Multiplex gradient immunochip for detection of post-vaccinal antibodies in poultry.

Multiplex analysis as an immunochip-in-a well format for simultaneous detection of post-vaccinal antibodies to three poultry infections (Newcastle disease, infectious bronchitis and bursal disease) in one chicken sera was developed. The immunochip had a microarray format printed on the bottom of a standard microtiter plate well and consisted of 36 microspots (d = 400 µm each) with three lines of viral antigens absorbed in a gradient of five decreasing concentrations. Optimization of assay conditions revealed the necessity of careful choice of the reaction buffer due to the high tendency of chicken IgY to exhibit unspecific binding. The best results were obtained for PBS buffer (pH 6.0) supplied with 0.1% Tween 20. Assay results were visualized by a number of coloured microspots that were correlated with the specific antibody titre in the analysed serum. High (> 8000), medium (3000-8000) or low (1000-3000) antibody titre level for each of three infections could be quickly assessed in one probe visually or with the help of smartphone. ELISA results (antibody titres) and visual gradient immunochip results interpretation (high, medium, low antibody level/titre) for 63 chicken sera with multiple levels of post-vaccinal antibodies against Newcastle disease, infectious bronchitis and bursal disease were in good correlation.

Dried Blood Spots technology for veterinary applications and biological investigations: technical aspects, retrospective analysis, ongoing status and future perspectives.

Dried Blood Spots (DBS) technology has become a valuable tool in medical studies, however, in veterinary and biological research DBS technology applications are still limited. Up-to-date no review has comprehensively integrated all the evidence existing across the fields, technologies and animal species. In this paper we summarize the current applications of DBS technology in the mentioned areas, and provide a scope of different types of dried sample carriers (cellulose and non-cellulose), sampling devices, applicable methods for analyte extraction and detection. Mammals, birds, insects and other species are represented as the study objects. Besides the blood, the review considers a variety of specimens, such as milk, saliva, tissue samples and others. The main applications of dried samples highlighted in the review include epidemiological surveys and monitoring for infections agents or specific antibodies for disease/vaccination control in households and wildlife. Besides the genetic investigations, the paper describes detection of environmental contaminants, pregnancy diagnosis and many other useful applications of animal dried samples. The paper also analyses dried sample stability and storage conditions for antibodies, viruses and other substances. Finally, recent developments and future research for DBS technology in veterinary medicine and biological sciences are discussed.

A semi-quantitative rapid multi-range gradient lateral flow immunoassay for procalcitonin.

A rapid semi-quantitative gradient lateral flow immunoassay (LFIA) of procalcitonin (PCT), a peptide precursor of the hormone calcitonin, was developed. The method is based on particular analyte cut-offs by immobilizing specific antibodies on the test strip with a consistent (gradient) increase in concentration from line to line. Semi-quantitative multi-range analysis is evaluated visually by counting the number of colored test lines corresponding to a certain concentration range of sepsis marker: [PCT]˂0.25; 0.25 ≤ [PCT] < 0.5; 0.5 ≤ [PCT] < 2; 2 ≤ [PCT] < 10; [PCT] ≥ 10 ng·mL-1. This multi-range gradient LFIA was implemented by using two types of label: spherical gold nanoparticles (35 nm) and hierarchical popcorn-like gold nanoparticles (100 nm). The comparison of this LFIA with an ELISA (for n = 82) yielded 87.5% and 76.6% sensitivities, and 92.3% and 92.3% specificities, respectively. Thus, multi-range gradient LFIA performs well at PCT thresholds, which is important for early diagnosis of sepsis and severe bacterial infection. In our perception, this method has a wide scope in that it may be implemented in numerous other LFIA based test systems. Graphical abstract Schematic of the gradient lateral flow immunoassay for determination of clinically relevant procalcitonin ranges. It allows to reach the correlation between the number of developed test lines and procalcitonin concentration range in serum by pre-immobilization of capture antibodies in a consistently (gradient) increasing concentration.

Strip-dried blood sampling: applicability for bovine leukemia virus detection with ELISA and real-time PCR.

We recently proposed a new so-called strip-dried format aimed for convenient use of dried biomaterial in diagnostic purposes. In this work, 334 blood samples obtained in strip-dried form were used for bovine leucosis analysis with ELISA and real-time PCR methods. High percentage of seropositive animals (18.3%) let us estimate both indirect (serological) and direct methods applicability for the analysis of strip-dried blood samples and also to compare them (PCR results concurred with ELISA in 93.4% cases). Parallel analysis of native and corresponding strip-dried samples approved the proposed format as a reliable analytical way of sampling being in 100% concordance with conventional serum/whole blood ELISA and PCR analysis. Even distribution of antibodies against bovine leukemia virus along the membrane carrier was demonstrated by square-to-square analyzing of the sample strip (CV not exceeded 7%). Also, strip-dried blood samples showed enhanced stability at elevated temperatures comparing to liquid serum. The proposed strip-blood format is a promising way of sampling, storage and transportation and can find application in veterinary practice for infectious disease monitoring.

Detection of Antibodies Against Foot-and-Mouth Disease Virus Serotypes A, O and Asia-1 by ELISA in Strip-Dried Samples from Vaccinated Bovines.

Antibodies against foot-and-mouth disease virus serotypes A, O and Asia-1 were detected by ELISA in liquid and strip-dried samples from vaccinated bovines. The results showed high concordance of the results in both types of serum samples (coefficient of correlation varied from 0.89 to 0.96). Stability studies showed that anti-foot-and-mouth disease virus antibodies can be detected in strip-dried serum samples stored at different temperatures on a level of that in native samples. Preliminary study in strip-dried whole blood samples demonstrated good potential of this sample pretreatment procedure for the following anti-foot-and-mouth virus antibodies testing.

Rapid flow-through enzyme immunoassay of progesterone in whole cows' milk.

A rapid flow-through immunoassay using an enzyme (horseradish peroxidase) as a label for quantitative and semi-quantitative determination of progesterone in whole cows' milk was developed. The flow-through test device consisted of a porous nitrocellulose membrane coated with antibodies and an absorbent membrane. The substrate solution containing 3,3',5,5' -tetramethylbenzidine was used for colour visualization. The detection limit of 0.4 ng/mL P4 was obtained by this method; analysis time did not exceed 15 min. To eliminate matrix interference a simple sample preparation procedure was used. Results of analysis of whole cows' milk samples with flow-through method were in good correlation with ELISA results (R = 0.96, n = 34). The developed rapid flow-through test system showed high efficiency for the determination of progesterone level in whole cow's milk and can be used on-site for quick identification of milk samples with low and high progesterone concentration.

A new dried milk sampling technique and its application for progesterone detection in cows.

A new method for milk sample collection and storage, based on a dried milk sampling technique, is proposed. The method includes application of a whole milk sample to a porous membrane followed by drying. One hundred whole milk samples (dried and liquid) taken on day 21 post insemination were analysed for progesterone by ELISA and results for both dried and liquid samples were well correlated (r=0.911). Milk progesterone ELISA accuracy for pregnancy diagnosis in cows was 87%.

Highly sensitive field test lateral flow immunodiagnostics of PVX infection.

A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1-0.2 ml (3-5 drops) of tested solution only (extracted from 10-20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor.

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.