We extracted proteins from the P. insidiosum strain Pi-S [21, 22], using the established method [17, 23]. In short, the organism was maintained on Sabouraud dextrose agar. Ten excised agar cubes (~0.5 cm3) with P. insidiosum colony were incubated with shaking (150 rpm) at 37 °C for 10 days in 100 ml of Sabouraud dextrose broth. The growing organism was removed from the cultured broth by consecutive filtrations through a filter paper (Whatman No.1) and a 0.22-μm-pore-size Durapore membrane (Merck Millipore). The filtrated broth was concentrated by centrifugation (10,000x g) with an Amicon Ultra 15M tube (Merck Millipore). The concentrated broth, now called culture filtrate antigen (CFA), was stored at -30 °C.
We developed a multiple host-specific ELISA by modifying the human-specific ELISA protocols of Chareonsirisuthigul et al [15] and Lohnoo et al [23]. In brief, a 96-well flat-bottom polystyrene plate (Corning) was coated overnight at 4 °C with 100 µl/well of CFA (5 µg/ml) in 0.1 M carbonate buffer (pH 9.6) and 1.5% NaCl. The plate was washed 4 times with the washing buffer (phosphate-buffered saline pH 7.4 [PBS] with 0.05% Tween-20) and blocked with 250 µl of 0.5% bovine serum albumin (Merck) in PBS at 37 °C for 60 min. After the washing step (as above), a serum sample (1:1,600 in PBS) was added (100 µl/ well) and incubated at 37 °C for 60 min. After washing, 100 µl of protein A/G conjugated with peroxidase (Bio-Rad) (1:100,000 in PBS) was added to each well and stored at 37 °C for 60 min. The ELISA plate was washed once again. Chromogenic substrate (3,3´,5,5´-Tetramethylbenzidine and H2O2; Thermo Scientific) was applied to each well (100 µl/well) and incubated at room temperature for 3 min in a dark chamber. Sulfuric acid (0.5 N; 100 µl/well) was used to stop the enzymatic reaction. Optical density (OD) of each serum sample was measured at the 450-nm wavelength.
A total of 25 serum samples from 20 humans, 4 horses and a dog with pythiosis, diagnosed in Thailand, were available for an assay evaluation. Control sera were derived from 50 healthy individuals with no sign of pythiosis (10 humans, 10 horses, 10 dogs, 10 cats, and 10 cows). Positive (from a known pythiosis patient) and negative (from a healthy blood donor) control sera were analyzed in parallel. All serum samples were tested in duplicate. ELISA value (EV) was an OD value of a serum sample divided by an OD value of the negative control serum. ELISA cutoff value was derived from the mean EV of the control sera plus 2, 3, 4, or 5 standard deviations (SD). Diagnostic performance (i.e., specificity, sensitivity, and accuracy) were calculated as described previously [15] (Table 1). Distribution of EVs (representing a detectable level of anti-P. insidiosum antibodies) of the pythiosis (n = 25) and control (n = 50) sera from both humans and animals was shown in Figure 1. Mean EV of the pythiosis sera was 41.0 (range, 23.1-46.9; SD, 5.2), whereas that of the control sera was 3.0 (range, 0.4-12.2; SD, 2.1). The “mean EV+5SDs” cutoff point provided the best sensitivity, specificity, and accuracy for the detection of the anti-P. insidiosum antibodies (Table 1).
With a proper ELISA cutoff point, all pythiosis sera can ultimately be distinguished from the control sera (Table 1; Figure 1). Unlike the species-specific ELISAs established by other investigators [10–15], the protein A/G-based ELISA is capable of detecting not only the anti-P. insidiosum antibodies in a single animal species, but also in humans and various animals (i.e., horses and dogs) those are the affected hosts of pythiosis. A key feature of our ELISA is the use of protein A/G (rather than a species-specific IgG) as a conjugate, which can bind the anti-P. insidiosum immunoglobulins from humans and other animal species. Recently, Intaramat et al. have developed the protein A/G-based ICT, as a rapid and user-friendly assay for diagnosis of pythiosis in humans and animals [16]. They performed a side-by-side performance comparison of their ICT [16] and the species-specific ELISA [11, 13, 15] and showed that ICT has equivalent specificity but relatively-lower sensitivity. One major obstacle for clinical use of ICT is the assay production, which is very complicated and requires a skilled developer and some special reagents that are not available in general clinical laboratories. As opposed to ICT, although ELISA is a multi-step assay and has a longer turnaround time, the production of ELISA is more feasible and requires widely-available reagents. In conclusion, protein A/G-based ELISA has been successfully developed for immunodiagnosis of both human and animal pythiosis. It is a versatile, feasible-to-develop, and functional immunodiagnostic assay.