In this study, C. albicans strain SC5314 was used (He et al., 2015). C. tropicalis ATCC1369, N. glabrata ATCC15126, C. parapsilosis ATCC22019, and C. krusei (P. kudriavzevii) ATCC6258 were obtained from the American Type Culture Collection (ATCC). The cells were subcultured on sabouraud dextrose agar (SDA) and incubated for 24 h at 35°C for all experiments. An inoculum from SDA plates was resuspended in the yeast extract peptone dextrose medium (YPD, 1% yeast extract, 2% peptone, and 2% dextrose) and incubated at 35°C for 48 h. The yeast cells were centrifuged, washed twice with the phosphate buffered saline (PBS; pH7.4, 0.01 M), and densities were determined by plate counting or measuring optical density values at 600 nm (OD600).

Whole cell ELISA was used to determine the specificity of mAbs 9H8 and 10H8 across different clinically important pathogens. The production of 9H8 and 10H8 mAbs has been described previously (He et al., 2022).

Candida albicans cells were adjusted to an OD600 of 0.5 in a 0.05 M carbonate buffer solution (CBS) that contained 15 mM Na₂CO₃ and 35 mM NaHCO₃ at pH9.6. The prepared suspensions were then used to coat the ELISA plates (100 μl/well) overnight at 4°C. As the negative controls, a panel of clinically relevant organisms including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecium, Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermis, Cryptococcus neoformans, C. tropicalis, N. glabrata, C. parapsilosis, C. krusei (P. kudriavzevii), Saccharomyces cerevisiae, and Candida dublinensis were used. These strains were grown, resuspended in CBS (OD600 = 0.5) and then coated onto ELISA plates. The plates were washed three times with PBS containing 0.05% Tween 20 (PBST), blocked with 100 μl of PBST plus 3% (w/v) bovine serum albumin (BSA), and incubated for 2 h at 37°C. The plates were treated for 1 h at 37°C with 10H8 or 9H8 that were diluted to 1 μg/ml by PBST plus 1% (w/v) BSA, followed by addition of 1:5,000 diluted horseradish peroxidase conjugated (HRP) goat anti-mouse IgG (Solarbio, Beijing China). After washing, 100 μl of the 3,3′,5,5′-tetramethylbenzedine (TMB) solution (Beyotime, Shanghai, China) was added to each well and incubated at 37°C for 10 min. The reaction was stopped with 50 μl of 2 N H₂SO₄, and the OD450 values were finally measured with the VersaMax plate reader (Molecular Devices, CA, United States).

The recombinant CaEno1 was used as a standard (He et al., 2015). The ELISA assay uses the Sandwich-ELISA principle and checker-board analyses were performed to determine the optimal concentrations of both the capture and detection antibodies.

Figure 1 depicts the protocol scheme for the DAS-ELISA. Briefly, mAb 9H8 (3 μg/ml in CBS, pH 9.6, 100 μl) was coated overnight at 4°C in each well of a 96-well microplate. After washing trice with PBST, the wells were blocked with 100 μl of PBST plus 3% (w/v) BSA, and incubated for 30 min at 37°C. The plasma samples (or diluted standards, 50 μl/well) and 50 μl of an optimized dilution (1:2,000) of HRP-10H8 were thereafter added to the wells and incubated at 37°C for 45 min. After washing, TMB substrate was applied to each well and incubated for 15 min at room temperature. The reaction was terminated, and the OD450 values were measured. The results were analyzed by using the software of ELISACalc V0.2.

The calibration curves were generated by calculating the ratios between the OD450 values and the corresponding standard concentration. The linearity was determined by serial dilution of a mock plasma sample with high CaEno1 concentration. The limitation of detection (LOD) was calculated by signal blank ± 3SD (Dixit et al., 2011). The recovery tests were conducted to evaluate the accuracy of the DAS-ELISA (Shen et al., 2020).

Three female New Zealand white rabbits weighing approximately 2.5 kg were procured from Hebei Medical University’s Experimental Animal Center (Shijiazhuang, China). The Bethune International Peace Hospital’s Ethics Committee authorized the animal care and use methods (No.2019-KY-23), and all the applicable institutional and governmental standards regulating the ethical use of animals were followed.

Each rabbit was inoculated with 1.5 × 10⁶ CFU of C. albicans SC5314 blastospores suspended in PBS intravenously through the marginal ear vein to cause the systemic candidiasis. Both the body temperature and weight of the rabbits were measured before the inoculation and on the 1st, 2nd, 4th, 7th, 10th, 15th, 20th, and 27th days post inoculation. Approximately 3.0 ml of anticoagulant blood was obtained from the model rabbits at each time point for different tests. Among them, 100 μl of whole blood was used for the blood hematology that was carried out with Sysmex 2100 (Sysmex Corporation, Kobe, Japan). The platelet rich plasma was obtained from 400 μl of blood and used to analyze the (1,3)-β-D-glucan concentration with a commercial kit by using the kinetic turbidimetry method (Xiamen Bioendo Technology Co., Ltd., Xiamen, China). The remaining 2.5 ml of blood was thereafter centrifuged to separate the plasma and blood cells. The plasma was utilized for biochemical analysis, as well as the detection of CaEno1 antigen and antibodies. Blood biochemistry analysis was performed by an Olympus AU5400 system (Olympus, Shinjuku, Japan). Six biochemical tests were used to assess the liver, kidney, and heart function of the rabbits: total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), plasma blood urea nitrogen (BUN), and phosphocreatine kinase (CK). The residual blood cells were resuspended in the normal saline to 2.5 ml and injected into the children’s blood culture bottles, where they were cultivated by using the BacT/ALERT 3D automated system (bioM é rieux, Craponne, France). The positive samples were subcultured on the standard media by using the routine microbiological techniques.

Candida albicans enolase1 IgG antibody in rabbit plasma was measured by indirect ELISA, as previously described (He et al., 2015). Recombinant CaEno1 was then coated onto each well of blank ELISA plates for overnight at 4°C after being diluted to 1 μg/ml in CBS. After washing trice with PBST, the plates were then blocked with PBST plus 3% (w/v) BSA solution, and incubated for 2 h at 37°C. After washing, rabbit plasma samples with a 1:500 dilution by PBST plus 1% (w/v) BSA were added and incubated at 37°C for 1 h. After three washes with PBST, 1:5,000 diluted HRP-conjugated goat anti-mouse IgG (Solarbio, Beijing, China) was added and incubated for 1 h at 37°C. After another round of washing, 100 μl of TMB substrate solution was applied to each well and incubated for 10 min at 37°C. Thereafter, the reaction was terminated and the OD450 values were determined.

We have previously demonstrated that 9H8 and 10H8 can specifically recognize CaEno1 in total lysate from C. albicans. We have also developed whole-cell ELISA assays to determine the response specificity of the 9H8, 10H8 mAbs against the different clinically important pathogens including a panel of Candida spp. and non-Candida organisms. The specificity of the DAS-ELISA was determined by detecting CaEno1 in the supernatant of various Candida spp. and grown for 48 h in YPD medium. The other negative control organisms were grown in enrichment broth and the supernatant was applied to the DAS-ELISA test as well. Table 1 depicts that 9H8 mAb can react specifically with C. albicans, with no cross interaction with other organisms that were evaluated. Remarkably, 10H8 mAb exhibited distinct response characteristics and could cross-react with all the Candida spp. and S. cerevisiae. The two mAbs’ DAS-ELISA could specifically react with the C. albicans culture supernatant.

Figure 2A depicts a standard curve generated with the recombinant CaEno1 as the standard. Normal rabbit plasma serial dilution of the recombinant CaEno1 was used as mock clinical samples, and the result indicated good linearity of DAS-ELISA across the range that we have investigated (top limit was close to 50 ng/ml, bottom limit was about 1 ng/ml, Figure 2B). The LOD of CaEno1 detection by this method was 0.33 ng/ml. The recovery test was conducted by addition of a high value standard sample into a low value matrix sample, with the volume ratio of 1:9. The recovery rate was determined by using the following formula: Recovery (in %) = [(amount detected − amount sample)/amount standard spiked] × 100. The recovery experiment was repeated trice, with an average recovery rate of 103.26% (95% CI = 85–115%).

All the rabbits displayed infection symptoms after being inoculated with C. albicans SC5314, such as loss of appetite and reduced activity. The body weight decreased significantly after infection (Figure 3A), and the body temperature increased dramatically on the first day, then gradually recovered to normal (Figure 3B). The number of peripheral white blood cells (WBC) rose steadily beginning on the first day after the injection and peaked on the seventh day. The dynamics of the neutrophil lymphocyte ratio (NLR) were remarkably comparable to the dynamics of the body temperature. It increased rapidly on the first day and then was gradually restored to the normal value (Figure 3C). The biochemical tests showed that once rabbits were infected with C. albicans, the heart, liver, and kidney functions were impaired. The peak of ALT emerged on the second day after Candida inoculation, the peak of AST level was reached on the seventh day, whereas the peaks of both CK and BUN level appeared on the fourth day (Figures 3D,E).

Candida albicans enolase1 and (1,3)-β-D-glucan were analyzed in rabbit plasma samples, and their dynamics have been depicted in Figure 4A. CaEno1 and (1,3)-β-D-glucan were found in the peripheral blood of rabbit following fungal inoculation. Both the levels in the blood steadily declined over time. On the seventh day after inoculation, (1,3)-β-D-glucan could not be detected in the rabbit model’s blood, and only one rabbit assessed positive for CaEno1. The blood culture findings revealed that all the rabbits had positive results after fungal inoculation. One rabbit remained positive for 24 h, whereas the two others remained positive for 48 h. In addition, upon microscopic analysis, blastospores and hyphal Candida could be observed in all blood positive cultures that were recognized as C. albicans by CHROMagar.

We next compared the diagnostic efficiency CaEno1, G-test, and blood culture. Each rabbit was assigned a value of 1 for each positive result and a value of 0 for each negative result, yielding the heat map as displayed in Figure 4B. On the first day after inoculation, all the tests performed identically to diagnose IC; on the second day, the CaEno1 detection and the G-test still diagnosed all rabbits, and better than the blood cultures; on the fourth and seventh days, CaEno1 detection diagnostic efficiency was slightly better than the G-test, while the blood culture diagnostic efficiency was 0.

All rabbits generated anti-CaEno1 IgG after being inoculated with C. albicans. The antibodies began to appear around day 7 after inoculation and steadily rose subsequently, and continued to grow until the experiment ended on day 27 (Figure 5A).

Indirect ELISA exhibited a significant background when detecting anti-CaEno1 IgG antibodies in rabbit plasma samples. We used rabbit plasma before inoculation as a control and tested it 20 times, with the OD450 > mean OD450_control + 2SD_control set as the threshold for determining positive samples. The heat map in Figure 5B was produced by assigning a value of one to each positive result and a value of zero to each negative result for each rabbit. The results demonstrated that the CaEno1 antigen and antibody test exhibited high complementarily for the diagnosis of Candida infection. The positive antigen findings were mostly found in the initial stages, whereas the positive antibody results were mostly observed in the late stages of infection. The combined antigen–antibody tests resulted in the successful diagnosis in all rabbits from days 1 to 4 and 20 to 27 after Candida inoculation, as well as in a portion of rabbits from days 4 to 15.