Development of a Monoclonal Antibody and a Sandwich ELISA for the Detection of Mucormycosis in Humans

Abstract

Background: Mucormycosis is a rapidly progressive and often fatal invasive fungal infection caused by moulds in the order, Mucorales. Early diagnosis is essential for effective clinical management; however, conventional diagnostic approaches such as culture and histopathology are slow, insensitive, and require specialist mycological expertise. Although molecular methods are available for disease detection, they are not widely accessible. At present, no enzyme immunoassay (EIA) exists for the detection of mucormycosis. Methods: A murine IgG1 monoclonal antibody (mAb), FH12, was generated against extracellular polysaccharides (EPSs) produced by Mucorales pathogens during active growth. The antibody was characterised for specificity, epitope stability, and antigen localisation using ELISA, immunoblotting, and immunofluorescence techniques. The mAb was incorporated into a sandwich ELISA and evaluated using culture filtrates, purified EPSs spiked into human serum, and tissue homogenates from a patient with cutaneous mucormycosis caused by Lichtheimia ramosa. Results: mAb FH12 demonstrated pan-Mucorales specificity and no cross-reactivity with other clinically relevant yeasts and moulds. The epitope recognised by FH12 is periodate-insensitive and moderately heat-stable. The sandwich ELISA detected EPS antigens in human serum with limits of detection ranging from pg/mL to low ng/mL levels, and successfully identified the EPS biomarker in patient tissue homogenates. Conclusion: The FH12-based sandwich ELISA shows high sensitivity and specificity, and has the potential to be used as a laboratory-based adjunct diagnostic test for the detection of mucormycosis in humans.

1. Introduction

Mucormycosis is a rapidly progressive angio-invasive disease of humans caused by fungi in the Zygomycete order, Mucorales, manifesting as life-threatening rhino–orbital–cerebral, pulmonary, cutaneous, gastrointestinal, and disseminated infections (Figure 1A) [1]. The species most commonly reported as pathogens of humans are Apophysomyces variabilis, Cunninghamella bertholletiae, Lichtheimia corymbifera and Lichtheimia ramosa, Mucor circinelloides, Rhizomucor pusillus, Rhizopus arrhizus, and Rhizopus microsporus [2]. Identified as a global high-priority pathogen group by the World Health Organisation [3], and a critical-priority pathogen group in Southeast Asia by others [4], mortality rates from Mucorales infections of up to 80% have been reported [2].

Figure 1.

(A) Diseases caused by Mucorales fungi. The predominant disease in humans is rhino–orbital–cerebral mucormycosis (ROCM) involving the nose, eye and brain, and is typically reported in the settings of poorly controlled diabetes and haematological malignancy. A surge of ROCM was reported during the second wave of the coronavirus pandemic, especially in diabetic patients and/or those receiving corticosteroids for COVID-19 infection. Pulmonary mucormycosis is most commonly seen in haematological malignancy and transplant patients, and is related to desferrioxamine therapy with renal impairment. Cutaneous (skin and soft tissue) mucormycosis can occur in burn wounds, and following contaminated traumatic injury, including tornado-type and combat-related injuries. Less common manifestations include gastro-intestinal and disseminated mucormycosis. (B) Sandwich ELISA protocol. Step 1: Wells of microtiter plates coated with 50 μL of protein A-purified mAb FH12 (4 μg/mL) are blocked for 15 min with 100 μL of PBS containing 1.0% (w/v) BSA, and then incubated at 23 °C for 1 h with 50 µL of antigen solution. Step 2: After washing, the wells are incubated for 50 min at 23 °C with FH12-HRP conjugate diluted 1 in 10,000 (v/v) in PBST containing 0.25% (w/v) BSA. Step 3: After washing, the wells are incubated with substrate solution for 15 min and the absorbance values determined at 450 nm. (C) Procedure for heat treatment of human serum. Step 1: Human serum is mixed 1:2 (v/v) with PBS buffer containing Na₂EDTA, and then heated for 3 min at 100 °C in a boiling water bath. Step 2: The heated serum is centrifuged at 16,000× g for 5 min to pellet insoluble serum proteins. Step 3: Following centrifugation, the clear supernatant is mixed 1:1 (v/v) with running buffer (RB) and 50 μL assayed by sandwich ELISA. Figure 1 was created with BioRender.com.

The prompt detection and differentiation of Mucorales species from other mould pathogens such as Aspergillus fumigatus is critical to patient survival, enabling timely treatment with surgery and with Mucorales-active antifungal drugs [5]. Detection of Mucorales pathogens has traditionally relied on histopathology and fungal culture from a biopsy sample, and the identification by microscopy of characteristic morphological structures, but these procedures are laborious, insensitive and require mycological expertise [5,6]. For this reason, more sophisticated and costly diagnostic modalities such as metagenomic next-generation sequencing (NGS) [7] or matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) [8] can be used for their identification following culture in vitro, but access to such technology is limited.

While polymerase chain reaction (PCR) tests have been developed for the detection of circulating Mucorales nucleic acid in human serum and bronchoalveolar lavage fluid [9,10], there is currently no enzyme immunoassay (EIA) that can be used as adjunct tests to detect mucormycosis [10]. Despite this, we have recently reported the development of species-specific [11], genus-specific [12] and pan-Mucorales-specific [13,14] monoclonal antibodies (mAbs) that detect Mucorales extracellular polysaccharides (EPSs), and their incorporation into lateral-flow immunoassays [11,13] for the detection of these antigenic biomarkers of mucormycosis in humans [15].

Here, we report the development of a murine IgG1 mAb, FH12, specific to EPSs produced during active growth of Mucorales pathogens. When incorporated into a sandwich-enzyme-linked immunosorbent assay (sandwich ELISA), mAb FH12 is able to detect Mucorales EPSs in human serum with limits of detection of pg/mL. We show how the sandwich ELISA can be used to detect mucormycosis using tissue homogenates from a patient with a cutaneous and soft-tissue infection caused by Lichtheimia ramosa. This is the first time that a pan-Mucorales-specific sandwich ELISA has been reported for the detection of mucormycosis, enabling the laboratory-based detection of EPS antigens in human biofluids, which can act as signature molecules of invasive Mucorales infections.

2. Materials and Methods

2.1. Ethics Statement

Hybridoma work described in this study was conducted under a Home Office Project Licence, and was reviewed by the institution’s Animal Welfare Ethical Review Board (AWERB) for approval on 20 January 2022. The work was carried out in accordance with the Animals (Scientific Procedures) Act 1986 and Directive 2010/63/EU, and followed all the codes of practice which reinforce this law, including all elements of housing, care, and euthanasia of the animals.

2.2. Fungal Culture

Fungi (Table 1 and Table 2) were routinely cultured on malt extract agar (MEA; 70145, Sigma, Poole, UK). The medium was autoclaved at 121 °C for 15 min prior to use, and fungi were grown at 30 °C. To induce sporulation of Apophysomyces and Saksenaea species, the methods described in Thornton and Davies [12] were used.

Table 1.

Details of human pathogenic fungi used to determine the specificity of mAb FH12, and results of direct ELISA tests using purified EPS antigens. ¹ CBS; Westerdijk Fungal Biodiversity Institute, The Netherlands. CRT; C. R. Thornton, University of Exeter, UK. FGSC; Fungal Genetic Stock Centre, Kansas City University, USA. NCPF; National Centre for Pathogenic Fungi, UK. ² For direct ELISA tests, the absorbance values are the means of two replicate values ±SE, and the threshold absorbance value for test positivity is ≥0.100.

Species	Isolate Number	Source 1	Direct ELISA (Abs 450 nm) 2
Apophysomyces elegans	477.78	CBS	1.518 ± 0.050
Apophysomyces mexicanus	136361	CBS	0.445 ± 0.012
Apophysomyces ossiformis	125533	CBS	1.432 ± 0.012
Apophysomyces variabilis	658.93	CBS	1.450 ± 0.018
Aspergillus fumigatus	Af293	FGSC	0.099 ± 0.002
Cunninghamella bertholletiae	151.80	CBS	1.324 ± 0.034
Fusarium solani	224.34	CBS	0.066 ± 0.004
Lichtheimia corymbifera	109940	CBS	1.421 ± 0.019
Lichtheimia hyalospora	146576	CBS	1.190 ± 0.069
Lichtheimia ornata	142195	CBS	1.393 ± 0.016
Lichtheimia ramosa	124197	CBS	1.507 ± 0.007
Lichtheimia ramosa	112528	CBS	1.460 ± 0.063
Lichtheimia ramosa	2845	NCPF	1.410 ± 0.028
Lomentospora prolificans	3.1	CRT	0.074 ± 0.006
Mucor circinelloides	B5-2	CRT	1.545 ± 0.031
Mucor racemosus f. racemosus	222.81	CBS	1.426 ± 0.063
Rhizomucor pusillus	120587	CBS	1.327 ± 0.016
Rhizopus arrhizus	111233	CBS	1.320 ± 0.060
Rhizopus arrhizus var. arrhizus	112.07	CBS	1.346 ± 0.022
Rhizopus microsporus var. rhizopodiformis	102277	CBS	1.550 ± 0.044
Saksenaea vasiformis	113.96	CBS	0.566 ± 0.018
Scedosporium aurantiacum	121926	CBS	0.088 ± 0.001
Scedosporium boydii	835.96	CBS	0.084 ± 0.001

Table 2.

Details of fungi used in this study, and results of sandwich ELISA tests using culture filtrates from fungi grown as shake cultures for 72 h in YNB+G medium. ¹ CBS; Westerdijk Fungal Biodiversity Institute, The Netherlands. CRT; C. R. Thornton, University of Exeter, UK. FGSC; Fungal Genetic Stock Centre, Kansas City University, USA. NCPF; National Centre for Pathogenic Fungi, UK. ² For sandwich ELISA tests, the absorbance values are the means of three replicate values ±SE, and the threshold absorbance value for test positivity is ≥0.100.

Species	Isolate Number	Source 1	Sandwich ELISA (Abs 450 nm) 2
Absidia glauca	2	CRT	0.802 ± 0.007
Absidia spinosa	3	CRT	0.681 ± 0.013
Actinomucor elegans var. kuwaitensis	117697	CBS	0.769 ± 0.055
Apophysomyces elegans	477.78	CBS	0.625 ± 0.034
Apophysomyces mexicanus	136361	CBS	0.891 ± 0.004
Apophysomyces ossiformis	125533	CBS	0.827 ± 0.052
Apophysomyces variabilis	658.93	CBS	0.790 ± 0.038
Aspergillus fumigatus	Af293	FGSC	0.087 ± 0.014
Aspergillus flavus	144B	CRT	0.065 ± 0.020
Aspergillus nidulans	A4	FGSC	0.075 ± 0.009
Aspergillus niger	102.4	CBS	0.063 ± 0.003
Aspergillus terreus var. terreus	601.65	CBS	0.057 ± 0.009
Candida albicans	SC5314	SB	0.048 ± 0.002
Cryptococcus neoformans	8710	CBS	0.075 ± 0.025
Cunninghamella bertholletiae	151.80	CBS	0.821 ± 0.042
Fusarium oxysporum	167.3	CBS	0.035 ± 0.002
Lichtheimia corymbifera	109940	CBS	0.835 ± 0.020
Lichtheimia corymbifera	120580	CBS	0.853 ± 0.020
Lichtheimia hyalospora	146576	CBS	0.867 ± 0.009
Lichtheimia ornata	142195	CBS	0.845 ± 0.026
Lichtheimia ramosa	124197	CBS	0.790 ± 0.016
Lichtheimia ramosa	112528	CBS	0.790 ± 0.016
Lichtheimia ramosa	2845	NCPF	0.777 ± 0.074
Lomentospora prolificans	3.1	CRT	0.049 ± 0.004
Mucor ardhlaengiktus	126271	CBS	0.682 ± 0.005
Mucor circinelloides	124429	CBS	0.799 ± 0.054
Mucor circinelloides	B5-2	CRT	0.840 ± 0.044
Mucor circinelloides f. circinelloides	E2A	CRT	0.776 ± 0.046
Mucor circinelloides f. circinelloides	120582	CBS	0.757 ± 0.035
Mucor indicus	120.08	CBS	0.734 ± 0.005
Mucor irregularis	103.93	CBS	0.725 ± 0.078
Mucor plumbeus	96	CRT	0.847 ± 0.025
Mucor racemosus f. racemosus	112382	CBS	0.404 ± 0.049
Mucor velutinosus	126272	CBS	0.725 ± 0.036
Phycomyces nitens	133	CRT	0.709 ± 0.013
Rhizomucor pusillus	120586	CBS	0.731 ± 0.022
Rhizomucor pusillus	120587	CBS	0.500 ± 0.026
Rhizopus arrhizus	TV4	CRT	0.751 ± 0.089
Rhizopus arrhizus	2634	CBS	0.570 ± 0.095
Rhizopus arrhizus var. arrhizus	112.07	CBS	0.405 ± 0.004
Rhizopus arrhizus var. arrhizus	118614	CBS	0.644 ± 0.032
Rhizopus arrhizus var. delemar	2601	NCPF	0.606 ± 0.014
Rhizopus arrhizus var. delemar	607.68	CBS	0.313 ± 0.004
Rhizopus arrhizus var. delemar	544.80	CBS	0.677 ± 0.039
Rhizopus azygosporus	357.93	CBS	0.533 ± 0.001
Rhizopus homothallicus	336.62	CBS	0.481 ± 0.004
Rhizopus microsporus var. rhizopodiformis	102277	CBS	0.650 ± 0.088
Rhizopus microsporus var. rhizopodiformis	220.92	CBS	0.727 ± 0.016
Rhizopus microsporus var. rhizopodiformis	118987	CBS	0.612 ± 0.073
Rhizopus schipperae	138.95	CBS	0.474 ± 0.014
Rhizopus stolonifer var. stolonifer	389.95	CBS	0.734 ± 0.040
Saksenaea erythrospora	138279	CBS	0.792 ± 0.092
Saksenaea vasiformis	113.96	CBS	0.703 ± 0.050
Scedosporium apiospermum	117407	CBS	0.056 ± 0.011
Scedosporium aurantiacum	118934	CBS	0.070 ± 0.005
Scedosporium aurantiacum	121926	CBS	0.071 ± 0.006
Scedosporium boydii	835.96	CBS	0.038 ± 0.002
Scedosporium boydii	100870	CBS	0.075 ± 0.012
Scedosporium boydii	100393	CBS	0.083 ± 0.007
Scedosporium dehoogii	117406	CBS	0.067 ± 0.003
Scedosporium fusoideum	106.53	CBS	0.064 ± 0.003
Syncephalastrum racemosum	155	CRT	0.765 ± 0.035
YNB+G only	-	-	0.067 ± 0.001

2.3. Production of Hybridomas and Screening by ELISA

2.3.1. Hybridoma Production

Extracellular polysaccharides (EPSs) were prepared as described elsewhere [11,12,13]. For hybridoma production, the immunogen comprised a 1 mg/mL solution of EPS from Apophysomyces variabilis CBS658.93, with six-week-old BALB/c white mice each given four intra-peritoneal injections (300 µL per injection) of immunogen at 2 wk intervals, and a single booster injection 5 d before fusion. Hybridoma cells were produced by the method described elsewhere using SP2/0 myeloma cells as the fusion partner, and monoclonal antibody (mAb)-producing clones identified in indirect ELISA tests.

2.3.2. Indirect-Enzyme-Linked Immunosorbent Assay

For indirect ELISA, EPS at a concentration of 20 mg/mL phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄ [pH 7.2]) was immobilised to the wells of Maxisorp microtiter plates (Nunc) at 50 µL/well. Wells containing immobilised antigen were incubated with 50 µL of mAb hybridoma tissue culture supernatant (TCS) for 1 h, after which wells were washed three times, for 5 min each, with PBST (PBS containing 0.05% (v/v) Tween-20). Goat anti-mouse polyvalent immunoglobulin (G, A, M) peroxidase conjugate (PA1-84388; Invitrogen, Loughborough, UK), diluted 1:5000 (v/v) in PBST, was added to the wells and incubated for a further hour. The plates were washed with PBST as described, given a final 5 min wash with PBS, and bound antibody visualised by incubating wells with tetramethyl benzidine (TMB) substrate solution for 30 min, after which reactions were stopped by the addition of 3 M H₂SO₄. Absorbance values were determined at 450 nm using a microplate reader (infinite F50, Tecan Austria GmbH, Reading, UK). Control wells were incubated with tissue culture medium (TCM) containing 10% (v/v) foetal bovine serum (F7254, Sigma, Poole, UK) only. All incubation steps were performed at 23 °C in sealed plastic bags. The threshold for detection of the antigen in indirect ELISA was determined from control means (2 × TCM absorbance values). These values were consistently in the range of 0.050–0.100. Consequently, absorbance values ≥0.100 were considered as positive for the detection of antigen.

2.4. Determination of Ig Class and Sub-Cloning Procedure

The Ig class of mAbs was determined by using antigen-mediated ELISA. Wells of three replicate microtiter plates coated with 20 μg EPS/mL PBS were incubated successively with hybridoma TCS for 1 h; goat anti-mouse IgG1-, IgG2a-, IgG2b-, IgG3-, IgM- or IgA-specific antiserum (ISO-2, Sigma, Poole, UK) diluted 1:3000 (v/v) in PBST for 30 min, and rabbit anti-goat peroxidase conjugate (A5420, Sigma, Poole, UK) diluted 1:1000 (v/v) for a further 30 min. Bound antibody was visualised with TMB substrate as described. Hybridoma cell lines were sub-cloned three times by limiting dilution, and cell lines were grown in bulk in a non-selective medium, preserved by slowly freezing in FBS/dimethyl sulfoxide (92:8 v/v), and stored in liquid N₂.

2.5. Antibody Purification and Enzyme Conjugation

Hybridoma TCS of mAb FH12 was harvested by centrifugation at 2150× g for 40 min at 4 °C, followed by filtration through a 0.8 μM cellulose acetate filter (10462240, GE Healthcare Life Sciences, Little Chalfont, UK). Culture supernatant was loaded onto a HiTrap protein A column (17-0402-01, GE Healthcare Life Sciences, Little Chalfont, UK) using a peristaltic pump P-1 (18-1110-91, GE Healthcare Life Sciences, Little Chalfont, UK) with a low pulsation flow of 1 mL/min. Columns were equilibrated with 10 mL of PBS and column-bound antibody was eluted with 5 mL of 0.1 M glycine-HCl buffer (pH 2.5) with a flow rate of 0.5 mL/min. The buffer of the purified antibody was exchanged to PBS using a disposable PD-10 desalting column (17-0851-01, GE Healthcare Life Sciences, Little Chalfont, UK). Following purification, the antibody was sterile-filtered with a 0.24 µm syringe filter (85037-574-44, Sartorius, Göttinghen, Germany) and stored at 4 °C. Protein concentrations were determined using a Nanodrop spectrophotometer with protein concentration calculated using the mass extinction coefficient of 13.7 at 280 nm for a 1% (10 mg/mL) IgG solution. Antibody purity was confirmed by SDS-PAGE and gel staining using Coomassie Brilliant Blue R-250 dye (Thermo Fisher Scientific, Loughborough, UK). Protein A-purified mAb FH12 was conjugated to horseradish peroxidase (HRP) for ELISA studies using a Lightning-Link horseradish peroxidase conjugation kit (701-0000, Bio-Techne Ltd., Abingdon, UK), or to alkaline phosphatase (AKP) for Western blotting studies using a Lightning-Link alkaline phosphatase conjugation kit (702-0010, Bio-Techne Ltd., Abingdon, UK).

2.6. Determination of Antibody Specificity

For antibody specificity tests, wells of two replicate plates were coated with purified EPS antigens at a concentration of 20 μg/mL PBS from different Mucorales fungi and from unrelated moulds of clinical importance (Table 1). The wells were assayed by direct ELISA using the FH12-HRP conjugate diluted 1:5000 (v/v) in PBST for 1 h, followed by TMB substrate solution for 30 min.

2.7. Epitope Characterisation by Periodate Oxidation and Heat Treatment

For periodate oxidation, three replicate microtiter plates containing immobilised EPS from Apophysomyces variabilis strain CBS658.93 at a concentration of 20 μg/mL PBS were incubated with 50 μL of sodium meta-periodate solution (20 mM NaIO₄ in 50 mM sodium acetate buffer, pH 4.5) or acetate buffer only (control) for 24, 4, 3, 2, 1, or 0 h, at 4 °C in sealed plastic bags. The plates were given four 3 min washes with PBS before assay by direct ELISA using the FH12-HRP conjugate as described. The heat stability of the FH12 epitope was determined by heating three replicate EPS samples from Apophysomyces variabilis strain CBS658.93 at a concentration of 20 μg/mL PBS in a boiling water bath. At 5 min and 10 min intervals, 50 μL volumes were removed and, after cooling, were transferred to the wells of microtiter plates for assay by direct ELISA using the FH12-HRP conjugate as described.

2.8. Polyacrylamide Gel Electrophoresis and Western Blotting

Sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 4–20% gradient polyacrylamide gels (161-1159, Bio-Rad, Watford, UK) under denaturing conditions. Antigens were separated electrophoretically at 165 V and pre-stained markers (161-0318, Bio-Rad, Watford, UK) were used for molecular weight determinations. For Western blotting, separated antigens were transferred electrophoretically onto a PVDF membrane (162-0175, Bio-Rad, Watford, UK) for 2 h at 75 V, and the membrane was blocked for 16 h at 4 °C in PBS containing 1% (w/v) bovine serum albumin (BSA). Blocked membranes were incubated with FH12-AKP conjugate diluted 1:5000 (v/v) in PBS containing 0.5% (wt/vol) BSA for 2 h at 23 °C. Membranes were washed three times with PBS, once with PBST and bound antibody visualised by incubation in substrate solution. Reactions were stopped by immersing membranes in dH₂O, and membranes were then air dried between sheets of Whatman filter paper.

2.9. Spatio-Temporal Production of EPS Antigens In Vitro

2.9.1. Colony Immunoblotting

For colony immunoblots [13], MEA was inoculated centrally with 5 μL of a 10³ spores/mL spore suspension of Apophysomyces variabilis strain CBS658.93 and incubated for 16 h at 30 °C, after which colonies were overlaid with PVDF membrane (162-0175, Bio-Rad, Watford, UK) for 8 h to bind extracellular antigens. The membranes were removed and discarded, the colonies incubated for a further 16 h, and the blotting procedure repeated. The membranes were blocked and processed with FH12-AKP conjugate as described for Western blotting, with mAb TG11-AKP conjugate [12] diluted 1:5000 (v/v) used as the control.

2.9.2. Immunofluorescence

Immunofluorescence was carried out as described elsewhere [16] using spores of Mucor circinelloides f. circinelloides CBS120582 germinated on glass slides at 30 °C in autoclaved YNB+G liquid medium [10,11,12], and with a 1:500 (v/v) dilution in PBS of mAb FH12 directly conjugated to the fluorophore Cy3 using a Lightning-Link (R) Rapid Cy3 conjugation kit (340-0030, Bio-Techne Ltd., Abingdon, UK). Epifluorescence imaging was conducted with an SP8 confocal laser microscope under 550 nm absorbance and 570 nm emission spectra.

2.9.3. Shake Culture

Conical flasks (250 mL), each containing 100 mL of autoclaved YNB+G liquid medium [11,12,13], were inoculated with spores of Mucorales fungi and unrelated moulds of clinical importance (Table 2) to a final concentration of 10³ spores/mL. The cultures were incubated at 30 °C with shaking (120 RPM) in a New Brunswick orbital shaker. At 24 h intervals, three replicate flasks were harvested, the culture fluids separated from mycelium by filtration through Miracloth (475855-1R, Merck Millipore, Germany), and then stored at −20 °C. Mycelial biomass was dried for 4 d at 80 °C and weighed. On thawing, culture fluids were centrifuged for 5 min at 16,000× g, and then assayed by sandwich ELISA and by Western blotting.

2.10. Sandwich ELISA

The sandwich ELISA procedure is illustrated in Figure 1B. Wells of Maxisorp microtiter plates (Nunc) were coated with 50 µL volumes of protein A-purified mAb FH12 at a concentration of 4 μg/mL PBS. After incubation for 16 h at 4 °C, the wells were washed three times (5 min each wash) with PBST, once with PBS for 5 min, and then given a final rinse with dH₂O before air-drying at 23 °C. Antibody-coated wells were blocked for 15 min with 100 μL of PBS containing 1.0% (w/v) BSA, and then incubated at 23 °C for 1 h with 50 µL of culture filtrates diluted 1:1 (v/v) with PBST (control wells incubated with YNB+G medium only diluted 1:1 (v/v) with PBST). The wells were given four 5 min washes with PBST, and then incubated for 50 min at 23 °C with FH12-HRP conjugate diluted 1 in 10,000 (v/v) in PBST containing 0.25% (w/v) BSA. The wells were washed four times with PBST as described, given a final 5 min wash with PBS, and bound antibody visualised by incubating wells with tetramethyl benzidine (TMB) substrate solution for 15 min. Enzyme–substrate reactions were stopped by the addition of 3 M H₂SO₄, and absorbance values were determined at 450 nm using a microplate reader (infinite F50, Tecan Austria GmbH, Reading, UK). All incubation steps were performed at 23 °C in sealed plastic bags.

2.11. Detection of EPS Antigens in Human Serum and Limits of Detection of Sandwich ELISA

The limit of detection (LOD) of the sandwich ELISA was determined in running buffer (RB) and in human serum (HS). For HS, normal serum from healthy AB blood group males (H6914, Sigma, Poole, UK) was spiked with purified EPS from Apophysomyes variabilis CBS658.93, Cunninghamella bertholletiae CBS151.80, Mucor circinelloides B5-2, Lichtheimia corymbifera CBS109940, Lichtheimia ramosa CBS112528, Rhizomucor pusillus CBS120587, Rhizopus arrhizus var. arrhizus CBS112.07, and Rhizopus microsporus var. rhizopodiformis CBS102277, and stored as aliquots at −20 °C prior to use. The standard operating procedure for serum pre-treatment and testing using the sandwich ELISA is illustrated in Figure 1C. On thawing, 50 μL spiked or control (unspiked) serum was mixed 1:2 (v/v) with PBS containing 0.5% (w/v) Na₂-EDTA (D/0700/53, Thermo Fisher Scientific, Loughborough, UK)(pH 6.0), and heated in a boiling water bath for 3 min. The heated mixture was centrifuged at 16,000× g for 5 min, the clear supernatant mixed 1:1 (v/v) with PBST, and the resultant solution assayed by sandwich ELISA (50 μL per well) as described. The LOD for each pathogen was determined from the mean absorbance values of 4 replicate samples showing significant differences (Student’s t-test (p < 0.05)) compared with matched replicate controls (RB only or normal serum only) in each dilution series, with an absorbance value of 0.100 used as the threshold absorbance value for test positivity.

2.12. Detection of Cutaneous Mucormycosis

The ability of the sandwich ELISA to detect Mucorales infections in humans was determined using clinical samples from a single case of cutaneous mucormycosis. Further details of this case can be found in Hudson et al. [17]. The samples comprised tissue homogenates from an immunocompetent male patient with cutaneous and soft tissue mucormycosis of the right hand caused by the Mucorales pathogen Lichtheimia ramosa strain 266533346 [17]. The tissue homogenates, prepared in dH₂0 [17], were centrifuged at 17,000× g for 5 min to remove cellular debris, and undiluted 50 μL samples of supernatants tested in the sandwich ELISA (Figure 1B) as described. Sterile dH₂0 acted as the control.

2.13. Statistical Analysis

Numerical data were analysed using the statistical programme Minitab (Minitab 16; Minitab, Coventry, UK). Analysis of variance (ANOVA) was used to compare means, and post hoc Tukey–Kramer analysis was then performed to determine statistical significance.

3. Results

3.1. Production of Hybridomas and mAb Isotyping

A single hybridoma fusion was performed, and 327 hybridoma cell lines were tested in indirect ELISA tests for recognition of the immunogen. Seven cell lines produced EPS-reactive antibodies, five of which produced mAbs of the immunoglobulin class, G1 (IgG1). The remaining two cell lines produced mAbs of the class G2b (IgG2b). The cell line FH12 (an IgG1) was selected for further evaluation due to its broad specificity for Mucorales species and lack of cross-reactivity with non-Mucorales fungi.

3.2. Specificity of mAb FH12 and Epitope Characterisation

In direct ELISA tests of purified EPSs from the most common Mucorales and non-Mucorales mould pathogens of humans, mAb FH12 was found to be pan-Mucorales-specific, reacting with all of the Mucorales fungi tested (Table 1). No cross-reactivity was found with the unrelated mould pathogens of clinical importance (Aspergillus, Fusarium, Lomentospora, and Scedosporium species) [18]. Direct ELISA tests showed that mAb FH12 binds to a periodate-insensitive epitope (Figure 2A), with no loss in mAb binding due to periodate treatment over the 24 h experimental period. Heating of the EPS antigen led to a progressive loss in antibody binding compared with the control (no heat treatment; 0 min) over the 60 min experimental period. However, the loss in mAb binding, determined by successive decreases in ELISA absorbance values compared with the control, was significant (p < 0.05) at time points beyond the first 10 min of heating only (Figure 2B).

Figure 2.

Epitope characterisation by periodate oxidation and heat treatment. (A) Effect of periodate oxidation on mAb FH12 binding in direct ELISA to EPS from Apophysomyces variabilis CBS658.93. There was no significant effect of periodate treatment (shaded bars) compared with the control (open bars) over the 24 h period of treatment. (B) Effect of heat treatment at 100 °C on binding of mAb FH12 to EPS from Apophysomyces variabilis CBS658.93. There was no significant effect on mAb binding for 5 min or 10 min heating compared with the control (no heat treatment; 0 min), but there were successive and significant decreases in mAb binding between 20 min and 60 min compared with the control. For both the periodate and heat treatments, bars and data points are the means of 3 replicates ± SE, and bars (A) with the same letters show no significant differences (p < 0.05) between periodate treatment and matched controls (buffer only) at each time point. Data points (B) with an asterisk * show significant differences (Student’s t-test (p < 0.05)) between heat treatment and the control (no heat treatment; 0 min) at each time point.

3.3. Colony Immunoblotting and Immunofluorescence

In colony immunoblots, production of the FH12 antigen was shown to be extracellular and was associated with the growing margins of the fungal colony (Figure 3A). This staining pattern was similar to the staining pattern found with the pan-Mucorales-specific IgG1 control mAb TG11 (Figure 3B). In immunofluorescence studies of germinated cells of Mucor circinelloides f. circinelloides CBS120582, the FH12 antigen was localised to the outer cell wall of hyphae and germinated spores (Figure 3C,E), but was not found on the outer cell wall of the ungerminated conidium (indicated by the asterisk * in Figure 3F).

Figure 3.

Colony immunoblots and immunofluorescence imaging of the Mucorales pathogens Apophysomyces variabilis CBS658.93, and Mucor circinelloides f. circinelloides CBS120582. Colony immunoblots of Apophysomyces variabilis CBS658.93, showing extracellular production of EPSs bound by (A) mAb FH12 and (B) mAb TG11 ([13]) EPS antigens. Note the intense immuno-staining of extracellular antigens produced at the colony margins during active growth of the pathogen. Scale bars = 0.5 cm. (C,E) Epifluorescence images of germinated spores of Mucor circinelloides f. circinelloides CBS120582 immuno-stained with mAb FH12-Cy3 conjugate, showing intense immunofluorescence of the hyphal and spore cell walls of germinated conidiospores. The corresponding brightfield images are shown in (D) and (F), respectively. Note the lack of immunofluorescence of the ungerminated spore in (E), indicated by an asterisk in the corresponding brightfield image (F). Scale bar = 7 μm.

3.4. Sandwich ELISA and Western Blotting of Culture Filtrates

Using culture filtrates from yeasts and moulds grown for 72 h in YNB+G liquid medium, the sandwich ELISA was shown to be pan-Mucorales-specific (Table 2), reacting strongly with filtrates from Mucorales fungi only (sandwich ELISA absorbance values >0.100, the threshold absorbance value for test positivity). There was no cross-reaction of mAb FH12 with filtrates from unrelated yeasts and moulds of clinical importance, including Aspergillus spp., Candida albicans, Cryptococcus neoformans, Fusarium spp., Lomentospora prolificans, and Scedosporium spp. (all absorbance values <0.100, the threshold for test positivity).

A study of antigen production in YNB+G shake culture by Rhizomucor pusillus CBS120587, a Mucorales pathogen responsible for gastro-intestinal mucormycosis in humans, showed that growth of the pathogen (dry weight (g) of hyphal biomass) plateaued between 72 h and 96 h, while growth of the unrelated mould pathogen Aspergillus fumigatus Af2923 increased up to the final sampling point at 168 h (Figure 4A). Sandwich ELISA tests of the culture filtrates showed that the FH12 EPS antigen was secreted into the culture medium and was first detectable in the sandwich ELISA 24 h post-inoculation (Figure 4B). Antigen production continued to rise and, similar to hyphal biomass, plateaued between 72 h and 96 h post-inoculation. Western blots of culture filtrates showed that mAb FH12 reacted strongly with immunoreactive EPS antigens with molecular weights of between ~25 kDa and ~250 kDa (Figure 4C), which were first detectable at 48 h post-inoculation. The antibody did not cross-react with 72 h old and 96 h old culture filtrates of Aspergillus fumigatus Af293 (Figure 4C).

Figure 4.

Production of the FH12 EPS antigen by the Mucorales pathogen, Mucor indicus CBS120.08. (A) Dry weights of the pathogen over the 168 h experimental period. (B) Sandwich ELISA of YNB+G culture filtrates. Each data point is the mean of three replicates ± SE (A,B). The threshold absorbance value for detection of the antigen in the sandwich ELISA is 0.1 (indicated by the line in (B)), with values above this threshold positive for the detection of the FH12 antigen. (C) Western blot of culture filtrates using mAb FH12, showing the presence of immunoreactive EPS antigens with molecular weights of between ~25 kDa and ~250 kDa which are absent in the 72 h and 96 h culture filtrates of the unrelated human pathogen, Aspergillus fumigatus Af293. The Western blot is representative of three experimental repeats.

3.5. Sandwich ELISA of Human Serum and Limits of Detection

Using running buffer (RB) spiked with purified Mucorales EPSs (Table 3), the sandwich ELISA was shown to have limits of detection of pg EPS/mL (Apophysomyces variabilis, Cunninghamella bertholletiae, Lichtheimia corymbifera, Mucor circinelloides, and Rhizomucor pusillus) and in the ng EPS/mL range (Rhizopus arrhizus and Rhizopus microsporus). The sandwich ELISA is compatible with human serum (HS), with limits of detection in the pg/mL serum range (A. variabilis, C. bertholletiae, M. circinelloides, and R. pusillus) and in the ng/mL range (Lichtheimia corymbifera, Rhizopus arrhizus, and Rhizopus microsporus).

Table 3.

Limits of detection (LODs) of the sandwich ELISA in ng/mL using running buffer (RB) or human serum (HS) spiked with purified EPSs from Mucorales fungi. The LOD values are determined from the mean absorbance values of 4 replicate samples showing significant differences (Student’s t-test (p < 0.05)) compared with matched replicate controls (RB only or normal serum only) in each dilution series, with an absorbance value of 0.100 used as the threshold absorbance value for test positivity.

Mucorales Species	Isolate Number	Limit of Detection in RB (ng/mL)	Limit of Detection in HS (ng/mL)
Apophysomyces variabilis	658.93	0.45	0.57
Cunninghamella bertholletiae	151.80	0.07	0.35
Lichtheimia corymbifera	109940	0.46	4.59
Lichtheimia ramosa	124197	3.23	1.01
Mucor circinelloides	B5-2	0.44	0.88
Rhizomucor pusillus	120587	0.19	0.24
Rhizopus arrhizus var. arrhizus	112.07	36.13	72.25
Rhizopus microsporus var. rhizopodiformis	102277	10.20	65.25

3.6. Detection of Cutaneous Mucormycosis

In sandwich ELISA tests, the soluble FH12 EPS antigen was readily detectable in tissue homogenates from a cutaneous and soft tissue mucormycosis caused by the Mucorales pathogen Lichtheimia ramosa strain 266533346 (Figure 5) recovered during surgical debridement of the patient’s hand on day 5 (D5) and day 14 (D14) of hospital admission [17]. The mean absorbance values for the D5 (1.806 ± 0.076) and D14 (0.157 ± 0.006) tissue homogenate samples were significantly greater than the mean value for the control (0.065 ± 0.004), showing the presence of the Mucorales-specific FH12 antigen in the homogenate samples.

Figure 5.

Detection of cutaneous mucormycosis by sandwich ELISA. The mean absorbance values for the day 5 (D5) and day 14 (D14) tissue homogenate samples are significantly greater than the mean absorbance value for the negative control comprising sterile dH₂O (C), showing the presence of soluble Mucorales-specific FH12 antigen in the homogenate samples. Bars are the means of 3 technical replicates ± SE, and bars with different letters show significant differences in absorbance values between samples (p < 0.05).

4. Discussion

In this paper, we describe the development of a pan-Mucorales-specific IgG1 monoclonal antibody (mAb), FH12, and a sandwich ELISA for the detection of Mucorales-specific extracellular polysaccharides (EPSs). While an indirect ELISA has been reported for the serological detection of mucormycosis using preparations of heterogeneous antigens from Rhizopus arrhizus [19], this is the first time, to our knowledge, that a mAb-based sandwich ELISA has been reported for the diagnosis of mucormycosis in humans.

As shown by the immunoblotting of growing colonies and the immunofluorescence of germinating spores, the EPSs bound by mAb FH12 are excreted by Mucorales fungi during active hyphal growth, while Western blotting studies of culture filtrates demonstrate production of EPSs with molecular weights (~25 kDa to ~250 kDa) similar to the Mucorales cell wall EPSs bound by mAb TG11 [13,14]. However, unlike mAb TG11, which binds to a single epitope present in Mucorales EPSs and thereby restricts its use to a competitive format lateral-flow immunoassay [13], the repeat epitopes bound by mAb FH12 enable its use in a sandwich-format immunoassay (a sandwich ELISA). We have shown that the epitope bound by mAb FH12 is insensitive to mild periodate oxidation at acid pH, a modifying procedure commonly used for detecting anti-carbohydrate mAbs when used in conjunction with ELISA [20]. However, not all carbohydrate epitopes bound by mAbs are sensitive to periodate cleavage, particularly those dependent on a core trisaccharide rather than terminal residues. Consequently, we conclude that mAb FH12 binds to a core carbohydrate epitope rather than terminal sugar moieties in Mucorales EPSs.

Of the different types of ELISA format, the sandwich format is the most sensitive [21], and forms the basis of commercial ELISA tests for the detection of invasive pulmonary aspergillosis using human serum and bronchoalveolar lavage fluid (BALf) samples [22,23]. The pan-Mucorales-specific sandwich ELISA described here is similarly compatible with human serum. The moderate heat stability of the FH12 epitope enables the heat treatment of serum with EDTA to prevent immunoassay prozone effects, enabling detection of Mucorales EPS antigens in spiked human serum with detection in the pg/mL to low ng/mL range. This range of sensitivity is similar to that of enzyme immunoassays for aspergillosis detection [22,23].

The specificity of mAb FH12 enables the differentiation of pathogenic Mucorales species from other moulds and yeasts (Candida albicans and Cryptococcus neoformans) of clinical importance. This is especially significant in mixed-species infections with Aspergillus species [24], the cause of invasive pulmonary aspergillosis and the most common differential diagnosis with mucormycosis, and where other moulds such as Fusarium, Lomentospora, and Scedosporium are present in polymicrobial infections [25,26,27,28,29,30].

While the FH12 sandwich ELISA has yet to be evaluated with clinical serum and BALf specimens, we were able to demonstrate its potential clinical utility to detect invasive Mucorales infection in a single patient with skin and soft-tissue mucormycosis caused by Lichtheimia ramosa following a farming injury [17]. The soluble EPS biomarker bound by mAb FH12 was readily detectable in fluids from homogenised tissue removed during an early-stage debridement (day 5) of the infected wound. The biomarker was still detectable on day 14 of the infection, albeit significantly reduced compared with day 5, which was likely due to treatment with the antifungal drug amphotericin B and cessation of active Mucorales infection [17]. The results with the FH12 sandwich ELISA mirror those obtained with the TG11-LFD test [17], confirming the utility of Mucorales EPSs as signature molecules of mucormycosis in humans. Importantly, the sandwich ELISA was able to detect the EPS biomarker days before conventional microbiological tests (culture and histopathology of tissue biopsies) confirmed cutaneous infection by the Mucorales pathogen [17].

In conclusion, we have developed a sensitive and specific sandwich ELISA for the detection of mucormycosis, which has the potential to be used as a laboratory-based adjunct test for Mucorales infections in humans. A commercial EIA kit, ISCA Diagnostics Limited, Exeter, UK for the diagnosis of mucormycosis is currently in development and will be evaluated for clinical performance using serum samples from a large cohort of patients with proven and probable mucormycosis, and other invasive mould infections.

5. Trademark

The word ZygoDx^® (EU018696066) is protected by ISCA Diagnostics Ltd. through the European Union Intellectual Property Office (EUIPO).

Author Contributions

Conceptualisation, C.R.T. and G.E.D.; methodology, C.R.T. and G.E.D.; investigation, C.R.T. and G.E.D.; formal analysis, C.R.T. and G.E.D.; resources, C.R.T.; data curation, C.R.T. and G.E.D.; writing—original draft preparation, C.R.T.; writing—review and editing, C.R.T. and G.E.D.; supervision, C.R.T.; project administration, C.R.T.; funding acquisition, C.R.T. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Hybridoma work described in this study was conducted under a Home Office Project Licence, and was reviewed by the institution’s Animal Welfare Ethical Review Board (AWERB) for approval on 20 January 2022.

Informed Consent Statement

Human tissue samples were obtained for diagnostic purposes with informed written consent. Samples were tested, examined and disposed of, with informed written consent and in accordance with the Human Tissue Act 2004, and the Human Tissue Authority Code of Practice.

Data Availability Statement

The data presented in this study are available on request from the corresponding author, but are not publicly available due to commercial confidentialities.

Conflicts of Interest

CRT is founder director and an employee of ISCA Diagnostics Ltd. GED is an employee of ISCA Diagnostics Ltd.

Funding Statement

This work was funded by ISCA Diagnostics Ltd. (project title: A Monoclonal Antibody and Sandwich ELISA for Mucormycosis Detection).