Immunological Separation of Bioactive Natural Compounds from Crude Drug Extract and Its Application for Cell-Based Studies

In this study, we present a review on a useful approach, namely, immunoaffinity column coupled with monoclonal antibodies (MAbs), to separate natural compounds and its application for cell-based studies. The immunoaffinity column aids in separating the specific target compound from the crude extract. The column capacity was stable even after more than 10 purification cycles of use under the same conditions. After applying the crude extract to the column, the column was washed with washing buffer and eluted with elution buffer. The elution fraction contained the target compound bound to MAb, whereas the washing fraction was the crude extract, which contained all compounds except a group of target compounds; therefore, the washing fraction was referred to as a knockout (KO) crude extract. Cell-based studies using the KO extract revealed the actual effects of the natural compounds in the crude extract. One-step separation of natural compounds using the immunoaffinity column coupled with MAbs may help in determining the potential functions of natural compounds in crude extracts.

Assessment of Citrinin in Spices and Infant Cereals Using Immunoaffinity Column Clean-Up with HPLC-Fluorescence Detection

Historically, the analysis of citrinin has mainly been performed on cereals such as red yeast rice; however, in recent years, more complex and abnormal commodities such as spices and infant foods are becoming more widely assessed. The aim of this study was to develop and validate clean-up methods for spices and cereal-based infant foods using a citrinin immunoaffinity column before HPLC analysis with fluorescence detection. Each method developed was validated with a representative matrix, spiked at various citrinin concentrations, based around European Union (EU) regulations set for ochratoxin A (OTA), with recoveries >80% and % RSD <9% in all cases. The limit of detection (LOD) and the limit of quantification (LOQ) were established at 1 and 3 µg/kg for spices and 0.1 and 0.25 µg/kg for infant cereals, respectively. These methods were then tested across a variety of spices and infant food products to establish efficacy with high recoveries >75% and % RSD <5% across all matrices assessed. Therefore, these methods proved suitable for providing effective clean-up of spices and infant cereals, enabling reliable quantification of citrinin detected. Samples such as nutmeg and infant multigrain porridge had higher levels of citrinin contamination than anticipated, indicating that citrinin could be a concern for public health. This highlighted the need for close monitoring of citrinin contamination in these commodities, which may become regulated in the future.

Determination of Aflatoxins and Ochratoxin a in Animal Liver Using HPLC-FD Method with Immunoaffinity Column Clean-Up

Analytical methods based on immunoaffinity column clean-up and quantitative determination with liquid chromatography-fluorescence detection were used to determine aflatoxins and ochratoxin A in liver samples. The validation of the procedures was performed. The linearity of the methods was checked, and a good coefficient of correlation was found for all aflatoxins and OTA as well. The LOD and LOQ were acceptable: 0.003 µg/kg and 0.009 µg/kg for AFB1; 0.001 µg/kg and 0.005 µg/kg for AFB2; 0.006 µg/kg and 0.020 µg/kg for AFG1; 0.007 µg/kg and 0.022 µg/kg for AFG2; 0.08 µg/kg and 0.27 µg/kg for OTA. The results for the repeatability estimated by the relative standard deviation (RSDr) were satisfactory and the obtained values were in the acceptable range (1.97–14.41% for all aflatoxins and 3.76-8.31% for OTA) at three proposed concentration levels. RSDR values showed acceptable correlation between two analysts for all four aflatoxins and OTA. The RSDR values were as followed: 2.37% and 5.60% for AFB1, 6.71% and 8.78% for AFB2, 4.40% and 7.00% for AFG1 and 10.30% and 13.91% for AFG2 (for the first and second analyst, respectively). The RSDR values for OTA were 4.91% and 3.15% (1 µg/kg); 3.76% and 4.12% (5 µg/kg) and 8.31% and 8.21% (10 µg/kg). The mean recovery for total aflatoxins and OTA were 78.10% and 93.34%, respectively. All validation parameters were in accordance to European legislation. They indicate that the proposed analytical procedures are suitable and they could be methods of choice for the determination of aflatoxins and OTA in liver samples.

STUDY OF AFLATOXINS IN CASHEW NUTS PRODUCED IN COTE DIVOIRE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (LCHP)

The objective of this study is to isolate, identify and quantify four types of aflatoxins noted AFB1, AFB2, AFG1, and AFG2 that can be found in cashews grown in Cote dIvoire. These carcinogenic mycotoxins (AF) are secondary metabolite toxins produced by Aspergillus molds in plant foods.This work involved eleven (11) samples of 500 g of cashew nuts from eleven (11) cities of Cote dIvoire for the 2018-2019 campaign. These cities are: Beoumi, Bondoukou, Dabakala, Daloa, Douekoue, Ferkessedougou, Korhogo, Mignignan, Odienne, Sinematiali, and Touba. The test were carried out by high performance liquid chromatography (HPLC) after extraction of the four (4) mycotoxins on an immunoaffinity column at a flow rate of 3 mL / minute. These aflatoxins were identified and quantified from the following pairs of Retention time (Rt) in minutes and Limit of Detection (LD) in µg / kg: (13.777 0.00143), (10.583 0.00136), (9.901 0.00151), and (8.184 0.00564) respectively for AFB1, AFB2, AFG1, and AFG2. Our results show that all eleven (11) samples from these eleven (11) different cities contain aflatoxin (AFB1, AFB2, AFG2 and AFG1) contents below the national standard (2 µg / kg), that of the CODEX Alimentarius (1.4 µg / kg) and that of the European Union (2 µg / kg) indicating that cashews produced in Cote dIvoire comply with international standards and their consumption does not pose any risk to human health caused by the studied aflatoxins.

Preparation and Characterization of scFv-Coupled Immunoaffinity Column for Purification of Fibrinolytic Enzyme from Bacillus subtilis Natto-89

Background: The focus of this study was to prepare and characterize the single-chain variable fragment antibody (scFv)-coupled immunoaffinity column for purification of subtilisin BRC. Methods: The scFv against subtilisin BRC was immobilized onto CNBr-activated Sepharose 4B. Adsorption isotherm for subtilisin BRC on scFv-BRC-coupled Sepharose 4B was obtained and calculated the maximum binding capacity. The extraction conditions, including eluting solution, the concentration of eluting solution and flow rate, were optimized. Under the optimized eluting conditions, the dynamic binding capacity of the immunoaffinity column was determined. Results: The scFv-BRC-coupled Sepharose 4B for immunoaffinity purification of subtilisin BRC was prepared. The coupling efficiency was about 78.4%, e.g. about 8 mg of scFv-BRC was covalently coupled to 1 g CNBr-activated Sepharose 4B. The maximum equilibrium binding capacity (qm) and dissociation constant (Kd) of the immunoaffinity column for subtilisin BRC were 3.01 mg/mL and 0.465 mg/mL, respectively. The immunoaffinity chromatography conditions were optimized and the subtilisin BRC was purified 3.29-fold with 55.6%. Conclusion: The subtilisin BRC was effectively purified with high purity using scFv-BRC-coupled Sepharose 4B and the dynamic binding capacity of the column was determined. These results suggested that scFv-BRC can be used as a ligand for affinity purification of subtilisin BRC.

Comparison of aflatoxins contamination levels in betel nuts (Areca catechu L.) imported from Asian countries

Background Aspergillus and their linked metabolites such as aflatoxins (AFs) are one of the extremely significant contaminants affecting food production around the world. The contamination of AFs has been identified in various food commodities, which have been recognized as carcinogenic, mutagenic, teratogenic and immunosuppressive. The present study was undertaken to assess the AFs contamination in betel nuts (Areca catechu L.) being imported to Pakistan from South Asian countries during 2018–2019. Methods A total of 143 betel nuts consignments (India = 80, Indonesia = 39 and Sri Lanka = 24) were obtained and analyzed for the AF contamination using immunoaffinity column (IAC) clean-up procedure subsequent by liquid chromatography with fluorescence detection. Results: In Indian-origin betel nuts, about 96.3% samples were contaminated with AFs, ranging from 1.18‒331.57 µg/kg with mean contamination of 76.11 ± 1.12 µg/kg; whereas, in Indonesian and Sri Lankan shipments, 100% samples of betel nuts were found infected with AFs, ranging between 1.88‒378.94 and 4.74‒106.58 µg/kg with an average level of 123.76 ± 1.25 and 47.95 ± 0.98 µg/kg, respectively. Conclusions In conclusion, the AFs levels present an acute toxicity to human health and also hazard factors for the economy since contaminated foodstuffs do not fulfill the requirements of export/import. Therefore, instant actions should be engaged and re-evaluate agricultural procedures and regular monitoring of AFs level in food stuffs to minimize the chances of various diseases such as oral pre-cancerous oral wounds, submucous fibrosis and squamous cell carcinoma (cancer).

Determination of Free Bisphenol A in Commercially Packaged Ready-to-Consume Carbonated/Non-Carbonated and Non-Alcoholic Beverages with Immunoaffinity Column Purification and UPLC with Fluorescence Detector, First Action 2019.07

Background Bisphenol A (BPA) is a chemical of concern in the food industry. There is a need for a sensitive analytical method for the determination of BPA in beverages. Objective To develop a method for the determination of BPA in carbonated, non-carbonated, and non-alcoholic drinks. Method Replicates of a carbonated soft drink, orange juice with pulp, and a dairy-based coffee drink at spiking levels ranging from 0 to 32 ng/mL were analyzed. The carbonated soft drink was adjusted to pH 7.4 and diluted with phosphate buffered saline (PBS). The orange juice with pulp and the dairy-based coffee drink were extracted with methanol and sodium chloride, then diluted with PBS. Results LOD ranged from 0.06 to 0.08 ng/mL and LOQ ranged from 0.10 to 0.14 ng/mL. Recoveries of BPA from all sample types at 1 to 16 ng/mL spiked levels were between 93 and 100%; relative standard deviation (RSDr, %) ranged from 0.71 to 8.38% depending on matrix and spiking levels. Conclusions The results indicate that the method for determination of BPA in carbonated, non-carbonated, and non-alcoholic drinks is reproducible and meets AOAC Official MethodSM performance criteria. Highlights The test portions were filtered and the filtrates applied to an immunoaffinity column (IAC) containing antibodies specific for BPA. After the column was washed with water, BPA was eluted from the IAC with 80% methanol and the eluate was directly injected, or concentrated and injected, into ultra-performance liquid chromatography (UPLC) with fluorescence detector (FLD) for separation, detection, and quantitation.