scholarly journals Development of new immunoanalytical test systems for diagnostics of potato blackleg caused by Dickeya spp. bacteria

2021 ◽  
Vol 16 (3) ◽  
pp. 198-214
Author(s):  
Shyatesa Razo ◽  
Pavel A. Galushka ◽  
Yuri A. Varitsev ◽  
Anatoly V. Zherdev ◽  
Irina V. Safenkova ◽  
...  

Potato blackleg caused by Dickeya spp. bacteria is one of the most important bacterial diseases of potatoes. The rapid spread of this disease in the territory of Russia requires new effective diagnostic tools for the timely detection of infection. To solve this problem, antisera specific to Dickeya spp. were obtained. Polyclonal antibodies isolated from antisera have shown high affinity for the main species of Dickeya spp. ( D. solani, D. dianthicola, D. chrysanthemi, D. dadantii, D. paradisiaca ). Enzyme linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) test systems have been developed based on specific and high affinity antibodies that were obtained. For ELISA, the detection limit was 0.8 105 cells/mL for D. solani and 2 104 cells/mL for D. dianthicola . For LFIA, suitable for use in non-laboratory conditions, the detection limit of D. solani was 2 105 cells/mL and the analysis time was 15 minutes. When testing potato seed material, LFIA test system confirmed positive results of ELISA determination in 75 % of samples, and negative - in 100 % of samples.

1993 ◽  
Vol 76 (2) ◽  
pp. 381-386 ◽  
Author(s):  
W Harvey Newsome ◽  
Jupiter M Yeung ◽  
Peter G Collins

Abstract A simple, sensitive, and precise enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of captan as its degradation product tetrahydrophthalimide (THPI) in foods using polyclonal antibodies. Three hapten analogues of THPI with different alky I spacer arm lengths were synthesized. Immunogens and coating proteins were prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. A 5-carbon spacer arm appeared to be optimum for the production of antibodies. Heterologous coating proteins did not improve the sensitivity, but reduction of homologous coating protein concentration did improve the sensitivity, resulting in a concentration of test compound required to inhibit binding by 50% of 15.5 ng/mL The antiserum is specific for captan, captafol, and THPI, but not other structurally related compounds. The minimum detection limit was 1 ng/mL; the linearity was 1-200 ng/mL. The overall recoveries of captan and THPI from 11 commodities spiked at 4 levels were 92 and 100%, respectively. The intra-assay and interassay coefficients of variation were 9.1 and 16.8% for apple blanks and 5.9 and 4.2% for apple spiked with 3 ppm THPI, respectively. The ELISA described is suitable for measuring captan and THPI at levels comparable to those typically found in fruit.


1996 ◽  
Vol 59 (9) ◽  
pp. 992-997 ◽  
Author(s):  
FENG-YIR YU ◽  
FUN S. CHU

Polyclonal antibodies against fumonisin B1 (FmB1) were produced in rabbits after immunizing the animals with either FmBl-keyhole limpet hemocyanin (KLH) or FmB1 bovine serum albumin (BSA). A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were used for the characterization of the antibodies and for analysis of the toxin in corn samples. The antibody titers in the serum of rabbits immunized with FmBl-KLH were considerably higher than in those immunized with FmBl-BSA. The antibodies from the rabbits immunized with FmBl-KLH were further characterized. The concentrations causing 50% inhibition of binding of FmB1-horseradish peroxidase (HRP) to the antibodies by FmB1, FmB2 and FmB3 in the ELISA were found to be 0.45, 0.72, and 25 ng/ml, respectively. The detection limit of FmBl, based on 95% confidence at 5% of inhibition of binding of FmBl-HRP conjugate, in buffer of the dc-ELISA was found to be 0.05 ng/ml. In the presence of a matrix such as corn, the detection limit was less than 50 ppb. The overall analytical recoveries of FmBl (50 to 1,000 ng/g) added to the ground corn and then extracted with CH3CN/H2O (1/1, vol/vol) with cleanup and without cleanup in the dc ELISA were found to be 70.5 and 85.9%, respectively. A good correlation was found between the FmBl levels in 2 starch and 10 naturally contaminated corn samples analyzed by the dc-ELISA and the high-pressure liquid chromatography (HPLC) method. The correlation coefficients between ELISA and HPLC were found to be 0.955 (y [ELISA] = 1.3 1x [HPLC] + 77 ppb; P < 0.001) and 0.811 (y = 1.13x + 34 ppb; P < 0.01) for the sample without and with cleanup treatment, respectively.


Author(s):  
Roy F. M. van der Putten ◽  
Henk te Velthuis ◽  
Lucien A. Aarden ◽  
Hugo ten Cate ◽  
Jan F. C. Glatz ◽  
...  

AbstractTissue factor, the main initiator of blood coagulation, is shed into plasma by blood cells and endothelium. While studying such circulating plasma tissue factor with a commercially available immunoassay, we found unsatisfactory results and therefore developed a new and highly sensitive enzyme-linked immunosorbent assay (ELISA). High-affinity monoclonal antibodies raised against recombinant soluble tissue factor were used and the new assay had a detection limit of 40fmol/L, approximately six-fold lower than existing assays. Normal ranges in 20 healthy donors were established in serum and in citrated EDTA and heparinized plasma. Tissue factor was also measured in three successive plasma samples from 43 patients with type 2 diabetes mellitus. In citrated plasma from healthy donors, tissue factor concentrations were 2.5 (1.0–9.3) pmol/L (median with range) and were not significantly different in diabetics. With a commercially available immunoassay, seven plasma samples were below the detection limit. Use of the new assay reduced intra-individual variation in diabetics from 49% to 14% and we conclude that high-affinity antibodies may markedly improve immunoassay performance.


2011 ◽  
Vol 347-353 ◽  
pp. 1537-1541 ◽  
Author(s):  
Han Yu Chen ◽  
Hui Sheng Zhuang

A novel immunoassay has been developed for the quantitative determination of polybrominated biphenyls (PBBs) using indirect competitive format. A new method was developed to synthesize PBBs congener (PCB15) hapten and its artificial immunogen, then the polyclonal antibodies. The assay was optimized concerning the coating conjugate and antibody concentration, incubation time and temperature, the tolerance to organic solvents and so on. Under optimized conditions, PBB15 can be determined in the concentration range of 0.01-100 μg L-1 with a detection limit of 0.02 μg L-1. The cross-reactivities of the assays were below 8%. While water samples could be analyzed directly.


2000 ◽  
Vol 83 (1) ◽  
pp. 139-143 ◽  
Author(s):  
Jupiter M Yeung ◽  
W Harvey Newsome ◽  
Michael A Abbott

Abstract An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of egg proteins in foods. The polyclonal antibodies developed were specific to whole egg proteins and did not cross-react with any of the 38 nuts, legumes, or other common food ingredients tested. The concentrations of egg proteins that will inhibit 50% of antibody–antigen binding, IC50, were 3–7 ng/mL, and the linear range was 0.5–62.5 ng/mL. The detection limit was 0.2 ppm for various foods. Recoveries ranged from 67 to 96%. The intra- and inter-assay coefficients of variation in this procedure were 10–13% for ice cream spiked at 0.8 and 1.6 ppm. The ELISA has been applied to ice creams, noodles, pasta, and breads. Egg proteins were identified in all declared egg products, and no false positives were found.


Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2622
Author(s):  
Aliah Zannierah Mohsin ◽  
Rashidah Sukor ◽  
Jinap Selamat ◽  
Anis Shobirin Meor Hussin ◽  
Intan Hakimah Ismail ◽  
...  

The chemical, technological and allergy properties of goat’s milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10−3 μM compared to 9.046 × 10−3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.


Polymers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1497 ◽  
Author(s):  
Ewa Moczko ◽  
Richard Díaz ◽  
Bernabé Rivas ◽  
Camilo García ◽  
Eduardo Pereira ◽  
...  

In 2004, octopamine was added to the list of drugs banned by the world anti-doping agency (WADA) and prohibited in any sport competition. This work aims to develop a new analytical method to detect octopamine in water and human urine samples. We proposed a pseudo-enzyme-linked immunosorbent assay (pseudo-ELISA) by replacing traditional monoclonal antibodies with molecularly imprinted polymer nanoparticles (nanoMIPs). NanoMIPs were synthesised by a solid-phase approach using a persulfate initiated polymerisation in water. Their performance was analysed in pseudo competitive ELISA based on the competition between free octopamine and octopamine-HRP conjugated. The final assay was able to detect octopamine in water within the range 1 nmol·L−1–0.1 mol·L−1 with a detection limit of 0.047 ± 0.00231 µg·mL−1 and in human urine samples within the range 1 nmol·L−1–0.0001 mol·L−1 with a detection limit of 0.059 ± 0.00281 µg·mL−1. In all experiments, nanoMIPs presented high affinity to the target molecules and almost no cross-reactivity with analogues of octopamine such as pseudophedrine or l-Tyrosine. Only slight interference was observed from the human urine matrix. The high affinity and specificity of nanoMIPs and no need to maintain a cold chain logistics makes the nanoMIPs a competitive alternative to antibodies. Furthermore, this work is the first attempt to use nanoMIPs in pseudo-ELISA assays to detect octopamine.


2021 ◽  
pp. 24-31
Author(s):  
O.V. Tsinoviy ◽  
◽  
L.I. Nalyvayko ◽  

In the absence of diagnostic kits for the detection of antibodies to metapneumovirus infection (MPVI) epizootological monitoring in Ukrainian farms is practically not carried out, imported test systems have a "sky-high" price, so there is a need for domestic methods of diagnosing this disease. The most accurate, easy-to-use method is ELISA-based test systems (enzyme-linked immunosorbent assay). A diagnostic ELISA test system for the detection of antibodies to MPVI has been developed and it has been established that this diagnosticum should be used in the practice of veterinary medicine for serological control of metaviral virus infection. The optimal ratios of components for the manufacture of ELISA test system have been worked out. The form of calculation of antibody titers in blood sera of chickens when testing them in one dilution is calculated. The sensitivity and specificity of the ELISA test system were determined (comparative analysis of serum testing results in ELISA, RNGA and RN). Scientific documentation has been developed – instructions for the manufacture and control of ELISA test systems for the detection of antibodies to metapneumovirus infection in the serum of chickens and instructions for its use. Indication and identification of the obtained virus isolate was carried out using polymerase chain reaction (PCR) and nucleotide sequencing. Based on the studies carried out in the suspension of the internal organs of turkeys (trachea, lungs), a virus belonging to subtype B of the genus Metapneumovirus, subfamily Pneumovirinae, family Paramyxoviridae of the order Mononegavirales was revealed. By comparing the nucleotide sequence of the G gene fragment of the PVT-09/B strain with the sequences of strains and isolates of the avian metapneumovirus subtype B published in the GenBank database, it was found that the metapneumovirus isolated from sick turkeys is phylogenetically close to the Brazilian strains 27A-07 2007 and MPV/B/Brazil-07/USP-08 G


Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 3975 ◽  
Author(s):  
Shyatesa Razo ◽  
Vasily Panferov ◽  
Irina Safenkova ◽  
Yuri Varitsev ◽  
Anatoly Zherdev ◽  
...  

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL−1 to 5.4 ng∙mL−1 in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method.


1996 ◽  
Vol 63 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Edith Laloy ◽  
Jean-Christophe Vuillemard ◽  
Mouhsine El Abboudi ◽  
Ismael Fliss ◽  
Eric De Grace ◽  
...  

SummaryPolyclonal antibodies raised against partly purified aminopeptidase were specific to cell-free extracts ofLactobacillus caseisubsp.pseudoplantarumUL 137, and did not cross react with any other proteins in Cheddar cheese, at least during the first week of maturation. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed in order to quantify cell-free extracts in the cheese curd, and was also used to determine the efficiency of encapsulation of cell-free extracts in liposomes. This method was very sensitive and exhibited a detection limit of ∼10 μg total protein/g cheese and ∼1 μg total protein/ml liposome suspension. The efficiency of encapsulation of cell-free extracts into liposomes was ∼55–60%. The retention of liposome-encapsulated cell-free extracts was ∼14 times that of non-encapsulated extracts.


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