Isolation and Characterization of Chicken Serum Albumin (Hen Egg Alpha-Livetin, Gal d 5).

Chicken serum albumin, i.e., hen egg alpha-livetin, is a recognized food allergen in chicken meat and hen eggs. Currently, there is no immunoassay available for its detection from food matrices. The characterization of chicken serum albumin-specific antibodies and the extraction of the target protein are essential for immunoassay development. One monoclonal antibody (mAb), 3H4, was used in this study due to its selectivity to a linear epitope on avian serum albumin. To study the extraction of chicken serum albumin, phosphate-buffered saline (PBS) with two additives, i.e., sodium dodecyl sulfate (SDS) and dithiothreitol (DTT), was used for its extraction from chicken blood plasma and hen egg yolk. SDS and DTT improved the chicken serum albumin's recovery and enhanced chicken serum albumin's immunodetection. In addition, chicken serum albumin retained the best solubility and immunoreactivity after heat treatment in a neutral condition. It experienced degradation and aggregation in acidic and alkaline conditions, respectively. Overall, PBS containing 0.1% SDS and 1 mM DTT (pH 7.2) was a better extraction buffer for chicken serum albumin. However, the complexity of the food matrix and elevated temperature could reduce its solubility and immunoreactivity.

Characterization of a 36 kDa antigenic protein of fish-specific monoclonal-antibody 8F5.

The 36 kDa antigenic protein of a fish-specific monoclonal antibody (mAb), 8F5, previously developed to detect fish in foodstuffs to protect fish allergic individuals, was characterized to establish its identity and to identify the fish-specific epitope. We hypothesized that this antigenic protein is tropomyosin based on its thermal stability and molecular weight. Western blot showed that both the 36 kDa protein and fish tropomyosin were recognized by mAb 8F5, and their molecular weight migration in urea gel electrophoresis was identical. In addition to matching the amino acid composition profile, this 36 kDa protein's sequences precisely correspond to those in fish tropomyosin fragments. Further analysis revealed the sequence of the fish-specific epitope bound by mAb 8F5 to be EDDLVALQKK. These results confirm that the 36 kDa protein is indeed tropomyosin and will be a suitable biomarker for the immunodetection of fish in cooked food.

Development and Characterization of Monoclonal Antibodies for the Detection of Fish Protein.

This study developed and characterized anti-fish monoclonal antibodies (mAbs) capable of detecting fish, a major allergenic food, in processed food products to protect fish sensitized individuals. Of the three mAbs raised against crude protein extract of cooked fish muscle, mAb 8F5 exhibited a positive reaction to all 50 common food fish species tested with no cross-reactions to shellfish, land animals, or food additives. Although the ELISA results were negative against swordfish and yellowfin tuna, western blot clearly detected both after cooking. The ~36 kDa antigenic protein of mAb 8F5, which was found in all fish species, was detectable by mAb 8F5 in all of the fish samples even after prolonged heat treatment (100 °C, up to 60 min). These findings suggest that mAb 8F5 has great potential utility as a probe for the immunochemical detection of fish tissue in cooked food.

Matrix effect on food allergen detection - A case study of fish parvalbumin.

Two fish parvalbumin models were established to study relationships among matrix effect, extractability, and thermostability during in vitro immunodetection using two parvalbumin-specific monoclonal antibodies (3E1 and PARV19). Our results illustrated that matrix-induced thermal instability of parvalbumin was due mainly to physical (hydrophobic effect) and chemical (thiol-disulfide interchange) interactions. The addition of sodium dodecyl sulfate (SDS, surfactant), ß-mercaptoethanol (reducing agent) or ethylenediaminetetraacetic acid (EDTA, metal chelator) during sample preparation could not only increase the extractability of parvalbumin but also enhanced its immunodetection. Our findings demonstrated excess EDTA completely chelated Ca2+ in parvalbumin and rendered it undetectable using PARV19 (a Ca2+-dependent antibody). Overall, our resulted showed that matrix effect on in vitro analyte quantification cannot be underestimated. Any false negative or positive results could lead to severe or life-threatening allergic reactions.

Monoclonal antibody-based ELISA for the quantification of porcine hemoglobin in meat products.

Misusage of porcine blood proteins, such as misbranding and substitution, can cause religious objections, law violation, and food quality concerns. These issues highlight the need for detecting unlabeled or overuse of porcine blood in foods. Compared with acidic and neutral pHs, porcine hemoglobin (PHb) at alkaline pH retained the best solubility, molecular integrity, and immunoreactivity after heat treatment. PHb at acidic and alkaline pHs remained stable during storage at 4 °C for 29 days. A monoclonal antibody (mAb) specific to mammalian hemoglobin, 13F7, was developed. A mAb13F7-based indirect competitive ELISA (icELISA) was optimized for the quantification of PHb in meat products. This assay had a wide working range from 0.5 ppm to 1000 ppm. It was sensitive (limit of detection: 0.5 ppm), precise and reproducible with low inter- and intra-coefficient of variances (<20%). This assay is suitable for government, food industry, and third-party authority to surveillance food quality.

Speciation of animal fat: Needs and challenges.

The use of pork fat is a concern for Muslims and Jews, who for religious reasons avoid consuming anything that is pig-derived. The use of bovine materials, including beef fat, is prohibited in Hinduism and may also pose a risk of carrying the infectious agent for bovine spongiform encephalopathy. Vegetable oils are sometimes adulterated with animal fat or pork fat with beef fat for economic gain. The development of methods to determine the species origin of fat has therefore become a priority due to the complex and global nature of the food trade, which creates opportunities for the fraudulent use of these animal fats as food ingredients. However, determining the species origin of fats in processed foods or composite blends is an arduous task as the adulterant has a composition that is very similar to that of the original fat or oil. This review examines some of the methods that have been developed for fat speciation, including both fat-based and DNA-based methods, their shortcomings, and the need for additional alternatives. Protein-based methods, specifically immunoassays targeting residual proteins in adipose tissue, that are being explored by researchers as a new tool for fat speciation will also be discussed.

Immunoassay for the Detection of Animal Central Nervous Tissue in Processed Meat and Feed Products.

An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the detection of the thermal-stable central nervous tissue (CNT) marker protein, myelin basic protein (MBP), was developed to detect animal CNT in processed meat and feedstuffs. Two meat samples (cooked at 100 °C for 30 min and autoclaved at 133 °C for 20 min) of bovine brain in beef and two feed samples (bovine brain meal in beef meal and in soybean meal) were prepared at levels of 0.0008, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6%. An anti-MBP monoclonal antibody (mAb3E3) was produced using the hybridoma technique and characterized using Western blot. The optimized icELISA was CNT-specific without cross-reactivity with either meat (beef and pork) or soybean meal samples and had low intra-assay (%CV ≤ 3.5) and interassay variability (%CV ≤ 3.3), with low detection limits for bovine MBP (6.4 ppb) and bovine CNT spiked in both meat (0.05%) and feed (0.0125%) samples. This assay is therefore suitable for the quantitative detection of trace amounts of contaminated animal CNT in processed food and feed products.

Real-time assessment of the microbial quality of retail shrimp using CO2 evolution rate.

A real-time CO(2) evolution rate (CER) method together with conventional cultural and sensory techniques were utilized to determine the microbial quality and shelf life of several types of shrimp products: chloramphenicol (CAP) treated, imported farm raised, and domestic wild caught. Treatment with CAP was used to create different bacterial loads in shrimp samples to demonstrate the ability and sensitivity of the CER method for differentiating the bacterial activity in samples. Samples were divided into control (nontreated) and 0, 10, and 30 ppm of CAP treatment groups and stored at 4°C. The CER was recorded with a microrespirometer, and aerobic plate counts (APCs), olfactory sensory analyses, and pH measurements were recorded daily until spoilage occurred. The real-time CER results were highly correlated with the APCs (R(2) = 0.93) and readily distinguished the onset of spoilage in each of the treatment groups. CAP treatment at 10 and 30 ppm increased the sample shelf life by 2 and 3 days, respectively, compared with the nontreated samples. Untreated domestic wild-caught shrimp had a shelf life 1 day longer than that of the untreated imported farm-raised shrimp. No pattern of change in pH was noted throughout the storage period. When the olfactory sensory scores reached the marginally acceptable level, the mean CER was 27.23 µl/h/g and the mean APC was 5.78 log CFU/g. A cutoff CER of 25.0 µl/h/g was therefore selected to define acceptable raw shrimp. The CER method was a highly effective and sensitive real-time method for determining the microbial quality of raw shrimp.

Characterization of a 12 kDa thermal-stable antigenic protein in bovine blood.

UNLABELLED: We have previously developed an immunoassay based on monoclonal antibody (MAb) Bb1H9 for quantitative detection of ruminant blood in processed food and feedstuffs. The purpose of this study was to characterize the unknown 12 kDa thermal-stable ruminant-specific antigenic protein recognized by MAb Bb1H9 in order to better define the application scope of the developed assay. Extracts obtained from raw and heat-treated bovine blood-derived products were analyzed with indirect ELISA and Western blot. Target proteins resolved by 2D electrophoresis were subjected to N-terminal sequencing. Results indicated that the 12 kDa protein is a monomer of the tetrameric hemoglobin molecule (64.5 kDa) and that the heme group is not required for its binding with MAb Bb1H9. This MAb can be utilized as a probe for red blood cell derived products of ruminant origin in raw or processed food and feedstuffs to enforce labeling regulations and to address consumer concerns. PRACTICAL APPLICATION: MAb Bb1H9 represents the first antibody with the capacity to recognize bovine hemoglobin both in the absence and presence of the heme group, regardless of the heat treatment. MAb Bb1H9 can therefore be utilized in immunoassays by manufacturers and regulators to detect any ingredients containing hemoglobin or globin (hemoglobin without the heme group) in both raw and processed food and feed materials for product quality control and labeling law enforcement.

Competitive enzyme-linked immunosorbent assay for quantitative detection of bovine blood in heat-processed meat and feed.

Animal blood is widely used as a functional additive in food and as a protein supplement in animal feed. Effective analytical methods are therefore needed to enforce labeling regulations for product quality control, as well as to address safety concerns. This study developed a monoclonal antibody (MAb)-based competitive enzyme-linked immunosorbent assay (ELISA) for quantitative detection of ruminant (bovine and ovine) blood in heat-processed meat and feedstuff. MAb BblH9 immunoglobulin G1, which recognizes a 12-kDa, thermostable, ruminant blood protein, was used as the detecting agent in the competitive ELISA. The immunoreactivity of MAb Bb1H9 was confirmed in both an indirect noncompetitive ELISA and a competitive ELISA, in which antigens are presented in immobilized form and free form, respectively. The competitive ELISA was optimized, and three spiked samples adulterated at 0 to 10% were tested to determine the detection limits of the optimized assay. Results showed that MAb Bb1H9 is specific to ruminant blood protein, with no cross-reaction with nonblood samples tested. The optimized competitive ELISA quantitatively detected heat-processed bovine blood over the tested range. The detection limit of the assay for bovine blood in beef (P < 0.001), bovine blood in porcine blood (P < 0.01), and bovine blood meal in soybean meal (P < 0.01) was found to be 0.5% in all cases. This MAb-based competitive ELISA is thus suitable for the sensitive and quantitative detection of ruminant blood proteins in heat processed meat and feed products.

Innovations in food technology for health.

Modern nutritional science is providing ever more information on the functions and mechanisms of specific food components in health promotion and/or disease prevention. In response to demands from increasingly health conscious consumers, the global trend is for food industries to translate nutritional information into consumer reality by developing food products that provide not only superior sensory appeal but also nutritional and health benefits. Today's busy life styles are also driving the development of healthy convenience foods. Recent innovations in food technologies have led to the use of many traditional technologies, such as fermentation, extraction, encapsulation, fat replacement, and enzyme technology, to produce new health food ingredients, reduce or remove undesirable food components, add specific nutrient or functional ingredients, modify food compositions, mask undesirable flavors or stabilize ingredients. Modern biotechnology has even revolutionized the way foods are created. Recent discoveries in gene science are making it possible to manipulate the components in natural foods. In combination with biofermentation, desirable natural compounds can now be produced in large amounts at a low cost and with little environmental impact. Nanotechnology is also beginning to find potential applications in the area of food and agriculture. Although the use of new technologies in the production of health foods is often a cause for concern, the possibility that innovative food technology will allow us to produce a wide variety of food with enhanced flavor and texture, while at the same time conferring multiple health benefits on the consumer, is very exciting.