
ELISA to detect anti c-myc-ChBD antibody activity compared with the antibody 9E10 from hybridoma
A microplate coated with 2 μg/mL c-myc-GST-human IL-8 antigen was blocked with PBS containing 1% (w/v) BSA. Then serially dilution of the anti-c-myc-ChBD antibody from CHO-S or anti c-myc 9E10 antibody from hybridoma were added and incubated at 37 °C for 1 h. Both antibodies concentrations were ranging from 100 to 0.078 ng/mL. After microplates washing, anti-mouse HRP (Sigma A2554) and TMB were sequentially added to them. Enzyme reaction was stopped with 1 M HCl and color development was measured at 450 nm on a microtiter reader.
Preparation of microtiter plates with acetylated chitosan
The procedure described by Bernard et al., 2004 with modifications was used. Deacetylated chitosan (30 mg) were dissolved in 5 mL of 0.1 M Sodium acetate pH 3. Two deacetylated chitosans with different grades of deacetylation were used: chitosan from white mushroom (60% deacetylation, Sigma 740,063) and chitosan from crab shells (80% deacetylation, Sigma 48,165). Each solution was diluted 20-fold in sodium acetate pH 5, and the polystyrene plates filled with 50 μL per well. Afterward, 8 μL per well of acetic anhydride (Sigma 320,102) were added. The plate was placed in a fume hood and allowed to dry overnight. Eventually, the plate was washed using PBS 10X pH 7.4 and blocked with 0.3 mL PBS (10x) pH 7.4: BSA (1 μg/mL).
Comparison of microplates coated with two different deacetylated chitosans by binding of the chimeric anti-c-myc-ChBD antibody
Two different chitosan acetylated microplates prepared using white mushroom and crab shells chitosan. Anti-cmyc-ChBD antibody in serial dilutions ranging from 0.039 to 50 ng/mL were added. Microplates were incubated at 37 °C for 2 h. After washing, anti-mouse HRP (Sigma A2554) and TMB were sequentially added to them. Enzyme reaction was stopped with 1 M HCl and color development was measured at 450 nm on a microtiter reader.
Optimization of protein c-myc-GST-human IL-8 detection in acetylated chitosan microplates
Anti c-myc-ChBD antibody concentration, incubation time (1, 2, 4, 8 and 16 h) and the binding buffer for the binding reaction chitin- chitin binding domain were established. Concentrations ranging 0.312 to 1 μg/mL of anti c-myc-ChBD antibody and PBS pH 7.4 or NaHCO3 pH 9 buffers were used. ELISA assays were carried out on chitosan acetylated surfaces. Anti c-myc antibody from 9E10 hybridoma was used as negative control of the chitin specific binding. ELISA was revealed with an anti-mouse HRP (Sigma A2554).
Comparative ELISA between standard polystyrene and chitosan acetylated surfaces
ELISA assays were performed simultaneously in standard polystyrene (Maxisorp, Thermo 442,404) and in chitosan acetylated microplates (Fig. 6). Standard microplates were coated with the anti-c-myc antibody while chitosan acetylated plates were coated with the anti-c-myc-ChBD antibody. Afterwards, a concentration range of the chimeric protein His6-c-myc-GST- human IL-8 from 100 to 0.39 ng/mL, was prepared. 100 μL of samples per well were added and incubated at 37 °C for 1 h. After washing steps, the biotinylated antibody anti IL-8 (clone MT8F19) and streptavidin- conjugated horseradish peroxidase were used. TMB was added for color development and plates were measured at 450 nm on a microtiter reader. Both ELISAs were performed simultaneously under the same conditions, on 5 different days, using three replicates of each concentration and 10 replicates of the target.
Influence of serum, plasma and cell culture medium using acetylated chitosan microplates
The c-myc-GST-human IL-8 spiked samples were prepared by mixing two different concentrations of protein, 100 ng/mL and 10 ng/mL in human serum, human plasma, PBS: BSA 0.5% and cell culture medium (RPMI 1640 medium). The sample concentrations were determined in chitosan acetylated microplates plotting in control curve performed in PBS-BSA 0.5%.
The recovery percentages were calculated using the concentrations measured in PBS: BSA as 100% of recovery or expected concentration. We used the equation:
% recovery = (Sample concentration observed- medium concentration unspiked)/ Concentration obtained in PBS: BSA 0.5%.
Assay performance analysis
All datasets obtained from the developed ELISA procedures were subjected to four-parameter logistic function- based standard curve analysis in the GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA): EC50, R2, to determine limit of detection (LOD) and limit of quantitation (LOQ). The EC50 indicates the concentration at which half of the test signal is obtained or the half maximal effective concentration, and R-squared, is a statistical measure of how close the data are to the fitted curve. Both parameters were determined from the report data generated by the software.
Absorbance corresponding to the detection limit was the result of the average absorbance of the blank from all the repeats plus 3 standard deviations of the blank while for quantification limit was the same but 10 standard deviations. Then, by plotting the absorbance on the standard curve, the values of the LOD and LOQ concentration are obtained.