Gentaur Pub

#33385362   2020/12/29 To Up

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.
Imen Tayachi, Yousr Galai, Meriem Ben-Abid, Nasreddine Saidi, Ines Ben-Sghaier, Karim Aoun, Aïda Bouratbine

1438 related Products with: Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

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#33001312   2020/10/01 To Up

Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR.

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.
Aghdas Ramezani, Mohammad Javad Rasaee, Amirmohsen Jalaeefar, Ali Hatef Salmanian

1170 related Products with: Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR.

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#32930214   // To Up

A one-step potentiometric immunoassay for plasma cardiac troponin I using an antibody-functionalized bis-MPA-COOH dendrimer as a competitor with improved sensitivity.

Herein, we have reported a new one-step potentiometric immunoassay for the sensitive and specific detection of human plasma cardiac troponin I (cTnI), a biomarker of cardio-cerebrovascular diseases. Initially, the cTnI biomolecules were immobilized on the surface of a gold nanoparticle-functionalized screen-printed graphite electrode (SPGE). Thereafter, rabbit polyclonal antibodies to cTnI were covalently conjugated to the bis-MPA-COOH dendrimers through typical carbodiimide coupling. The introduction of the target analyte caused a competitive immunoreaction between the immobilized cTnI on the electrode and the conjugated antibody on the dendrimers. The potentiometric measurement was mainly derived from the change in the surface charge on the surface of the modified electrode due to the negatively charged bis-MPA-COOH dendrimers after the immunoreaction. On increasing target cTcI, the number of charged dendrimers on the immunosensor decreased, resulting in a change in the electric potential. Under optimum conditions, the potentiometric immunosensor exhibited good potentiometric responses for the detection of cTcI and allowed the determination of the target analyte at a concentration as low as 7.3 pg mL-1. An intermediate precision of ≤8.7% was accomplished with batch-to-batch identification. Meanwhile, the potentiometric immunosensor showed good anti-interfering capacity and selectivity against other proteins and biomarkers. Importantly, our system displayed high accuracy for the analysis of human plasma serum samples containing target cTcI relative to commercial human cTcI enzyme-linked immunosorbent assay (ELISA) kits.
Erru Ni, Yizhen Fang, Fangfang Ma, Gaoshun Ge, Jingyi Wu, Yingying Wang, Yao Lin, Huabin Xie

1070 related Products with: A one-step potentiometric immunoassay for plasma cardiac troponin I using an antibody-functionalized bis-MPA-COOH dendrimer as a competitor with improved sensitivity.

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#32822033   // To Up

Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.

5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.
Peter Jessop, Martin Gering

2082 related Products with: Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.

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#32762697   2020/08/06 To Up

Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach.

Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests.
Chanakan Areewong, Amarin Rittipornlertrak, Boondarika Nambooppha, Itsarapan Fhaikrue, Tawatchai Singhla, Chollada Sodarat, Worapat Prachasilchai, Preeyanat Vongchan, Nattawooti Sthitmatee

2779 related Products with: Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach.

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#32734120   2020/05/16 To Up

Assessment of antigenic specificity of polyclonal antisera raised against by ELISA.

Lack of availability of commercial antibodies against whole-cell antigen or an antigenic epitope of () has hindered the development of novel immunoassays for the diagnose infectious coryza (IC). In this study, we raised polyclonal antisera against and evaluated its antigenic-specificity using enzyme linked immunosorbent assay (ELISA). We standardized antigen coating concentration(s), antibody detection limit, and optimal range of dilutions of primary antisera and secondary conjugated antibody. Our results show the development of antigen-specific antibody response in rabbits following repeated antigenic exposure with 0.5% formalinized antigen over a period of four weeks. Further, we showed its possible applicability in detection of pathogens in tissues by immunohistochemistry for confirmatory disease diagnosis and disease pathogenetic study.
Ajaz Ahmed, Sidhartha Deshmukh, Harmanjit Singh Banga, Sandeep Sodhi, Rajinder Singh Brar

1921 related Products with: Assessment of antigenic specificity of polyclonal antisera raised against by ELISA.

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#32663533   2020/07/11 To Up

A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants.

Banana bract mosaic virus (BBrMV) is a serious pathogen threatening the cultivation of banana and plantain worldwide. This study reports the development of a practical, rapid, sensitive, specific and user-friendly lateral flow immunoassay (LFIA) test for the on-site detection of BBrMV. The BBrMV coat protein (CP) was expressed in Escherichia coli and purified and used to immunize rabbits to produce a polyclonal antiserum (anti-BBrMVCP). The test was based on a double-antibody sandwich format. Protein-A affinity column-purified anti-BBrMVCP Immunoglobulins (IgG) (16 μg/mL), conjugated to ∼30 nm gold nanoparticles, was applied onto the conjugate pad. The anti-BBrMVCP IgG and goat anti-rabbit IgG were printed on the surface of a nitrocellulose filter membrane as the test line and control line, respectively. A positive result could be confirmed visually by the presence of a pink band that developed on the LFIA strip within 5-10 min. The detection limit of the test was 10 ng of the expressed recombinant BBrMV CP (rBBrMVCP), and a 1:20 dilution of the BBrMV-infected crude extract. This LFIA test was validated using 114 banana leaf samples randomly collected from the field and the results indicated a very high diagnostic sensitivity (99.04 %) and specificity (100 %) for the test. A Cohen's kappa coefficient of 0.861 obtained also indicated a very good agreement between the LFIA developed in this study and ELISA. This assay could be adopted by farmers, tissue culture industries and quarantine departments for surveys and surveillance. This is the first report on the development of a LFIA-based test for BBrMV detection.
Ramasamy Selvarajan, Prasanya Selvam Kanichelvam, Velusamy Balasubramanian, Sundaram Sethurama Subramanian

2425 related Products with: A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants.

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#32598989   2020/06/27 To Up

Targeted identification of C-type lectins in snake venom by 2DE and Western blot.

C-type lectins (CTL) and CTL-like proteins (snaclecs) are important toxins found in snake venom which can disrupt hemostasis by binding platelet membrane glycoproteins. Traditional identification of these toxins usually relies on an "activity-directed fractionation" approach which is very arduous. Here, we report a new method for rapid screening of these proteins in snake venom.
Wang Ning, Li Yuanyuan, Zhong Lipeng, Ling Xiang, Huang Chunhong

1752 related Products with: Targeted identification of C-type lectins in snake venom by 2DE and Western blot.

10 96 wells (1 kit)1 mg100 μg0.1ml96T1 mL96T50 100 μg

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#32342306   2020/04/27 To Up

Obtaining and Characteristic of Antibodies to Vibrio cholerae Protective Antigens Conjugated with Gold Nanoparticles.

Gold nanoparticle conjugates with Vibrio cholerae antigens were synthesized. The animals were immunized with the obtained conjugates. Rabbit polyclonal antibodies to the antigens were obtained, which showed high specific activity. On the model of white laboratory mice, the protective activity of conjugates of cholera antigens with nanoparticles during infection of vaccinated animals was evaluated using a commercial vaccine as a control. It was shown that in terms of immunogenicity, the created prototypes of cholera vaccine using gold nanoparticles as a carrier and adjuvant complied with the production regulations for the Russian national cholera chemical vaccine.
L A Dykman, O A Volokh, O V Gromova, O S Durakova, S A Vorobeva, M N Kireev, L F Livanova, A K Nikiforov, S Y Shchyogolev, V V Kutyrev

1436 related Products with: Obtaining and Characteristic of Antibodies to Vibrio cholerae Protective Antigens Conjugated with Gold Nanoparticles.

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