Immunoaffinity High-performance Liquid Chromatography Research Articles

Aflatoxin contamination of household stored grains for smallholder farmers in Dodoma, Tanzania

Abstract Aflatoxins are toxic and carcinogenic secondary metabolites, produced by Aspergillus flavus and Aspergillus parasiticus, which contaminate stored grains including maize, groundnuts and sunflower seeds and cause negative health effects to both humans and animals. The aim of this study was to determine the level of aflatoxins contamination of household stored grains (maize, groundnuts and sunflower seeds) in Dodoma, Tanzania. Immuno-affinity high performance liquid chromatography with post column derivatization was used to analyse AFB1, AFB2, AFG1, AFG2 and total aflatoxins in 45 samples. Out of 45 samples, 38% were contaminated with aflatoxins; the highest mean levels of total aflatoxins were observed in groundnuts (269 μg/kg) followed by maize (74.9 μg/kg) and lastly sunflower (0.2 μg/kg). About 27% of maize and 67% of groundnut samples had higher levels of AFB1 and total aflatoxins beyond the East African Community limits of 5 and 10 μg/kg, respectively. The mean moisture contents were 9.6% for maize, 4.1% for groundnuts and 5.7% for sunflower seeds. These moisture levels were far below the recommended safe storage moisture levels of 13.5% for maize, 8% for groundnuts and 10% for sunflower seeds. Such moisture levels would not allow mould growth and subsequent aflatoxins contamination. It suggests that contamination could have occurred in the field or during the drying stages before reaching the observed moisture levels. Therefore, there is a need to conduct further study to assess aflatoxin contamination in the field, during drying operations and storage to identify the critical stage for intervention. Moreover, farmers should be trained on best management and handling practices to prevent contamination.

English

Maize (Zea mays) is the staple food for the majority of people in Tanzania which plays a key role in subsistence and a cash crop among actors of the maize value chain. Environmental factors such as soil contamination by fungi, water stress, warm and humid conditions are among several factors contributing to fungal growth and aflatoxins contamination in maize, leading to significant economic loss, reduced household income, health problems to humans and animals and interferes with food security to communities. Structured questionnaires were used to collect information on awareness associated with aflatoxin contamination in maize from 160 smallholder farmers, 160 consumers and 60 traders in Kondoa and Chemba districts in Dodoma Region. A total of 90 maize samples (40 from smallholder farmers, 30 from consumers and 20 from traders) were analyzed for AFB1 using immuno-affinity high-performance liquid chromatography (HPLC) type Agilent Technologies 1200 serial. Data were statistically analyzed to assess awareness levels among maize main stakeholder and to check the current levels of aflatoxins B1 contamination in the study community. AFB1 was detected in five samples. About 3.3% of the contaminated maize had AFB1 levels above TBS acceptable levels (5 µg/kg). The highest mean concentration of AFB1 was in maize samples taken from traders with a mean of 9.88±5.904 µg/kg. The majority 56% of smallholder farmers and 52% of traders were aware of aflatoxins contamination and associated health effects on animals and humans. However, 74% of consumers were unaware of aflatoxins contamination in maize. The levels of contamination are low in the sample taken along maize value chain. An effective and broad awareness programme for community especially consumers on good management for prevention of aflatoxins contamination is necessary, as maize is the most consumed grain in the study area.    Key words: Aflatoxins contamination, smallholder farmers, consumers.

English

The aim of this study was to determine the level of aflatoxin M1 (AFM1) in pasteurized and UHT milk marketed in Dar es Salaam, Tanzania.  AFM1 in pasteurized milk samples (75) and ultra-high temperature (UHT) milk (43) was determined by using immuno-affinity high performance liquid chromatography. AFM1 was detected in 97% (115/118) of samples that consisted of 96% (72/75) of pasteurized milk samples and 100% (43/43) of UHT milk samples. About 82% of the contaminated pasteurized and UHT had AFM1 levels above EU acceptable levels (0.05 µg/L). However, none of the contaminated pasteurized and UHT milk sample had levels of AFM1 above the maximum recommended Codex limits (0.5 µg/L). The results indicate that the contamination of the samples with AFM1 at such level could pose a serious public health problem. Thus, regular monitoring of AFM1 levels in milk is important in order to protect consumers.    Key words: Aflatoxin M1, ultra-high temperature (UHT), milk, pasteurized milk, food safety.

Proteomic identification of serum biomarkers for gastric cancer using multi-dimensional liquid chromatography and 2D differential gel electrophoresis

BackgroundEarly diagnosis and treatment of gastric cancer patients is essential for improving prognosis. However, no available serum-based test provides sufficient sensitivity or specificity for widespread use. Therefore, in this study we aimed to identify cancer biomarkers in human sera using 2-dimensional difference gel electrophoresis (2D-DIGE), and to characterize protein biomarkers with tandem mass spectrometry. MethodsWe compared the serum proteomic profiles of 20 gastric cancer patients and 10 healthy volunteers. Serum samples were first chromatographed using an immunoaffinity high-performance liquid chromatography (HPLC) column to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Differential protein analysis was then performed using DIGE. Significantly increased and decreased protein spot features were excised, trypsin digested, and analyzed by tandem matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF and a linear trap quadrupole (LTQ) mass spectrometer. ResultsSeventeen protein spot features were significantly increased and 7 were significantly decreased in cancer serum samples compared to healthy controls. We identified 7 unique proteins that were upregulated, including plasminogen, apolipoprotein A-IV, Kininogen-1, complex-forming glycoprotein HC, complement component C4A, apolipoprotein J, and clusterin, and 5 that were decreased. ConclusionsThese results suggest that the combination of multi-dimensional HPLC and 2D-DIGE provides a valuable tool for serum proteomics in gastric cancer.

Joint IDF-IUPAC-IAEA(FAO) interlaboratory validation for determining aflatoxin M1 in milk by using immunoaffinity clean-up before thin-layer chromatography

A collaborative study was conducted under the auspices of the International Dairy Federation (IDF), the International Union of Pure and Applied Chemistry (IUPAC) and the International Atomic Energy Agency (IAEA), a collaborative Food and Agricultural Organization (FAO) body fully to validate a method combining immunoaffinity clean-up to thin-layer chromatography for the determination of aflatoxin M1 in milk. Work was done in order to afford those laboratories not equipped with high-performance liquid chromatography, mainly from developing countries, with a simplified but fully validated method as an alternative to the European validated immunoaffinity-high performance liquid chromatography method published as an EN ISO Standard 14501, February 1999. The validation study was carried out on samples of aflatoxin M1-contaminated milk and milk powder at levels close to the tolerable level of 0.5 μg l−1 as recommended by the Codex Alimentarius and to the regulatory level of 0.05 μg l−1 as laid down by the European Commission. Fourteen laboratories representing 11 countries participated in the trial. The relative standard deviations for repeatability and reproducibility based on raw data were in the range 27–48 and 35–54%, respectively. The recovery rate varied from 32 to 120%. The mishandling of two crucial steps of the protocol such as matrix sample reconstitution and extract evaporation could explain the wide variation of the recovery rate. For this reason, data were then corrected for recovery. Consequently, the relative standard deviations for repeatability and reproducibility were recalculated after correction for recovery and were in the range 26–54 and 34–53%, respectively. The method will be published as a standard ISO/DIS 14674—IDF 190.

Isolation of immunoglobulins and their use in immunoaffinity HPLC.

For the isolation of monoclonal and polyclonal antibodies different high performance liquid chromatography (HPLC) and high performance affinity chromatography (HPAC) methods were investigated. Specially designed "mixed-bed" ion-exchange and hydroxylapatite columns as well as hydrophobic interaction columns were efficiently applied to the isolation of monoclonal antibodies. When these methods are used for the isolation of polyclonal antibodies from antiserum, the sample has to be pre-treated, e.g. by removal of serum albumin. Protein A HPAC is an easy method and quick to handle, especially for the preparative isolation of antibodies. The antibodies that do not bind to protein A, can be purified by protein G HPAC. If this method cannot be used because of the rather extreme elution conditions, hydroxylapatite, ion-exchange or hydrophobic interaction HPLC have to be considered as alternatives. We further concentrate on immunoaffinity HPLC with immobilized antibodies. This method has proved to be very effective for one-step isolation of antigens, even from very complex samples such as plasma membrane extracts. The problem with immunoaffinity HPLC is the quick deterioration of the columns, caused by increasing denaturing of the immobilized antibodies during elution. In order to solve this problem, an indirect method is recommended for analytical immunoaffinity HPLC. For this purpose, the antibodies are bound to a protein A HPAC column. The solution containing the antigens is then applied. After washing, the antigen-antibody complex is eluted from the column.

High-performance liquid affinity chromatography of liver plasma membrane proteins

Plasma membrane proteins from liver were analysed by concanavalin A affinity and immunoaffinity high-performance liquid chromatography. In the method, four peptide bands with apparent molecular weights of 140 000, 120 000, 80 000 and 60 000 could be isolated. In the second method, with two immobilized monoclonal antibodies, two corresponding antigens — the membrane proteins dipeptidyl-peptidase IV and GP 110— could be highly purified from plasma membrane extract with good yield in only one step