Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Antibody use is a critical component of cardiovascular physiology research, and antibodies are used to monitor protein abundance (immunoblot analysis) and protein expression and localization (in tissue by immunohistochemistry and in cells by immunocytochemistry). With ongoing discussions on how to improve reproducibility and rigor, the goal of this review is to provide best practice guidelines regarding how to optimize antibody use for increased rigor and reproducibility in both immunoblot analysis and immunohistochemistry approaches. Listen to this article\'s corresponding podcast at http://ajpheart.podbean.com/e/guidelines-on-antibody-use-in-physiology-studies/ .

Peanut nut and tree nut allergy are characterised by IgE mediated reactions to nut proteins. Nut allergy is a global disease. Limited epidemiological data suggest varying prevalence in different geographical areas. Primary nut allergy affects over 2% of children and 0.5% of adults in the UK. Infants with severe eczema and/or egg allergy have a higher risk of peanut allergy. Primary nut allergy presents most commonly in the first five years of life, often after the first known ingestion with typical rapid onset IgE-mediated symptoms. The clinical diagnosis of primary nut allergy can be made by the combination of a typical clinical presentation and evidence of nut specifc IgE shown by a positive skin prick test (SPT) or specific IgE (sIgE) test. Pollen food syndrome is a distinct disorder, usually mild, with oral/pharyngeal symptoms, in the context of hay fever or pollen sensitisation, which can be triggered by nuts. It can usually be distinguish clinically from primary nut allergy. The magnitude of a SPT or sIgE relates to the probability of clinical allergy, but does not relate to clinical severity. SPT of ≥ 8 mm or sIgE ≥ 15 KU/L to peanut is highly predictive of clinical allergy. Cut off values are not available for tree nuts. Test results must be interpreted in the context of the clinical history. Diagnostic food challenges are usually not necessary but may be used to confirm or refute a conflicting history and test result. As nut allergy is likely to be a long-lived disease, nut avoidance advice is the cornerstone of management. Patients should be provided with a comprehensive management plan including avoidance advice, patient specific emergency medication and an emergency treatment plan and training in administration of emergency medication. Regular re-training is required.

Hemagglutinin (HA) head has long been considered to be able to elicit only a narrow, strain-specific antibody response as it undergoes rapid antigenic drift. However, we previously showed that a heterologous prime-boost strategy, in which mice were primed twice with DNA encoding HA and boosted once with virus-like particles (VLP) from an H5N1 strain A/Thailand/1(KAN)-1/2004 (noted as TH DDV), induced anti-head broad cross-H5 neutralizing antibody response. To explain why TH DDV immunization could generate such breadth, we systemically compared the neutralization breadth and potency between TH DDV sera and immune sera elicited by TH DDD (three times of DNA immunizations), TH VVV (three times of VLP immunizations), TH DV (one DNA prime plus one VLP boost) and TK DDV (plasmid DNA and VLP derived from another H5N1 strain, A/Turkey/65596/2006). Then we determined the antigenic sites (AS) on TH HA head and the key residues of the main antigenic site. Through the comparison of different regiments, we found that the combination of the immunization with the sequence close to the consensus sequence and two DNA prime plus one VLP boost caused that TH DDV immunization generate broad neutralizing antibodies. Antigenic analysis showed that TH DDV, TH DV, TH DDD and TH VVV sera recognize the common antigenic site AS1. Antibodies directed to AS1 contribute to the largest proportion of the neutralizing activity of these immune sera. Residues 188 and 193 in AS1 are the key residues which are responsible for neutralization breadth of the immune sera. Interestingly, residues 188 and 193 locate in classical antigen sites but are relatively conserved among the 16 tested strains and 1,663 HA sequences from NCBI database. Thus, our results strongly indicate that it is feasible to develop broad cross-H5 influenza vaccines against HA head.

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Overexpression of the ATP-dependent drug efflux pump ABCG2 is a major molecular mechanism of multidrug resistance in cancer and might be a predictive biomarker for drug response. Contradictory results have been reported for immunohistochemical studies of ABCG2 protein expression in colorectal cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we established scoring guidelines for ABCG2 expression based on the clinically used guidelines for HER2 immunohistochemistry assessment in gastric cancer. The guidelines provide a semi-quantitative measure of the basolateral membrane staining of ABCG2 and disregard the apical membrane staining and the cytoplasmic signal. Intra-tumor heterogeneity in ABCG2 immunoreactivity was observed; however, statistical analyses of tissue microarrays (TMAs) and the corresponding whole sections from primary tumors of 57 metastatic CRC patients revealed a strong positive correlation between maximum TMA scores and whole sections, especially when more than one core was used. In conclusion, here, we provide validated results to guide future studies on the associations between ABCG2 immunoreactivity in tumor cells and the benefits of chemotherapeutic treatment in patients with CRC.

The main chemicals used against varoa are acaricides, and the antibiotics used for the control of bee bacterial diseases are mainly tetracyclines, streptomycins, sulfonamides and chloramphenicol. No maximum residue limits (MRLs) have been set for any antibiotics in honey. Therefore, in the European Union, minimum recommended concentrations (RC) for the analytical performance of methods to control a certain set of these non-authorised chemicals in honey were published by the European Union Reference Laboratory (EU-RL) in 2007. Concerning the strategy for the control for antibiotic residues in honey, there is still a great need for a cheap and single multi-residue method. Biochip array technology is an innovative assay technology for the multi-analyte screening of biological samples in a rapid and easy-to-use format. A multi-array system, called Evidence Investigator™ (Randox, Crumlin, Co., Antrim, UK), was evaluated in our laboratory. It is a semi-automated biochip system designed for research, clinical applications and veterinary use. A competitive chemiluminescent immunoassay is employed for the detection of antimicrobials. The MicroArray II kit (AM II) dedicated to the screening of six different families of antibiotic residues was validated according to the European guideline for the validation of screening methods for residues of veterinary medicines. The specificity was proven to be very satisfactory, and applicability to different kinds of honey was demonstrated. The detection capabilities (CCβ) of six antibiotic residues were determined and were below the RCs when exist. The AM II kit could detect at least six quinolones, four tetracyclines and three epimers, three aminoglycosides, three macrolides, thiamphenicol, florfenicol and ceftiofur along with one of its stabilised metabolites, the desfuroylceftiofurcysteine disulfide (DCCD).

BACKGROUND: In organ transplantation, the introduction of the solid phase immunoassay technology radically changed the practice of antibody monitoring against human leukocyte antigens (HLA).
OBJECTIVE: Precise identification of antibody specificities in complex sera of sensitized patients and monitoring of low levels of donor-specific HLA antibodies in the posttransplant phase became possible. However, at the same time, new technical problems and great variation emerged in the interpretation of test results, indicating a need for standardization.
CONCLUSIONS: In May 2012, The Transplantation Society (TTS) recruited a panel of laboratory and clinical experts to discuss emerging testing and clinical management issues that are associated with antibody testing in organ transplantation. In this article, we provide a summary of the TTS recommendations formulated in this international effort on the standardization of antibody monitoring in kidney transplantation.

There is significant interest in the development of methods with the potential to increase access to \'the interactome\' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.

Skin tests in patients with IgE-mediated immediate type allergy are performed with the intention to establish a contact between allergens and skin mast cells. The latter carry specific IgE antibodies on their surface. If mast cells get activated, mediators (mainly histamine) are released which induce a visible skin reaction (wheal and erythema).[nl]Skin tests are indicated, if an immediate type allergic disease is suspected. Systemic anaphylactic reactions at skin testing are very rare. However, it is necessary to take them into account and to provide emergency treatment. Relative contraindications comprise skin diseases in the test area, poor general condition and insufficiently treated severe asthma. If tests are used, which have a higher risk for a systemic anaphylactic reaction, pregnancy or beta-blocker therapy, are further contraindications.[nl]Skin test application does not depend on patient age. However, in pre-school age tests are reluctantly performed. It is essential to consider the half-life of drugs which may interfere with the test result, and which have to be discontinued early enough before testing. After anaphylactic reactions there may be a refractory period. Therefore, tests should not be done within the first week after such reactions. Skin prick tests are the procedures of first choice, intradermal tests are more sensitive than prick tests. Skin tests are performed at the flexor side of the forearm. As intradermal tests are more inconvenient, testing can be also done at a less susceptible site of the body (upper back).[nl]It is recommended to use standardized test extracts. However, if standardised extracts are not available or do not yield suitable test results, one may switch to other preparations. If the patient shows a positive reaction to a non-standardized substance, control tests should be performed in healthy subjects in order to exclude an unspecific reaction.[nl]The reaction is read after 15 to 20 min. Skin tests are regarded positive if the mean wheal diameter is ≥ 3 mm at the prick test, and ≥ 5 mm at the intradermal test.[nl]Skin test results may be negative although patients are allergic. If a skin test is positive, one will have to distinguish reactions, which are clinically relevant, from those, which are not. History and/or challenge tests help to clarify the relevance of a sensitization. Usually, a clinically irrelevant sensitization does not lead to practical consequences.