Materials
HRP, luminol, Cu(NO3)2, KNO3, CaCl2, NaCl, Mg(NO3)2, AlCl3, Zn(NO3)2, AgNO3, CoCl2, Pb(NO3)2, Ni(NO3)2, FeCl3, K2HPO4, KH2PO4, PPi, tris(hydroxymethyl) aminomethane (Tris), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 3-maleimidobenzoic N-hydroxysuccinimide ester (MBS), polyethylene glycol 4000 (PEG 4000) and gel extraction kit were purchased from Sigma-Aldrich Co. (Shanghai, China). Human PCSK9 protein as well as HRP-labelled goat anti-mouse IgG was obtained from Abcam Co. (Shanghai, China). PCSK9-Ab was obtained from FANTIBODY (Chongqing, China). PCSK9 peptide 1-KLH: EGRVMVTDFENVPEEDGTRFHRQAS was obtained from Jier Biochem Co. (Shanghai, China). Splint R ligase, phi 29 DNA polymerase, dNTPs, ATP and corresponding buffer were purchased from New England BioLabs (Beverly, MA, U.S.A.). Foetal bovine serum (FBS), RPMI-1640 medium, DMEM medium, BCA protein kit, fast silver stain kit, and TMB Kit were bought from KeyGEN BioTECH Corp., Ltd (Jiangsu, China). HAT supplement (100×), HEPES buffer, sodium pyruvate, L-glutamine and PS (10 U mL−1 penicillin and 10 mg mL−1 streptomycin) were purchased from ThermoFisher (Shanghai, China). Hydrogen peroxide (30%), bovine serum albumin (BSA), and all DNA oligonucleotides were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The DNA sequences were listed in Supplementary Table 1. The reaction buffer (pH 7.2) contained 500 mM Tris-HCl, 100 mM KCl, 100 mM (NH4)2SO4, 100 mM ATP and 40 mM DTT. Ultrapure water from a Millipore water purification system (Milli-Q, Millipore) was used for all experiments.
Apparatus
BioSpectrum 615 Imaging System with a cooled low-light CCD camera was used for chemiluminescent imaging to collect the mean pixel intensity within a circle. Chemiluminescence spectra was measured on F-7000 spectrometer (HITACHI, Japan). Absorption spectra was recorded on UV-3600 UV−vis-NIR spectrophotometer (Shimadzu Company, Japan). Chemiluminescent kinetic curves were obtained on MPI-A multifunctional electrochemical and chemiluminescent analytical system with PMT 600 (Xi’an Remex Analytical Instrument Co., Ltd. China). The gel electrophoresis was performed on Mini-PROTEAN Tetra System (Bio-RAD, USA) and imaged on Biorad ChemDoc XRS (Bio-Rad, USA). ELISA results were read out by Multiskan Sky (Thermo Fisher, USA).
Preparation of HRP-Cu2+ complex
After HRP (10 μg mL−1) was incubated with Cu2+ (6 mM) at 37 oC for 30 min, excess Cu2+ was removed by ultrafiltration (50 kDa). The concentration of HRP-Cu2+ complex was measured with BCA protein kit, which was then diluted to 1 μg mL−1 and stored at 4 oC for further use.
Preparation of PCSK9-DNA probes
Briefly, PCSK9 (40 μL, 1 mg mL−1) was firstly reacted with a 40-fold excess of MBS in PBS (10 mM, pH 7.2) with a total volume of 100 μL for 2 h at room temperature to obtain PCSK9-MBS by ultrafiltration. Meanwhile, 150-fold molar excess of TCEP was used to reduce thiolated DNA (DNA 1 or DNA 2, 10 μL, 100 μM) in PBS (10 mM, pH 5.5) with a total volume of 200 μL for 2 h at room temperature. After removing extra TCEP by ultrafiltration, the reduced DNA 1 or DNA 2 was mixed with PCSK9-MBS obtained-above to incubate for 2 h at room temperature, and the PCSK9-DNA probes were purified by ultrafiltration. The concentrations of PCSK9-DNA probes were calibrated with the BCA protein assay kit. The successful preparation of PCSK9-DNA probes was confirmed by native polyacrylamide gel electrophoresis (PAGE) analysis and protein-staining method.
Preparation of block-primer
After primer and block were mixed at a ratio of 1:2 to react at 95 oC for 5 min, block-primer was obtained, separated with gel extraction kit, and stored at 4 oC.
Preparation of hybridoma cells
Spleen cells, which were obtained from PCSK9 peptide1-KLH immunized BALB/c female mice, SP2/0 myeloma cells and BALB/c mice peritoneal macrophages (feeder cells) were supplied by FANTIBODY (Chongqing, China). After spleen cells were fused with SP2/0 myeloma cells by mixing them at a ratio of 1:5 and adding dropwise 0.8 mL PEG 4000 solution (1 g mL−1) within 1 min to incubate at 37 oC for 1.5 min, 29.2 mL of preheated serum-free RPMI-1640 was added into the mixture incubate at 37 oC for 5 min. The obtained hybrid cells were dispersed in 50 mL DMEM with 20% FBS, and mixed with feeder cells and then HAT supplement. After 1-week culture at 37 oC, heterogeneous hybridoma cells were obtained, which contained specific antibody-secreting cells, nonspecific antibody-secreting cells or nonsecretors. In order to establish the chemiluminescent screening procedure, the obtained heterogeneous hybridoma cells were conducted by subclone process with ELISA screening until they exhibited positive result, which were named as 6A6 hybridoma cells.
Electrophoresis analysis
8% native polyacrylamide gel was prepared using 1 × TBE buffer to load the mixtures, which were prepared by mixing 5 μL samples with 1.5 μL 5× loading buffer and 1 μL SYBRTMGold dye to stand for 3 min. The gel electrophoresis was run at 100 V for 60 min in 1 × TBE buffer and visualized by a Biorad ChemDoc XRS (Bio-Rad, USA).
Chemiluminescent response to primer
After the mixtures containing 10 μL primer at different concentrations and 1 μL 6 μM padlock were heated at 95 oC for 5 min and cooled to room temperature, 9 μL solution containing splint R ligase (25 U μL−1, 0.5 μL), phi29 polymerase (10 U μL−1, 0.5 μL), reaction buffer (1 μL), dNTPs (10 mM for each of dATP, dGTP, dCTP, and dTTP, 1 μL), BSA (20 mM, 1 μL) was added in the mixture to incubate at 37 oC for 1 h for performing the RCA reaction, which was terminated at 65 oC for 10 min. Afterward, 10 μL HRP-Cu2+ (1 μg mL−1) was added into the mixture for 5-min incubation to perform the chemiluminescence measurement with an exposure time of 1 min by addition of 20 μL freshly prepared mixture of H2O2 (2.5 mM) and luminol (0.5 mM).
Chemiluminescent response to PCSK9-Ab
10 μL PCSK9-Ab with different concentrations were firstly added into 10 μL mixture of PCSK9-DNA probes (10 μg mL−1, 2 μL), block-primer (2 μM, 1 μL), padlock DNA (6 μM, 1 μL), dNTPs (10 mM for each of dATP, dGTP, dCTP, and dTTP, 1 μL), reaction buffer (1 μL), BSA (20 mM, 1 μL), splint R ligase (25 U μL−1, 0.5 μL), phi 29 polymerase (10 U μL−1, 0.5 μL), and 2 μL water to incubate for 1 h at 37 oC for releasing the primer and performing the RCA reaction. After the reaction was terminated at 65 oC for 10 min, 10 μL HRP-Cu2+ (1 μg mL−1) was added to react for 5 min, and then 20 μL freshly prepared mixture of H2O2 (2.5 mM) and luminol (0.5 mM) was added to collect the chemiluminescent images by CCD with an exposure time of 1 min.
Calibration curve for CLA of PCSK9-Ab
10 μL DMEM culture media with different concentrations of PCSK9-Ab were added into 10 μL mixture of PCSK9-DNA probes (0.1 μg mL−1, 2 μL), block-primer (2 μM, 1 μL), padlock DNA (6 μM, 1 μL), dNTPs (10 mM for each of dATP, dGTP, dCTP, and dTTP, 1 μL), reaction buffer (1 μL), BSA (20 mM, 1 μL), splint R ligase (25 U μL−1, 0.5 μL), phi 29 polymerase (10 U μL−1, 0.5 μL) and 2 μL water to incubate at 37 oC for 1 h. After the RCA reaction was terminated at 65 oC for 10 min, 10 μL HRP-Cu2+ (1 μg mL−1) was added for 5-min reaction, and then 20 μL freshly prepared mixture of H2O2 (2.5 mM) and luminol (0.5 mM) was added to collect the chemiluminescent images by CCD with an exposure time of 1 min.
Calibration curve for ELISA of PCSK9-Ab
PCSK9 coated 96-well plate was firstly prepared by incubating the plate with 2 μg mL−1 PCSK9 for 6 h at 37 oC. After a vigorous washing, the plate was immersed in 10 mg mL−1 BSA for 2 h at 37 oC to block the unreacted sites. After the plate was washed, 50 μL PCSK9-Ab at different concentrations were added into wells to incubate at 37 oC for 2 h. After washing and drying the plate, 50 μL IgG-HRP (2 μg mL−1) was delivered into the wells to incubate for 2 h at 37 oC for performing ELISA with TMB kit at 450 nm on Multiskan Sky (Thermo Fisher, USA).
Assay of PCSK9-Ab secreted from 6A6 hybridoma cells
After 6A6 hybridoma cells were distributed on six 96-well plates to allow single cell in a well by limiting dilution with culture medium, they were cultured with DMEM medium (200 μL) supplemented with 20% FBS, HEPES buffer (10 mM), sodium pyruvate (1 mM), L-glutamine (2 mM) and PS in an atmosphere of 5% CO2 and 95% humidified air at 37 oC for 1, 2, 3, 4, 5 and 6 days, respectively. Then, the supernatant in each well was collected to detect the amount of PCSK9-Ab secreted from 6A6 hybridoma cells by CLA and ELISA.
Hela cells (KeyGEN Biotech, Nanjing, China), 3H2 hybridoma cells (secreting aminoterminal pro-brain natriuretic peptides monoclonal antibody, FANTIBODY, Chongqing, China), and nonsecretor hybridoma cells (FANTIBODY, Chongqing, China) were used as non-PCSK9-Ab-secreting cells for control experiment. The culture and CLA processes of these non-PCSK9-Ab-secreting cells were the same as that of 6A6 hybridoma cells.
Chemiluminescent screening of specific hybridoma cells
After heterogeneous hybridoma cells were distributed into the wells of two plates by limiting dilution, DMEM medium supplemented (200 μL) with 20% FBS, HEPES buffer (10 mM), sodium pyruvate (1 mM), L-glutamine (2 mM) and PS was added in each well to incubate in an atmosphere of 5% CO2 and 95% humidified air at 37 oC for 6 days. 10 μL supernatant was then collected from each well at Day-1 to perform chemiluminescent screening of specific hybridoma cells at single cell level, while 50 μL supernatant was collected at Day-6 to perform ELISA screening.
Statistics and reproducibility
All quantitative data in the article and Supplementary Information were independently repeated three times and displayed as the mean ± SD. All the calculations were done in Origin or Excel.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
