Microorganism and growth condition
C. albicans strain SC5314 was grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 37ºC, 5% CO2 to obtain blastospores. Hyphae were prepared by allowing SC5314 cells to germinate in culture at 37 °C, in 5% CO2 in RPMI medium supplemented with 10% fetal bovine serum (FBS). For standardization, 1 × 106 CFU of blastospores per mL of culture medium was fixedly seeded when performing experiments to measure the CaEno concentration in culture supernatants. C. albicans growth was monitored by turbidity measurement (McFarland units). The cultured blastospores/hyphae and its corresponding supernatants were then separated by centrifugation and prepared for analysis at the different time points if needed.
Generation of monoclonal anti-enolase antibodies
Recombinant CaEno (Walsh et al. 1991; He et al. 2015; He et al. 2016) was first injected into female BALB/c mice (n = 4) for mAb generation. The animals were obtained from the Experimental Animal Center of Hebei Medical University. Each mouse was administered 200 µg of recombinant CaEno protein emulsified in complete Freund’s adjuvant (i.p.). for the first immunization or 100 µg of protein emulsified in incomplete Freund’s adjuvant (i.p.) for the subsequent immunizations. Mice were immunized up to 4 times, with a 2-week interval between each immunization. Thereafter, antibody levels in the mice sera were determined by direct ELISA using recombinant CaEno and horseradish peroxidase-conjugated anti-mouse IgG (IgG–HRP) antibodies (Sangon Biotech, China). One mouse with a 1:100,000 serum titer of anti-CaEno was then sacrificed by cervical dislocation, and its spleen was removed for fusion with the SP2/0 myeloma cell line (Chinese Academy of Sciences, China). A total of 56 positive different hybridomas were screened out by ELISA and expanded. After verification the specificity by Western blot and preliminary antibody pairing experiment (data not shown), 2 were purified by passing the supernatants through protein G column, yielding specific antibodies of 9H8 and 10H8. The purity of the purified monoclonal antibodies was confirmed by SDS-PAGE.
Western blot analysis
Western blotting analysis was performed according to the standard protocols. The total lysates of C. albicans (Wiśniewski et al. 2009) and recombinant CaEno were transferred to polyvinylidene difluoride (PVDF) membrane (Merck millipore, Germany) after 12% SDS-PAGE electrophoresis. After blocking with tris buffer (pH 7.5) containing 3% bovine serum albumin (BSA), the membrane was incubated with primary 1:500 diluted 9H8 or 10H8 and then with HRP-conjugated goat anti-mouse IgG. Western blot was developed using diaminobenzidine (DAB) substrate (Solarbio, China).
The specificities of 9H8 and 10H8 antibodies were evaluated by an immunocapture assay and subsequently by using mass spectrometry analysis. The immunocapture assay was performed using anti-CaEno mAbs coupled Berpharose FF beads and C. albicans total lysates. Briefly, 15 mg of purified 9H8 or 10H8 solution was concentrated to 1.5 mL and dialyzed against the coupling buffer (0.2 M NaHCO3, 0.5 M NaCI, pH 8.3). Thereafter, the antibodies were coupled to N-hydroxysuccinimide (NHS) activated Berpharose FF beads (Bersee, China) at 4 °C overnight. After washing and blocking, the antibody-coupled beads were then packed into empty columns with 0.02 M phosphate-buffered saline (PBS). Affinity columns were equilibrated by passing 10 column volumes (CV) of 0.02 M PBS, and the total lysates of C. albicans were thereafter loaded onto the columns. The columns were washed with 40 volumes of PBS till the read value of the UV detector (Shimadzu, Japan) decreased to zero. Finally, the various bound fractions were eluted with 0.1 M glycine-HCI (pH 3.4) and neutralized with 1.5 M Tris-HCI (pH 9.5). Eluted proteins were collected and analyzed by SDS-PAGE and mass spectrometry.
The mass spectrometry identification of the immunocapture fraction of 9H8 mAb was performed by Sangon Biotech (Shanghai, China). The protein in the eluted solution was first precipitated by trichloroacetic acid (TCA) treatment and then treated with 8 M urea/100 mM Tris–HCl solution (pH 8.0) for causing denaturation and exposed to 10-mM dithiothreitol (DTT) to open the disulfide bond. Subsequently, the protein sample was digested by trypsin and desalinated by Sep-Pak C18. MS was performed using a Triple TOF 5600 System (AB SCIEX, USA). The mass spectrum data generated were thereafter retrieved by Protein Pilot (V4.5), and the database retrieval algorithm was Paragon. The database used for the retrieval purpose was the proteome reference database of Candida albicans in Uniprot.
The identification of the elute fraction generated by 10H8 mAb was performed by Shanghai Applied Protein Technology Co., Ltd. (China). The bands were excised from the silver-stained gels and digested with trypsin, and then, the peptides were separated by chromatography using an Easy nLC system (Thermo Scientific, Germany) and then used for mass spectrometry with a Q-Exactive (Thermo Scientific, Germany) mass spectrometer. The original raw files were imported into Max Quant software for the database retrieval. The database used for retrieval was “uniprot_candida_albicans_6040_20210421.fasta”.
Immunohistochemical staining analysis was done to acquire an overall image of CaEno distribution. After a 48-h culture, the collected blastospores/hyphae were first fixed with 10% phosphate-buffered formalin at 4ºC for 24 h. After paraffin embedding and sectioning, the slices were blocked by 5% BSA at room temperature for 20 min, followed by incubation with 1:25 diluted 10H8 at 37ºC for 1 h. After washing in PBS for three times, the sections were then incubated with 1:400 diluted anti-mouse IgG-HRP antibody (Solarbio, China) for 90 min at room temperature. Thereafter, the sections were reacted with 0.05% 3,3′-diaminobenzidine (DAB) and 0.003% H2O2 in 0.05 M tris buffer (pH 7.2–7.4). The stained sections were then evaluated under a BX43 microscope (Olympus, Japan). Digital images of the stained slides were captured at about 7 200 × magnification with an Olympus BX43 microscope equipped with a digital amplification system (DP26, Olympus).
Whole cell ELISA
The 24-h cultured C. albicans SC5314 cells were harvested and then washed three times with ice-cold 0.1 M PBS. After centrifugation, the cells were adjusted to a density of OD600 = 0.5 or 0.1 in pH 9.6 carbonate buffer solution (CBS). The prepared suspensions were thereafter used to coat ELISA plates (100 µL/well) at 4ºC overnight. CBS suspension of Escherichia coli (OD600 = 0.5) was used as a negative control. After blocking and washing, the plates were treated with serial dilutions of 10H8 (from 1:1000 to 1:128,000) at 37ºC for 1 h and then with 1:5000 diluted HRP goat anti-mouse IgG (Solarbio, China). One hundred microliters of the 3,3′,5,5′-tetramethylbenzedine (TMB) substrate was added into each well and incubated at 37ºC for 10 min after the washing. The reaction was terminated with 2 N H2SO4, and the optical density values at 450 nm (OD450) were finally measured using the Versa Max plate reader (Molecular Devices Co., USA).
Flow cytometry (FCM)
Flow cytometry was applied to determine the surface location of CaEno on live C. albicans cells. In brief, 24 h-cultured C. albicans cells were adjusted to a density of 1 × 107 CFU/mL and treated for 1 h with 1:100 diluted 10H8 mAb. Following that, cells were washed and treated for 1 h with 1:100 FITC goat anti mouse IgG, followed by 30 min of paraformaldehyde fixation. After washing, the immunofluorescence stained C. albicans cells were analyzed by an EPICS XL4 flow cytometry (Beckman coulter, USA).
The confocal microscopy analysis was completed by the Wuhan Servicebio Technology CO., LT., Wuhan, China. C. albicans cells were first centrifuged, washed, and fixed in 4% paraformaldehyde-PBS for 20 min. After washing with PBS, a 10 μl of cell suspension was dripped onto the glass slides to dry at room temperature overnight. The slides containing yeast were then blocked with 3% BSA and incubated with 1:200 diluted 10H8 overnight at 4 °C. The slides were subsequently incubated with Cy3-conjugated goat anti-mouse IgG (Servicebio, China) diluted 1:300 for 1 h in the dark after washing. Nikon Eclipse T1 confocal microscope (Nikon Inc., Japan) at 400 × magnification was used for image acquisition and a Nikon C2 confocal system (Nikon Inc., Japan) was employed to record images.
Double antibody sandwich ELISA (DAS-ELISA)
DAS-ELISA was developed to determine the levels of CaEno released in the culture medium. 9H8 mAb was diluted to 3 μg/ml in CBS and then used to coat ELISA plates (100 µL/well) at 4ºC overnight. After blocking and washing, culture supernatant collected from C. albicans blastospores or hyphea at different time points (0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 72, 96, and 120 h) were added (100 µL/well) and thereafter incubated at 37ºC for 1 h. Afterwards, the plate was washed and 100 µL of 1:5000 diluted HRP-10H8 was added to each well and incubated at 37ºC for 1 h. After incubation and washing, 100 µL of TMB substrate was added into each well and incubated at 37ºC for 10 min. The reaction was then terminated and the OD450 were determined with a plate reader. The diluted recombinant CaEno were used as the standard substances to establish a standard curve, and the results were analyzed by the software of ELISACalc V0.2.
The statistical analysis was performed using the GraphPad Prism software version 7.00. The continuous measures have been presented as the mean and standard deviation at each time point. The statistical significance of the differences between the groups at different times was determined by the one-way analysis of variance test, followed by the LSD t test. A P value of less than 0.05 (P < 0.05) was considered as statistically significant.