A highly sensitive immunoassay of pesticide and veterinary drug residues in food by tandem conjugation of bi-functional mesoporous silica nanospheres.

A novel type of enzyme-antibody conjugation using mesoporous silicon nanospheres (MSN) was developed, which amplified the labeling signal and highly increased the sensitivity of enzyme-linked immunosorbent assay (ELISA) for the determination of pesticide and veterinary drug residues in food. First, conjugates were prepared through layer-by-layer immobilization of an enzyme and an antibody on an MSN scaffold. Then the MSN scaffold was employed for labeling and signal amplification to develop a sensitive colorimetric immunoassay through the catalytic oxidation reaction of 5,50-tetramethylbenzidine (TMB). When this MSN-based ELISA was applied to detect chloramphenicol, avermectin, tetracycline and streptomycin in food samples, it provided linear ranges of 0.025 ng ml-1-25 ng ml-1, 0.05 ng ml-1-10 ng ml-1, 0.025 ng ml-1-10 ng ml-1 and 0.05 ng ml-1-25 ng ml-1, respectively, with low detection limits down to 0.011 ng mL-1, 0.134 ng mL-1, 0.015 ng ml-1 and 0.106 ng ml-1, respectively. For avermectin, it provided a 16.7-fold decrease of the limit of detection in contrast to that of standard ELISA without the loss of method specificity and accuracy. This novel immunoassay was hypersensitive, simple and easy-to-use, which made it high potential in applying for the accurate analysis of harmful substances in food.

Evaluation of the mucoadhesive strengths of Abelmoschus esculentus and Irvingia gabonensis gums for possible application in veterinary vaccine delivery: the effect of extraction methods.

This study evaluates the effects of different gum extraction methods on the mucoadhesive strengths of Abelmoschus esculentus (AE) and Irvingia gabonensis (IG) gums and the release of vaccine antigen in vaccine-gum formulations. AE and IG gums were extracted employing previously documented methods with acetone or sodium chloride (NaCl) and either oven-dried or freeze-dried. Gum extracts were analyzed for mucoadhesive strengths using a modified rotational cylinder method on animal mucosa. The time taken to detach from the mucosa was taken as the Peak Adhesion Time (PAT). The gum extracts were charged with Peste des petits ruminant vaccine and the antigen release was evaluated using agar gel immunodiffusion technique. The means of the PATS were analyzed using Mann-whitney t-test at p < .05. The NaCl extracted and freeze-dried IG gum showed sustained mean PATs of 1766 ± 73 s; 2116 ± 101 s; 7044 ± 117 s, while the oven-dried IG gum and both AE gums showed short-lived average PATs. Vaccine-gum formulations of IG at ratios 2:1, 1:1 & 1:2 had strong positive reactions while only that of AE at 2:1 showed a strong positive reaction. This study shows that NaCl extracted and freeze-dried IG gum has immunomodulatory potential for mucoadhesive vaccine delivery in ruminants.

Re-evaluating the LD50 requirements in the codified potency testing of veterinary vaccines containing Leptospira (L.) serogroup Icterohaemorrhagiae and L. serogroup Canicola in the United States.

Approximately one-third of the reportable USDA Category D and E laboratory animals in the United States are expended on the potency testing of leptospiral vaccines by the codified hamster vaccination-challenge assay. Valid tests require ≥80% of challenge controls to succumb to disease and an LD50 between 10 and 10,000. This work evaluates the risk associated with the removal of LD50 limits; thereby, eliminating back-titration hamsters from in vivo potency assays for Leptospira (L.) serogroups Canicola and Icterohaemorrhagiae. The impact was assessed through 1) retrospective analysis of industry and CVB serial release data from July 2011-April 2015 and 2) evaluation through vaccination-challenge assays. For the initial vaccination-challenge assays (n = 3/serogroup), one group received potent bacterin (PB) and six groups received subpotent bacterins (SB1-SB6). PB and SB1 were challenged with a single dilution of Leptospira between 10 and 10,000 LD50. SB2-SB6 received serial dilutions of more concentrated challenge. Based on the retrospective analysis and in vivo assays, 80% of the challenge controls succumbing to disease reasonably ensured the minimal LD50 was administered. Subpotent vaccines were not at increased risk for being deemed potent when challenged with >10,000 LD50, but potent vaccines were at risk of being deemed subpotent when challenged with >10,000 LD50.

An in vitro method for evaluating endotoxic activity using prostaglandin E(2) induction in bovine peripheral blood.

Severe side effects of veterinary vaccines, in particular Histophilus somni-containing vaccines for cows, have frequently been reported in Japan. These side effects are probably caused by endotoxins. Contamination levels of endotoxins could be monitored using the Limulus amebocyte lysate (LAL) test; however, the LAL test is not completely adequate for evaluation of in vivo endotoxic activities. In this study, we established a method for evaluating endotoxic activities using prostaglandin E2 (PGE2) induction in bovine peripheral blood. Blood and standard endotoxin, derived from Escherichia coli, were mixed and incubated. The concentration of induced PGE2 in the culture supernatant reached a maximum after 24-h incubation. A linear dose-response curve was observed for PGE2 concentration and the logarithmic transformed standard endotoxin concentration (5-5000 ng/ml). The endotoxic activity of H. somni in cows was the highest among those of several tested endotoxins. However, the LAL activities of H. somni were not as high as those of the other tested endotoxins. These results may provide a reason for the many report of side effects of H. somni-containing vaccines. The PGE2 detection assay described here could be a valuable method for evaluating the endotoxic activities of vaccines in cows.

Synthesis of novel haptens against ciprofloxacin and production of generic monoclonal antibodies for immunoscreening of fluoroquinolones in meat.

BACKGROUND: The residues of fluoroquinolone drugs in foods of animal origin are dangerous to the consumers. The objective of this study was to produce a generic monoclonal antibody for determination of fluoroquinolone residues in meat. RESULTS: Two novel haptens of ciprofloxacin containing a free amidogen group on the piperazinyl ring were synthesised that were used to produce the monoclonal antibodies. The antibodies obtained simultaneously recognised 12 fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, sarafloxacin, diflocaxin, danofloxacin, ofloxacin, marbofloxacin, pefloxacin, lomefloxacin, amifloxacin and enofloxacin). After evaluation of different coating antigen-antibody combinations, a heterologous competitive indirect ELISA was used to determine the 12 drugs. The cross-reactivities were in the range of 23-120% and the limits of detection were in the range of 1.0-4.5 ng mL(-1). Eight fluoroquinolone drugs licensed as veterinary drugs in China were fortified into blank chicken for analysis. The recoveries were in the range of 61.5-82.5% with coefficients of variation in the range of 7.5-15.2%. CONCLUSION: This method could be used as a rapid screening tool for routine monitoring the residues of these fluoroquinolone drugs in animal-derived foods.

Optical Fiber-Mediated Immunosensor with a Tunable Detection Range for Multiplexed Analysis of Veterinary Drug Residues.

We describe herein a newly developed chemiluminescent optical fiber immunosensor (OFIS) with a tunable detection range for multiplexed analysis of veterinary drug residues with vastly different concentrations in milk samples. The optical fiber probe is used as a carrier of biorecognition element as well as a transducer, enabling a low-cost compact design, which makes this system suitable for cost-effective on-site detection of the target analytes. Importantly, the synergy between modulation of the length of the optical fiber sensing region and the number of fibers allows performing multiplexed immunoassays in an easily controllable manner over a tunable detection range from pg/mL to µg/mL analyte concentrations. By combining the optical fiber sensor with a nanocomplex signal amplification system, a highly sensitive chemiluminescent OFIS system is demonstrated for the multiplexed assaying of veterinary drug residues in milk samples with linear ranges of 10-(2 × 104) pg/mL for chloramphenicol, 0.5-500 ng/mL for sulfadiazine, and 0.1-300 µg/mL for neomycin. This controllable strategy, based on modulation of the fiber probe, provides a versatile platform for multiplexed quantitative detection of both low-abundance and high-abundance targets, which shows great potential for on-site testing in food safety.

Comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs.

Two commercially available adjuvants, Gerbu LQ 3000 and Montanide ISA 50V, were assessed as potential replacements for Freund's adjuvant by evaluating their efficacy in the production of polyclonal antibodies to veterinary drugs in rabbits. The aim was to find an adjuvant that could produce a similar (or enhanced) immune response in the host animal without the undesirable side effects associated with Freund's complete and incomplete adjuvant. The assessment involved the examination of each injection site and the characterisation of the resultant antibodies with regards to antibody titre and sensitivity. It was found that the rabbits immunised with Gerbu adjuvant produced some of the most sensitive antibodies. However, titres were relatively low and adverse effects at injection sites were relatively common. Montanide adjuvant produced no adverse effects and the related antibodies were found to be of adequate sensitivity when compared to those from rabbits immunised with Freund's. It was concluded that Montanide ISA 50V could be considered as a suitable replacement to Freund's for the production of polyclonal antibodies, to low molecular weight compounds in rabbits.

Vaccine technologies: From whole organisms to rationally designed protein assemblies.

Vaccines have been the single most significant advancement in public health, preventing morbidity and mortality in millions of people annually. Vaccine development has traditionally focused on whole organism vaccines, either live attenuated or inactivated vaccines. While successful for many different infectious diseases whole organisms are expensive to produce, require culture of the infectious agent, and have the potential to cause vaccine associated disease in hosts. With advancing technology and a desire to develop safe, cost effective vaccine candidates, the field began to focus on the development of recombinantly expressed antigens known as subunit vaccines. While more tolerable, subunit vaccines tend to be less immunogenic. Attempts have been made to increase immunogenicity with the addition of adjuvants, either immunostimulatory molecules or an antigen delivery system that increases immune responses to vaccines. An area of extreme interest has been the application of nanotechnology to vaccine development, which allows for antigens to be expressed on a particulate delivery system. One of the most exciting examples of nanovaccines are rationally designed protein nanoparticles. These nanoparticles use some of the basic tenants of structural biology, biophysical chemistry, and vaccinology to develop protective, safe, and easily manufactured vaccines. Rationally developed nanoparticle vaccines are one of the most promising candidates for the future of vaccine development.

Collaborative study for the establishment of two European Pharmacopoeia Biological Reference Preparations for serological potency testing of tetanus vaccines for veterinary use.

The European Directorate for the Quality of Medicines (EDQM) has organised an international collaborative study, divided into two phases, aimed at producing and establishing two suitable reference sera for serological potency testing of tetanus vaccines for veterinary use for batch consistency demonstration. In phase I pools of sera were produced by immunising guinea pigs and rabbits with tetanus toxoid using the immunisation schedule prescribed by the European Pharmacopoeia (Ph. Eur.) for potency testing of tenanus vaccines for veterinary use. Following aliquoting and freeze-drying, characterization of the materials by immunochemical and biological assays enabled us to conclude that the sera should be suitable reference materials in respect of in-vitro assay methods for Clostridium (C.) tetani. The candidate (c) Ph. Eur. Biological Reference Preparations (BRP) were calibrated by Toxin Binding Inhibition test (ToBI) in phase II of the study by a large group of laboratories, including both manufacturers and official medicines control laboratories (OMCL). The activity of the proposed reference sera was determined by comparison with the existing equine monovalent World Health Organization (WHO) International Standard (IS). This study enabled us to provide a definitive value for the antitoxin activity of the reference preparations in respect of their anti-tetanus antibody content.

Developing country applications of molecular farming: case studies in South Africa and Argentina.

Molecular farming is a technology that is very well suited to being applied in developing countries, given the reasonably high level of expertise in recombinant plant development in many centers. In addition, there is an urgent need for products such as inexpensive vaccines and therapeutics for livestock and for some human diseases - and especially those that do not occur or are rare in developed regions. South Africa and Argentina have been at the fore in this area among developing nations, as researchers have been able to use plants to produce experimental therapeutics such as nanoantibodies against rotavirus and vaccines against a wide variety of diseases, including Rabbit haemorrhagic disease virus, Foot and mouth disease virus, Bovine viral diarrhoea virus, bovine rotaviruses, Newcastle disease virus, rabbit and human papillomaviruses, Bluetongue virus, and Beak and feather disease virus of psittacines. A combination of fortuitous scientific expertise in both places, coupled with association with veterinary and human disease research centers, has enabled the growth of research groups that have managed to compete successfully with others in Europe and the USA and elsewhere, to advance this field. This review will cover relevant work from both South Africa and Argentina, as well as a discussion about the perspectives in this field for developing nations.

Veterinary vaccines from transgenic plants: highlights of two decades of research and a promising example.

In the last two decades the development of efficient plant-based expression strategies and new concepts for the purification of recombinant proteins prompted the application of plant-derived vaccines for veterinary purposes. The expression of recombinant proteins in plants possesses advantages over conventional eukaryotic expression systems and therefore represents a versatile tool for the production of "edible" and "seasonal" vaccines. This review aims to provide an overview about the expression of vaccines using transgenic plants for veterinary medicine with the focus on increasing the amount of the recombinant proteins as well as concepts for their efficient purification. ELPylation of recombinant proteins is one strategy for on one side boosting the amount of the recombinant protein and on the other side simplifying its purification. This up-and-coming tool was applied for the development of effective production and purification strategies for antigens against Avian Flu, a very important animal disease with a strong economic impact. Future perspectives of plant-based veterinary vaccines in the context of purification and economy are also discussed within this article.

Nanovaccines: recent developments in vaccination.

In the past 100 years, vaccination has contributed immensely to public health by preventing a number of infectious diseases. Attenuated, killed or part of the microorganism is employed to stimulate the immune system against it. Progress in biotechnology has provided protective immunity through DNA vaccines. In recent years, nanovaccine is a novel approach to the methodology of vaccination. Nanomaterials are delivered in the form of microspheres, nanobeads or micro-nanoprojections. Painless, effective and safe needle-free routes such as the intranasal or the oral route, or patches of microprojections to the skin are some of the approaches which are in the experimental stage at present but may have a great future ahead in nanovaccination.

Selective recognition of veterinary drugs residues by artificial antibodies designed using a computational approach.

In this work we introduce a simple and inexpensive veterinary drugs residues detection method. Molecular dynamics simulations and computational screening were used to identify functional monomers capable of interacting with sulfadimidine (SM2). A library of 15 kinds of common functional monomers for preparing molecular imprinted polymer (MIP) was built and their interactions with SM2 in acetonitrile were calculated using the molecular dynamics software (GROMACS 3.3). According to the theoretical calculation results, the surface molecularly imprinted silica (MIP-silica) with SM2 as template was prepared by surface-imprinting technique using methacrylic acid (MAA) as functional monomer and divinylbenzene as cross-linker in acetonitrile. The surface composition of the MIP-silica was determined by Fourier transform infrared spectrometer (FT-IR) and energy dispersive X-ray spectrometer (EDS). Scanning electron microscopy (SEM) was used to characterize the morphological properties of the MIP-silica. The synthesized MIP-silica was then tested by equilibrium-adsorption method, and the MIP-silica demonstrated high binding specificity to the SM2. The molecular recognition of SM2 was analyzed in detail by using molecular modeling software (Gaussian 03).