Development of a Monoclonal Antibody-Based icELISA for the Detection of Ustiloxin B in Rice False Smut Balls and Rice Grains

Abstract

Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.

Development of Colloidal Gold‐Based Lateral Flow Immunoassay for Rapid Qualitative and SemiQuantitative Analysis of Ustiloxins A and B in Rice Samples

“…While the contents of UA and UB in rice FSBs (0.37–1.27 mg/g for UA; 0.16 to 0.66 mg/g for UB) (Table 1) and contaminated rice grains (1.99 to 107.90 µg/g for UA; 1.04 to 82.57 µg/g for UB) (Table 2) varied and were almost consisted with the results reported previously [26,27]. By analysis, the total content of UA and UB (0.58 to 1.93 mg/g) in rice FSBs was 3–640 fold greater than that (3 to 190 µg/g) in rice grains.…”

“…For LFIA of UB, 250–500 ng/mL of indicator range for UA (Figure S2A) displayed the cross reactivity was approximately 20%, which agreed with the results in icELISA [27]. No inhibition was observed for ustilaginoidins A, D, E and I at concentration of 50,000 ng/mL, whereas the test line was completely inhibited by UB at 100 ng/mL (Figure S2B), suggesting that this LFIA had no cross reactivity with ustilaginoidins in rice FSBs.…”

“…The detailed message of the rice FSB samples was shown in Table S2. The samples were extracted with distilled water according to the procedure described previously [15,26,27]. Briefly, 0.05 g of powdered sample was extracted with distilled water for three times (3 × 1.5 mL, 30 min for each time) in an ultrasonic bath at room temperature, followed by centrifugation at 8500× g for 10 min.…”

“…To develop such a LFIA, the antibody has been considered as the core reagent. Fortunately, two highly specific monoclonal antibodies (mAbs) 2D3G5 and 1B5A10 against UA and UB, respectively have been obtained [26,27]. In the present work, two LFIAs to UA and UB using the specific mAbs 2D3G5 and 1B5A10 were developed, respectively.…”

“…Conventional instrumental methods are unsuitable for high-throughput screening of a large number of samples as they are time consuming and labor intensive in addition to the need for expensive instruments. The enzyme-linked immunosorbent assay (ELISA) methods have also been reported for UA and UB [26,27]. However, ELISA method still requires labor-intensive operations, including incubation, washing, enzymatic reactions and instrument analysis.…”