Development of a sandwich enzyme-linked immunosorbent kit for reliable detection of milk allergens in processed food.

The inclusion of undeclared cow's milk proteins may cause health complications to milk-allergic consumers and is one of the leading cause of food recall in many countries all over the world. Therefore, to keep control on such incidences in processed products, we established a milk sandwich ELISA test kit by incorporating two polyclonal antibodies against milk proteins obtained from different species. Its analytical effectiveness in terms of sensitivity, specificity, accuracy, trueness, and precision were all analyzed. The limit of detection (LOD) of the test kit was 0.011 ppm, with high specificity for milk protein residues. The test kit was highly specific, apart from considerable cross-reactivity with goat milk and minor cross-reactivity with donkey and horse milk. The coefficient of variation of the test kit for intra-assay ranged from 4.02% to 14.62% and inter-assay ranged from 6.05% to 15.08% respectively. The sandwich ELISA was highly specific in detecting commercial food products. In a limited retail survey, 5/6 of the milk proteins declared on the ingredient labels tested positive for milk proteins. The study offers effective technical support for the sensitive detection of milk products both for food manufacturers and regulatory authorities.

Simultaneous detection of two herbicides in fruits and vegetables with nanoparticle-linked immunosorbent and lateral flow immunoassays.

Herbicides atrazine and acetochlor are used in crop production. Because of environmental and health hazards with respective maximum contamination levels of 3 and 20 ng/mL, quantifying these herbicides is important when considering presence in foods and vegetables. We utilized two Pd@Pt nanoparticle-amplified immunoassays, a colorimetric Pd@Pt nanoparticle-linked immunosorbent assay (NLISA) and differential pulse voltammetry (DPV) dependent on catalytic activity of Pd@Pt in a dual-lateral flow immunoassay (dual-LFIA-DPV). We achieved overall recoveries of 88.5-114 % in juice, fruit, and vegetable samples for both immunoassays. The NLISA yielded limits of detection (LODs) of 0.59 and 0.31 µg/kg and the dual-LFIA-DPV 0.27 and 0.51 µg/kg for the two respective species. Results for both immunoassays were validated by high-performance liquid chromatography (HPLC), for all food and drink samples though LODs are compromised when configuring the HPLC for both species with the same chromatogram. We expect Pd@Pt-based immunoassays to prove useful in various fields.

RNA-cleaving deoxyribozyme-linked immunosorbent assay for the ultrasensitive detection of chloramphenicol in milk.

In this presented work, an artificial deoxyribozyme was employed as the substitute for horseradish peroxidase (or alkaline phosphatase) in ELISA for generating amplified signals. The feasibility of the proposed deoxyribozyme-based ELISA (DLISA) was demonstrated in the detection of a forbidden veterinary drug, chloramphenicol. And its efficiency was praised since that ultrahigh sensitivity was accomplished with a detection limit of 0.1 ng/L. The wide linear range from 0.000001 µg/mL to 1.0 µg/mL, as well as good recoveries from 86 % to 104 % in whole milk samples showed its excellent practical performances. Besides, the DLISA was worth popularizing due to the easy connection of antibody and DNAzyme through a facile functionalization process of gold nanoparticles. These advantages showed the possibility of DLISA for developing commercial kits, and the utilization of flexible DNA fluorescent probes in DLISA would inspire more work on innovations.

Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin.

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.

Screening antibody and immunosorbent selectivity by two-dimensional liquid chromatography-MS/MS (2-D LC-MS/MS).

Selectivity of both peptide- and glycan-targeting antibodies was examined by 2-D LC-MS/MS. Proteins selected from biological extracts immunospecifically in a first chromatography dimension using antibodies immobilized by either covalent coupling or adsorption to protein G were desorbed with a denaturing mobile phase and transferred to a 1.5 microm nonporous particle RP chromatography (NP-RPC) column in a second dimension. Protein peak capacity of the NP-RPC column was approximately 50. Peaks collected from the RPC column were tryptic digested and the peptide fragments were identified by MALDI-MS/MS. The objective of this analytical strategy was to discriminate between protein antigens and nonantigens through identification of their peptides, leading to an evaluation of the selectivity of antibodies and immunosorbents. Quantification of the relative amount of antigen and nonantigen species captured by immunosorbents was achieved by absorbance, along with the likely capture mechanism. A limitation of the approach was in discriminating between isoforms of an antigen in which neither the antibody nor the LC-MS system targeted the differentiating feature in the isoforms.

Immunoaffinity Extraction and Alternative Approaches for the Analysis of Toxins in Environmental, Food or Biological Matrices.

The evolution of instrumentation in terms of separation and detection allowed a real improvement of the sensitivity and analysis time. However, the analysis of ultra-traces of toxins in complex samples requires often a step of purification and even preconcentration before their chromatographic analysis. Therefore, immunoaffinity sorbents based on specific antibodies thus providing a molecular recognition mechanism appear as powerful tools for the selective extraction of a target molecule and its structural analogs to obtain more reliable and sensitive quantitative analysis in environmental, food or biological matrices. This review focuses on immunosorbents that have proven their efficiency in selectively extracting various types of toxins of various sizes (from small mycotoxins to large proteins) and physicochemical properties. Immunosorbents are now commercially available, and their use has been validated for numerous applications. The wide variety of samples to be analyzed, as well as extraction conditions and their impact on extraction yields, is discussed. In addition, their potential for purification and thus suppression of matrix effects, responsible for quantification problems especially in mass spectrometry, is presented. Due to their similar properties, molecularly imprinted polymers and aptamer-based sorbents that appear to be an interesting alternative to antibodies are also briefly addressed by comparing their potential with that of immunosorbents.

Trace analysis by ion mobility spectrometry: From conventional to smart sample preconcentration methods. A review.

Ion mobility spectrometry (IMS) is a rapid and high sensitive technique widely used in security and forensic areas. However, a lack of selectivity is usually observed in the analysis of complex samples due to the scarce resolution of the technique. The literature concerning the use of conventional and novel smart materials in the pretreatment and preconcentration of samples previous to IMS determinations has been critically reviewed. The most relevant strategies to enhance selectivity and sensitivity of IMS determinations have been widely discussed, based in the use of smart materials, as immunosorbents, aptamers, molecularly imprinted polymers (MIPs), ionic liquids (ILs) and nanomaterial. The observed trend is focused on the development of IMS analytical methods in combination of selective sample treatments in order to achieve quick, reliable, sensitive, and selective methods for the analysis of complex samples such as biological fluids, food, or environmental samples.

Development of monoclonal antibody-based ultrasensitive enzyme-linked immunosorbent assay and fluorescence-linked immunosorbent assay for 1-aminohydantoin detection in aquatic animals.

Monitoring and rapid evaluation of nitrofurantoin metabolite, 1-aminohydantoin (AHD), are important for food safety and human health. Herein, we established the monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and quantum dots (QDs)-fabricated fluorescence-linked immunosorbent assay (FLISA). Monoclonal antibody specific to nitrophenyl derivative of AHD was derived from hybridoma cell lines 3.2.4/5A8. For another, CdTe core QDs with emission wavelength of 605nm were also synthesized. The performances of the proposed ic-ELISA and FLISA were further examined and the corresponding results were also validated by standard LC-MS/MS analysis. The obtained results indicated that both ic-ELISA and FLISA exhibited good dynamic linear detection for NPAHD over the range from 0.1 to 3.0ngmL-1. Meanwhile, proposed immunosorbent assays are characterized by satisfactory recovery rates of 81.5-113.7%. The experimental data suggested these two immunoassays could be facile, cost-effective and rapid tools for the prospective quantitative method for AHD analysis in food matrix.

Preparation of human antibodies to insulin.

Insulin was directly coupled and also indirectly coupled by a side chain to Sepharose 4B to form immunoabsorbents for affinity chromatography of insulin antibodies. The two direct derivatives were insulin lysyl and insulin phenylalanyl sepharose. The indirect derivative, insulin carboxyhexyl sepharose, was prepared in two ways: by a carbodiimide procedure and by an N-hydroxysuccinimide ester method. Insulin carboxyhexyl sepharose prepared by the latter method was found to be the most satisfactory material for affinity chromatography and was used to isolate insulin antibodies from human serum.

[Characteristics of erythrocyte diagnostic agents studied using optical methods]. / Kharakteristika éritrotsitarnykh diagnostikumov opticheskimi metodami.

The quantitative characterization of erythrocyte diagnosticums (ED) has been made by optical methods (light microscopy with the use of an image analyzer, model Magiscan 2, and the opacity spectrum technique). The following parameters of ED have been determined: the average of the major axis (5.25 +/- 0.57 micron for ED from Shigella sonnei and 5.53 +/- 0.50 micron for ED from Shigella flexneri), the ratio of semiaxes (p approximately equal to 3), the major axis length distribution, the refractive index (1.076 +/- 0.002). For controlling the concentration of ED the use of the opacity spectrum technique is recommended.

Comparison of antigen-type immunosorbents prepared by different ways.

7 types of IgG-containing immunosorbents were compared from different respects by using them for adsorption of antibodies. The human IgG was insolubilized by ethylchlorophormate and glutaraldehyde, thereafter it was bound to activated agarose and polyacrylamide gels and 3 new types of polyacrylamide immunosorbent were prepared by polymerizing the acrylamide in the protein solution and by coupling the protein to the matrix by different ways. Using batch technique, the ethylchlorophormate, the glutaraldehyde and some of the new polyacrylamide immunosorbents proved to be useful, but in almost every respect the immunosorbent prepared from Sepharose gel was the best.

Characterization of adsorption on the stationary phase using high-performance immunoaffinity chromatography.

Low-level adsorption on the stationary phase has been studied using immunochemical reagents. An immunoaffinity column has been evaluated using affinity-purified radioisotope-labeled monoclonal antibodies. Recovery experiments including continuous immunosorbent monitoring have been performed. Proper characterization of an immunoaffinity separation can result in the recovery of immunologically active material in high yield.

Immunosorbent consisting of DNA immobilized on oxirane-activated sepharose.

The preparation of adsorbents for DNA antibodies is described. The degree of immobilization of native DNA on Sepharoses activated with epichlorohydrin or bisoxirane was investigated as a function of pH, temperature, time, concentration of DNA, and oxirane content in the supports. The maximum amount of DNA bound was obtained after 8 h at 40-50 degrees C at pH 11-11.5. The amount bound was increased by raising either the concentration of DNA or the oxirane content of the supports, and could reach 300 mg/g dry support. The immobilized DNA was applied to the adsorption of DNA antibodies using either commercial human serum with anti-native DNA activity or the sera of patients with systemic lupus erythematosus. The amount of antibody adsorbed depended on the amount of DNA. The thermal stability of the immobilized DNA was also examined. After heating at 80 degrees C, the leakage of DNA was slight and the adsorption of antibodies was not affected.

Effects of preventing periparturient hypocalcemia in cows by parathyroid hormone administration on hematology, conglutinin, immunoglobulin, and shedding of Staphylococcus aureus in milk.

The effects of hypocalcemia at parturition on concentrations of serum immunoglobulin and conglutinin, number of bacteria shed into milk, and leukograms of dairy cows were investigated from -4 wk prepartum to 4 wk postpartum. Ten healthy multiparous Holstein cows were fed a high calcium diet to induce hypocalcemia at parturition. Five cows received intramuscular parathyroid hormone to prevent hypocalcemia at parturition. All cows experienced a leukopenia (attributable to an absolute and relative neutropenia) during the 1st wk after calving, decreased serum conglutinin activity during the first 3 wk postpartum, and decreased concentration of serum IgG1 during the 3 wk before calving. At parturition, a large increase in organisms was found in foremilk (1000 to 10,000 times more than prepartum values). Neither the hematological changes nor the decreased immunoglobulin concentration was influenced by hypocalcemia or the development of milk fever. This implies that the degree of hypocalcemia observed did not have a large or irreversible influence on bacterial infection, hematological, or humoral immunity changes in periparturient cows.