Development of a Monoclonal Antibody and a Serodiagnostic Lateral-Flow Device Specific to Rhizopus arrhizus (Syn. R. oryzae), the Principal Global Agent of Mucormycosis in Humans

Abstract

Mucormycosis is a highly aggressive angio-invasive disease of humans caused by fungi in the zygomycete order, Mucorales. Though a number of different species can cause mucormycosis, the principal agent of the disease worldwide is Rhizopus arrhizus, which accounts for the majority of rhino-orbital-cerebral, pulmonary, and disseminated infections in immunocompromised individuals. It is also the main cause of life-threatening infections in patients with poorly controlled diabetes mellitus, and in corticosteroid-treated patients with SARS-CoV-2 infection, where it causes the newly described disease, COVID-19-associated mucormycosis (CAM). Diagnosis currently relies on non-specific CT, a lengthy and insensitive culture from invasive biopsy, and a time-consuming histopathology of tissue samples. At present, there are no rapid antigen tests for the disease that detect biomarkers of infection, and which allow point-of-care diagnosis. Here, we report the development of an IgG1 monoclonal antibody (mAb), KC9, which is specific to Rhizopus arrhizus var. arrhizus (syn. Rhizopus oryzae) and Rhizopus arrhizus var. delemar (Rhizopus delemar), and which binds to a 15 kDa extracellular polysaccharide (EPS) antigen secreted during hyphal growth of the pathogen. Using the mAb, we have developed a competitive lateral-flow device (LFD) that allows rapid (30 min) and sensitive (~50 ng/mL running buffer) detection of the EPS biomarker, and which is compatible with human serum (limit of detection of ~500 ng/mL) and bronchoalveolar lavage fluid (limit of detection of ~100 ng/mL). The LFD, therefore, provides a potential novel opportunity for the non-invasive detection of mucormycosis caused by Rhizopus arrhizus.

Keywords: Rhizopus; biomarker; lateral-flow device; monoclonal antibody; mucormycosis.

Figure 1

Western blots of culture filtrates from Rhizopus species using pAb SK0078 (A,B) and mAb KC9 (C,D). Though pAb SK0078 binds to antigens with molecular weights of between ~18 kDa to 250 kDa from all Rhizopus spp., mAb KC9 reacts with a single antigen of ~15 kDa, and is species-specific, reacting with different strains of Rhizopus arrhizus var. arrhizus, Rhizopus arrhizus var. delemar, and Rhizopus oryzae only. The positive control, comprising 20 μg of purified EPS from the R. arrhizus var. arrhizus strain, CBS112.07 (B,D), also yields a single KC9-reactive band of ~15 kDa, whereas the negative control, comprising YNB culture medium only, is negative both for pAbSK0078 and for mAb KC9.

Figure 2

Specificity of the LFD. (A) Specificity of the LFD using 100 μg purified EPS/mL running buffer of the human-pathogenic mucoralean fungi, Apophysomyces variabilis (strain CBS658.93), Rhizopus arrhizus var. arrhizus (strain CBS112.07), Mucor circinelloides (strain B5-2), Cunninghamella bertholletiae (strain CBS115.80), Lichtheimia corymbifera (strain CBS109940), R. microsporus var. rhizopodiformis (strain CBS102277), Rhizopus oryzae (strain CBS 111233), and Rhizomucor pusillus (strain CBS120587). Species other than R. arrhizus var. arrhizus and R. oryzae had T lines similar to the control (running buffer only). EPS from R. arrhizus var. arrhizus and R. oryzae resulted in complete displacement of KC9-gold conjugate binding to the T line, demonstrating the species-specificity of the LFD. + indicates a positive test result, − indicates a negative test result. (B,C) ELISA of the purified EPS samples, showing specific binding of mAb KC9 to R. arrhizus var. arrhizus and R. oryzae (B), and broad reactivity of pAb SK0078 with all species (C). Each point is the mean of three replicates ± SE, and the threshold absorbance value for detection of antigen in ELISA is ≥0.100. (D,E) Western blots of the purified EPS samples, showing species-specific binding of mAb KC9 to an ~15 kDa antigen of R. arrhizus and R. oryzae (D), and the presence of pAb SK0078-reactive antigens (~15 kDa to ~250 kDa) in all samples (E). Each well contains 20 μg EPS.

Figure 3

(A) Visual score card used for determinations of LFD test (T) and control (C) line intensities, recorded on a scale of 0–10. (B) Cube reader used for determination of T and C line intensities, recorded as artificial units (a.u.); scale bar = 1.5 cm. (C,D) Sensitivities of the LFD using the visual score card and cube reader systems, respectively. Bars are the means of three replicates ± 2 × SE, and * indicates a significant difference (Student’s t-test (p < 0.05) of mean values compared to the control (running buffer only)). All samples had control (C) line scores of 8 using the score card, and >300 a.u. using the cube reader. (E) Detection of the EPS biomarker in human BALf and serum. Samples were spiked with purified EPS from R. arrhizus var. arrhizus (CBS112.07) to give a final concentration of 80 μg/mL. Note the displacement of the T line with spiked BALf and serum samples, indicating a positive (+) test result. Normal (unspiked) BALf and serum samples gave a negative (−) test result (T lines present).