Detection of Cystatin C biomarker for clinical measurement of renal disease by developed ELISA diagnostic kits

Nucleic acid gel imaging system, nucleic acid electrophoresis apparatus and protein gel electrophoresis apparatus were purchased from Shanghai Tanon company. Microplate reader was purchased from Thermo Fisher Technology Ltd (Shanghai, China). Polyethylene glycol(PEG) was purchased from Merck Co, Mouse typerisotyping panel kit from Bio-RAD Co, and RPMI MEDIEM 1640 medium, penicillin-Streptomycin double antibody solution, newborn calf serum and HEPES from Life Technologies Gibco Co. Hypoxanthine-Aminopterin-Thymidine (HAT) supplemented medium, Hypoxanthine- Thymidine (HT) supplemented medium, Freund's complete or incomplete adjutants were purchased from Sigma. Natural human cystatin C (N-HCC) was purchased from Enzo Life Sciences Ltd., Horseradish peroxidase-conjugated goat anti-mouse IgG from Santa Cruz Biotechnology Inc., or Tween-20 and Bovine serum albumin (BSA) from Amresco. BALB/c mice were from Shanghai Institutes for Biological Nutrition, according to the ethical permission approved by the committee of Animal Ethical Evaluation, Chinese Academy of Science. The natural camel single-domain heavy chain antibody library was kindly provided by Dr. Ario de Marco for Italian IFOM-IEO center.

The total RNA was extracted from renal epithelial 293 T cells using the TransZol Up RNA kit. The cDNA was synthesized from RNA using the Superscript II reverse transcriptase with OligodT (18) primers, as the template for the PCR reaction. The primers specific for HCC were used to introduce the restriction sites BamH I and Xho I (The primers: 5’-GGATCCAGTCCCGGCAAGCCG-3’ and 5’-CCTCGAGCTAGGCGTCCTGACAGGT-3’). PCR products (363 bp) corresponding to HCC fragments and then connected to pEASY-T1 simple T vectors [27‐29]. The cloning T vectors which contain purpose gene and the prokaryotic expression vector pET-32a was digested with BamH I and Xho I twice and dephosphorylated and gel purified before the ligation incubation. The ligation was performed overnight at 16°C by T4 DNA ligase. The recombinant plasmids were transformed into Rosetta and the transformants were selected on Luria-Bertani LB agar plates supplemented with 100 μg/ml ampicillin. Single bacterial colony was picked from the transformned plate and verified by PCR, and the positive bacteria were induced to express the target protein. The positive single colonies inoculated (1:100) into 10 ml of LB liquid media containing 100 μg/ml ampicillin as appropriate. Bacterial cultures were incubated at 37°C overnight with shaking and then inoculated into 1 L of fresh antibiotic-containing Luria-Bertani (LB). Isopropyl thio-β-D-galactose glycoside (IPTG) was added to a final concentration of 0.5 mM to induce protein expression. Bacteria were harvested by centrifugation (8000 r/min, 10 min, 4°C) and pellets were re-suspended in 100 ml pre-cold PBS, where phenylmethanesulfonyl fluoride(PMSF) was added at 1 mM. Bacterial lysate were centrifuged (12,000 r/min, 15 min, 4°C) and the supernatants were transferred to new tubes, while the precipitates were re-suspended in PBS [27, 30‐33]. An appropriate amount of both the supernatants and suspensions were mixed with an equal volume of 2 × SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) loading buffer. Expression levels of recombinant R-HCC in supernatants and precipitates were analyzed by SDS-PAGE analyses. Bacterial samples that were not induced with IPTG, wild-type Rosetta transformed with pET-32a were used as controls.

The proteins were expressed in an insoluble form as inclusion bodies, where 2 M urea was added, ultrasounded, and hen the lysates were centrifuged (12,000 r/min, 20 min, 4°C). The supernatants were transferred to dialysis bags in PBS and dialyzed overnight at 4°C thrice. The protein solution was dialyzed at the renaturation of His-R-HCC more than 80% (wt/wt). The Ni-NTA agarose (Qiagen) was used to purify His-R-HCC according to the manufacturer’s instructions. Two ml of Ni-NTA agarose pre-equilibrated with the lysis buffer, including 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, PH 8.0, was added to the supernatant and gently mixed at 4°C for 1 h. The lysate agarose mixture was loaded to the column and washed with the buffer, including 50 mM NaH₂PO4, 300 mM NaCl, and 0.05% (v/v) Tween 20, at pH 8.0, by the stepwise addition of graduate imidazole concentrations at 20 mM, 50 mM, 100 mM, 200 mM, or 500 mM, respectively. The protein was eluted using 10 ml of elution buffer with 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole at pH 8.0. Concentrations of purified recombinant proteins were determined by the Bradford assay [30]. His-tagged proteins were digested by thrombin and then the ultrafiltration concentrate was analyzed by SDS-PAGE analyses and Western blot.

Four BALB/c female mice (6-8 weeks old) were immunized with R-HCC by intraperitoneal injections of 100 μL at the concentration of 1 g/L of R-HCC and 100 μL of Freund's adjuvant. The first doses contained complete Freund's adjuvant, and subsequent doses were given a 3-weeks interval using incomplete Freund's adjuvant. Blood samples were collected one week after the fourth injection, and the titer and specificity of antibody response were determined with indirect ELISA [34, 35].

Murine myeloma cells were cultured in high-glucose Dulbecco's minimal Eagle's medium (DMEM) supplemented with 20% (v/v) FBS and 1% (wt/vol) penicillin-streptomycin. Cell fusion procedures were carried out according to the protocols of the Clonal Cell-HY™ kit. Mouse spleen lymphocytes were mixed with the myeloma cells at the ratio of 5:1 using 1 ml of PEG 4000. The fused cells were then transferred into a tissue culture plate containing the methycellulose-based selection medium and incubated. The individual hybridoma clones in the semisolid medium were transferred into a 96-well plate about 12 days after the cell fusion. The supernatants were collected when hybridoma cells were grown to approximately 10-20% (v/v), and initial screening was performed using indirect ELISA. The screen yielded positive hybridoma cells, which were subsequently sub-cloned thrice by the limiting dilution. Aliquots of first hybridomas and the clones were cryopreserved at several stages during the development.

Female BALB/c mice were intraperitoneally injected with 10⁶ hybridoma cells 7 days after intraperitoneal injection with 0.5 ml of liquid olefin. After 14 days, ascites fluid was collected 14 days after the injection with cells and centrifugated at 4000 r/min for 15 min. McAbs were purified from mouse ascites by ammonium sulfate precipitation followed by affinity chromatography on a protein G column [30, 34‐37]. Classes and subclasses of McAbs were identified by mouse monoclonal antibody isotyping reagents (Sigma-Aldrich) following the manufacturer’s instructions [33].

The affinity constants of McAbs were detected after sample preparation, chip surface pretreatment, sample injection, regeneration, and data analysis. Three μg of natural Human cystatin C (N-HCC) and 0.05 M PBS at 7.4 was prepared and filtered. Our pilot experiments showed that the isoelectric point of 9.0 HCC was bonded to the surface of CM5 chip in HCl-glycine buffer at pH = 4.5. Three μg HCC was dissolved in 200ul of HCl-glycine buffer to be fully integrated on the CM5 sensor, and then equilibrated with PBS overnight. The chip was regenerated and then finished, followed by data analysis.

R-HCC was coated overnight at 4°C in 4 ml Nunc-Immuno™ or Maxisorp™ tubes at a concentration of 30 μg/ml using 50 mM sodium carbonate buffer thrice before the addition of 3 × 10¹⁵ phages for the first round of panning after the blocking. Tubes were washed 10 times with PBS 0.05% Tween and PBS after 30 min rocking and 90 min standing upright at room temperature, and bound phages were eluted with 10 mM HCl, pH 2.0. Eluted phages were neutralized by Tris–HCl, pH 8.0 and then used to infect TG1 cells at 37ºC for 40 min. Infected cells were harvested by centrifugation and The new sublibrary of phages was resuspended, titrated, and used in the second round of panning [38, 39]. The procedure was repeated and the enrichment of the phage sublibrary obtained was calculated as the ratio of output/input phages.

Individual colonies from the dilution series were tested for antigen binding by phage ELISA [38, 39]. Tubes containing 1 ml of 2× TY medium were inoculated with 10 μl of the overnight culture. After then, 2 × 10¹² KM13 helper phages were added, mixed, and incubated for 1 h. Discard supernatant and resuspend pellets in 5 ml of 2 × TY medium, incubated for about 18 h, and then separated and transferred. The phage clones were tested as described previously [34, 39]. Maxisorp 96-well plates (Nunc) were coated with HCC and added the antigen at 1 μg/ml. Plates were washed with PBST and treated with anti-M13 McAb HRP conjugated for 1 h at 37°C, and added with 100 μl of TMB solution after PBST washing. Plats were analysed at 450 nm in a microplate reader. Clones with an absorbance value highest were considered positive and the sequences analyzed to identify unique binders.

Clones with unique sequence were subcloned into pET-28a vector to obtain His-tagged recombinant VHH binders after transformation into Rosetta competent cells. The supernatant was purified by nickel column affinity chromatography directly [30, 39]. The purity of the VHH solutions was confirmed by 12% SDS gel electrophoresis and coomassie blue staining. The property of the purified VHH was determined by ELISA and Western Blotting.

The affinity of VHHs was determined as described previously [34, 39]. Plates were coated with N-HCC after blocking and washing. The antibody solution at concentrations below dissociation constant(Kd), 0.5 nM was incubated with increasing concentrations of R-HCC from 0.1 nM to 1 μM. N-HCC-coated plates were added by 10 μl of the reaction mixtures, washed with PBST, and added with 100 μl of TMB solution for the readout at 450 nm.

The optimized concentrations of HCC paired antibody with McAb 5 F2 and 1E11, or phage displayed monoclonal VHHS P-3-2, P-3-24, P-3-33, and P-4-5, were determined by sandwich ELISA. McAbs at 5 μg/ml was added and the absorbance at 450 nm was measured.

The concentration of the coating conjugates with McAbs and VHH phages was optimized by orthogonal test titration. The capture antibody coated concentrations at 10.0, 5.0, 2.5, 1.0, 0.5, 0.2, and 0.1 μg/ml were used and the incubation concentrations of HCC antigen solution were 20 ng/ml. HRP-antibody solutions were diluted to 100, 200, 500, 1000, 2000, and 4000. The one with the OD450nm value closest to 1.0 was selected as the optimal detection concentration and for further assay development.

The properties of the developed assay, e.g. linear range, sensitivity, accuracy and precision, were validated. The linear range and sensitivity was determined according to the making of the standard curve. The HCC standard solution was diluted into 62.5, 31.3, 15.6, 7.8, 3.9, 2.0, 1.0, 0.5, 0.2, and 0 ng/ml, respectively. The standard curve was made using the screened 5 F2-P-3-2 detection kit to detect HCC concentrations. The accuracy of the kit was assayed and calculated by adding HCC to a 5-fold diluted preparation the urine or serum at standard concentrations, and detecting the recovery of HCC. The concentration of HCC in serum samples was also measured by the turbidimetric immunoassay as the reference. The concentration of HCC in serum samples measured by the commercialized kit was 0.63 μg/ml, while the DAS-ELISA could detect the serum levels of 10, 25, 50, 100, 250, 500, and 1000 fold dilution. The precision of the DAS-ELISA was calculated with the intra-variability at 30, 20, 10, or 5 ng/ml of HCC sample solution for 10 times and the coefficient of variation(CV) was used to represent the precision.