Development of ic-ELISA and lateral-flow immunochromatographic strip for detection of vitamin B2 in an energy drink and vitamin tablets

ABSTRACT Vitamin B2 (riboflavin) is a water-soluble vitamin that has important roles in human health. In this study, we developed a sensitive and specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip for the rapid detection of vitamin B2. Following routine fusion and selection, the optimum monoclonal antibody against vitamin B2 was obtained. The 50% inhibitory concentration and limit of detection of ic-ELISA were 8.18 and 1.80 ng/mL, respectively. The cut-off value of the lateral-flow ICA strip was 50 ng/mL. The results revealed that our developed methods are suitable for the on-site detection and mass screening of vitamin B2 in food and pharmaceutical products.


Introduction
Vitamin B 2 , or riboflavin, is one of the most widely distributed B vitamins, which participates in several cellular processes. Vitamin B 2 plays vital roles in biological systems such as cellular respiration and metabolism of fats, carbohydrates, and proteins (Basaranoglu et al., 2017;Henriques, Olsen, Bross, & Gomes, 2010;Lewicka et al., 2017;Powers, 2003). Vitamin B 2 is present in a wide variety of animal and plant foods, such as liver, dairy products, and leafy vegetables. Low intake of dairy products, excessive alcohol consumption, and certain diseases may contribute to vitamin B 2 deficiency, characterized by anemia, angular cheilitis, cataracts, seborrheic dermatitis, and growth retardation (Kennedy, 2016;MacMillan et al., 2017;Mazur-Bialy & Pocheć, 2017;Mazur-Bialy, Pochec, & Plytycz, 2015;Powers et al., 2011;Qi, Kniazeva, & Han, 2017;Thomas & Mirowski, 2010). Therefore, vitamin B 2 supplementation is particularly important for individuals with vitamin B 2 deficiency. In the US, the recommended daily allowance for vitamin B 2 is 1.7 mg/day for pregnant women (Fankhanel & Gassmann, 1998), 0.3-0.4 mg/day for infants, and 0.6-1.8 mg/day for adults. To reduce the risk of vitamin B 2 deficiency, vitamin B 2 is commonly added to several foods. Therefore, a highly sensitive and specific vitamin B 2 analytical method is critical for assessing the nutritional quality and safety of foods.
The enzyme-linked immunosorbent assay (ELISA) based on antibody-antigen interactions has received considerable attention due to its high sensitivity, specificity, and throughput (Berlina, Zherdev, Xu, Eremin, & Dzantiev, 2017;Guo et al., 2015;). An indirect competitive ELISA (ic-ELISA) based on polyclonal antibodies against vitamin B 2 has been used for the determination of vitamin B 2 in foods and pharmaceuticals . Even though this method is sensitive and specific, polyclonal antibodies have great limitations in commercial applications because they cannot be reused and have poor reproducibility. Therefore, a highly sensitive and specific monoclonal (mAb)-based ic-ELISA for vitamin B 2 determination is required. The lateral-flow immunochromatographic assay (ICA) strip, which is based on antigen-antibody interactions, have been widely used in the toxins, heavy metals, and chemicals analysis (Kong, Xie, Liu, Song, Kuang, Cui, et al., 2017;Kuang, Xing, et al., 2013;Peng et al., 2017). The lateral-flow ICA strip is simple, rapid (results can be obtained with the naked eye within 10 min), effective, and suitable for semi-quantitative detection, qualitative detection, and mass sample screenings.
In this study, we produced a mAb against vitamin B 2 . Highly sensitive and specific mAb-based ic-ELISA and lateral-flow ICA strip were developed for the determination of vitamin B 2 in food and pharmaceutical products.

MAb preparation
Vitamin B 2 -BSA-1, vitamin B 2 -BSA-2, and vitamin B 2 -BSA-3 were used as immunogens. Female BALB/c mice (8-10 weeks of age) were immunized by continuous multipoint subcutaneous injections with the immunogens to generate polyclonal antibodies against vitamin B 2 . The mouse with the highest serum antibody titer and lowest 50% inhibitory concentration (IC 50 ) was sacrificed, and its spleen was fused with Sp2/0 murine myeloma cells . The target cells were selected by ic-ELISA and obtained by the limiting dilution method. MAbs were purified by the caprylic acid-ammonium sulfate precipitation method.

Development of ic-ELISA
In this experiment, 96-well microplates were coated with coating antigen diluted in coating buffer (100 µL/well) and incubated at 37°C for 2 h. Subsequently, the wells were washed three times with washing buffer. After washing the plates, 200 µL blocking buffer was added to each well and incubated at 37°C for 2 h. After washing, 50 µL antivitamin B 2 mAb and 50 µL vitamin B 2 standard were added to each well and incubated for 0.5 h at 37°C. After washing, 100 µL HRP-labeled goat anti-mouse IgG was added to each well, and the plates were incubated for 0.5 h at 37°C. After washing the plates three times, 100 µL substrate solution was added to each well and incubated for 15 min at 37°C in the dark. The reaction was stopped with the addition of 2 M sulfuric acid (50 µL/well). Absorbance was measured at 450 nm (Ding, Liu, Song, Kuang, & Xu, 2017). IC 50 and LOD were calculated by a standard curve generated by plotting optical density at 450 nm (OD 450 ) on the y-axis against vitamin B 2 concentration on the x-axis.

Cross-reactivity
MAb specificity was evaluated by measuring cross-reactivity (CR). Vitamins B 1 , B 3 , B 5 , B 6 , and B 12 , biotin, and folic acid were analyzed by ic-ELISA. CR was calculated using the following equation: IC 50 of vitamin B 2 IC 50 of cross -reacting compound × 100%.

Preparation of the gold nanoparticle (GNP)-labeled mAb
GNPs and GNP-labeled mAb were synthesized in our laboratory. For the synthesis of GNPlabeled mAb, the pH value of a GNP solution was adjusted to 8.0 with 0.1 M K 2 CO 3 . Subsequently, 0.2 mg anti-vitamin B 2 mAb was slowly added to 10 mL GNP solution and maintained at room temperature for 1 h. BSA (0.5% w/v, 1 mL) was added dropwise. Following a 2-h incubation, the solution was centrifuged (7000g for 30 min at 4°C). Unconjugated gold nanoparticle and anti-vitamin B 2 mAb in the supernatant were discarded. The resulting precipitate was washed three times with 0.02 M PBS (containing 5% sucrose, 1% BSA, and 0.5% polyethylene glycol 6000, pH 7.4), dissolved in 5 mL of 0.02 M PBS (containing 0.02% NaN3), and stored at 4°C .

Preparation of the lateral-flow ICA strip
The lateral-flow ICA strip was developed in our laboratory as previously reported . PVC backing card, NC membrane, sample pad, and absorption pad were assembled in layers. The NC membrane was pasted onto the center of the PVC backing card. The sample and absorption pads were glued to the bottom and upper section of the PVC backing card, respectively, with a 2-mm overlap of the NC membrane. Using a membrane dispenser, goat anti-mouse IgG and coating antigen were sprayed onto the NC membrane at 1 µL/cm to form the control line and test line (C line and T line, respectively). After drying at 37°C, the card was cut into 3-mm wide individual test strips and stored in a desiccator for further experiments.

Principle of the lateral-flow ICA strip
The detection principle of the lateral-flow ICA strip is based on a competitive interaction between vitamin B 2 present in the sample and the coating antigen sprayed on the T line for GNP-labeled mAb. Sample solution (100 µL) was mixed with 50 µL of GNP-labeled mAb in the wells of a microtiter plate and allowed to react at room temperature for 5 min. The ends of the strips with sample pad were inserted into the mixture. By capillary action, the mixture migrates from the sample pad to the NC membrane and reacts with the coating antigen on the T line and the goat anti-mouse IgG on the C line, finally reaching the absorption pad. The results are visualized by the naked eye within 5 min. In vitamin B 2negative samples, red T and C lines are obtained because GNP-labeled mAbs are captured by the coating antigen. In vitamin B 2 -positive samples, only a red C line is obtained. Vitamin B 2 present in the sample combines with GNP-labeled mAbs; therefore, less GNP-labeled mAbs are available to combine with the coating antigen on the T line, resulting in a weak red color on the T line. With increasing vitamin B 2 concentrations, the T line color intensity decreases. When the concentration of vitamin B 2 in the sample exceeds a certain value, no color appears on the T line. This critical concentration is defined as the cut-off value of the strip (Isanga et al., 2017). GNP-labeled mAbs or vitamin B 2 -GNP-labeled mAbs continue to migrate and react with the goat-anti-mouse IgG antibody, resulting in a deep red color on the C line. The sensitivity of the lateral-flow ICA strip is determined by the detection of a series of vitamin B 2 standards. In this study, each test was repeated seven times.

Sample analysis
An energy drink and compound vitamin B tablet were analyzed. Following pH adjustment to 7-7.5, the energy drink (vitamin B 2 concentration: 4 mg/100 mL) was passed through a 0.2-µm filter membrane. The filtrate was diluted to a suitable concentration for analysis. Two pieces of compound vitamin B tablets (vitamin B 2 concentration: 1.25 mg/piece) were dissolved in pure water to generate a stock solution. The solution was centrifuged at 6500g for 25 min at 4°C. The lipid layer was removed, and the supernatant was collected. The solution was diluted to a suitable concentration for analysis.

UV spectroscopic characterization
Vitamin B 2 -BSA and vitamin B 2 -OVA conjugates were used as immunogen and coating antigen, respectively. Vitamin B 2 -BSA and vitamin B 2 -OVA conjugates were characterized by UV absorption spectroscopy (Figure 1). The carrier proteins (BSA and OVA) had a characteristic absorption peak at 278 nm. Vitamin B 2 -BSA and vitamin B 2 -OVA conjugates had absorption peaks at 364 and 445 nm, and vitamin B 2 had absorption peaks at 263, 364, and 445 nm. A significant blue shift was observed between the antigens and carrier proteins, probably due to vitamin B 2 coupling at 263 nm. These results confirmed that the antigens were successfully conjugated to the carrier proteins.

Development and characterization of ic-ELISA
Vitamin B 2 -BSA was used as immunogen in mice immunization, and vitamin B 2 -OVA was used as coating antigen in ic-ELISA. The mouse with the highest serum antibody titer and lowest IC 50 was selected for cell fusion. Following cell infusion, the hybridoma cell line 3H8 was obtained, and vitamin B 2 antibody was purified from ascites by the caprylic acid-ammonium sulfate precipitation method. The standard curve (Figure 2) of mAb 3H8 against vitamin B 2 concentration (y = 0.195 + 1.347/[1 + (x/8.180) × 1.691]) had a linear regression correlation coefficient (R 2 ) of 0.991. The IC 50 value was 8.18 ng/mL, and the LOD defined as IC 10 value, was 1.80 ng/mL with a linear range of 3.60-18.56 ng/mL. The results revealed that our  developed method was sensitive for the detection of vitamin B 2 . MAb specificity was evaluated by ic-ELISA using other B vitamins ( Table 1). The results showed that vitamin B 2 mAb did not significantly react with other B vitamins (CR values <0.1%). Therefore, the developed ic-ELISA was specific to vitamin B 2 .

Optimization and characterization of the lateral-flow ICA strip
The analytical performance and sensitivity of the lateral-flow ICA strip may be affected by a series of parameters, including coating antigen and suspension buffer. To determine the appropriate reaction ratio of vitamin B 2 -OVA, three kinds of coating antigens (reaction ratios 50:1, 100:1, and 150:1) were sprayed onto the NC membrane. The lateral-flow ICA strip was used to analyze a vitamin B 2 -negative sample (0 ng/mL) and a vitamin B 2 -positive sample (50 ng/mL). The results are presented in Figure 3  obtained with the vitamin B 2 -positive sample at any reaction ratio. A reaction ratio of 50:1 contributed to the deepest color on the T line with the vitamin B 2 -negative sample. Therefore, the coating antigen with reaction ratio 50:1 was used for subsequent experiments. The composition of the suspension buffer affects the flow rate, background color, sharpness, and intensity of the T line. The basic suspension buffer contained 20 mM Tris (pH 8.2), 0.1% PEG, 0.1% Tween, 5% sucrose, 5% trehalose, and 0.2% BSA. Different surfactants were subsequently added. The vitamin B 2 -negative sample and the vitamin B 2positive sample were analyzed (Figure 3(B)). When the basic suspension buffer containing 1% PEG, 1% PVA, or 1% casein were used, we obtained no color on the T line with the vitamin B 2 -negative sample. The basic suspension buffer and suspension buffer containing 1% PVP, 1% BSA, 1% sucrose, 1% trehalose, 1% sorbitol, or 1% mannitol contributed to a weak T color with the vitamin B 2 -positive sample. The basic suspension buffer containing brij 35 or triton X-100 contributed to a deep red T line color with the vitamin B2-negative sample. Therefore, subsequent experiments were performed using suspension buffer that contained brij 35 and triton X-100.

(A). No color on T line was
Figure 3(C) shows that when the vitamin B 2 concentration was decreased from 50 to 25 ng/mL, no red line on T line could be observed for both two kinds the suspension buffer. However, for vitamin B 2 -negative sample, the suspension buffer containing brij 35 contributed to a weak red T line color, while the basic suspension buffer containing triton X-100 contributed to a distinct red T line color. Therefore, the optimum conditions of the developed lateral-flow ICA strip consisted of a reaction ratio of 50:1 and a suspension buffer containing 1% triton X-100.

Sample analysis
An energy drink and compound vitamin B 2 tablet were analyzed. The energy drink and compound vitamin B 2 tablet samples were analyzed at 6400-, 3200-, 1600-, 800-, 400-, 200-, and 100-fold dilution ( Figure 5). Ultrapure water was used as the negative control. For the energy drink sample, a lighter color was observed on the T line with the 1600-fold diluted sample, and the T line disappeared with the 800-fold diluted sample (50 ng/mL). For the compound vitamin B tablet, a lighter color was observed on the T line with the 3200-fold diluted sample, and the T line disappeared with the 1600-fold diluted sample (78.13 ng/mL). The results obtained from the energy drink sample were more sensitive compared with the results obtained from the compound vitamin B tablet sample, which is probably due to substrate conditions and matrix interference effects. More sensitive results could be obtained by repeated measurements of different dilution ratios. The results revealed that our developed lateral-flow ICA strip assay was sensitive, accurate, and suitable for the detection and screening of vitamin B 2 in real samples.

Conclusions
In this study, we developed a highly sensitive and specific mAb-based ic-ELISA and lateralflow ICA strip for the detection and screening of vitamin B 2 in food and pharmaceutical products. The IC 50 and LOD of ic-ELISA was 8.18 and 1.80 ng/mL, respectively, with a linear dynamic range of 3.60-18.56 ng/mL. The cut-off value of the lateral-flow ICA strip was 50 ng/mL. These methods were effective for the analysis of both simple and complex matrices. Therefore, the developed mAb-based ic-ELISA and lateral-flow ICA strip are effective for on-site detection and mass sample screening of food and pharmaceutical products.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Lu Zeng got her bachelor degree from Zhejiang Chinese Medical University, Hangzhou, China in 2015 and then she began to study in Jiangnan University (Wuxi, China) for her Master Degree in food science. Her research interests are immunoassay applications in food.
Wei Jiang got her bachelor degree from Yangzhou University, Yangzhou, China in 2015 and then she began to study in Jiangnan University (Wuxi, China) for her Master Degree in food science. Her research interest includes immunoassay development for food safety. Liqiang Liu got his Ph. D in Food science in 2014 from Jiangnan University, Wuxi, China and then became a faculty in the college of Food science and technology of Jiangnan University. His research interests are immunochromatographic strip design and application.
Shanshan Song got her Master degree in Food science in 2012 from Jiangnan University, Wuxi, China and then became a research assistant in the college of Food science and technology of Jiangnan University. Her research interests are monoclonal antibody development. Hua Kuang got her Ph. D from China Agricultural University in 2009 and then began to work as a faculty in the college of Food science and technology of Jiangnan University. She is currently a full professor in food safety. Her research interest is biosensor development.