scholarly journals Development of a Kit for Rapid Immunochromatographic Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Based on Polyclonal and Monoclonal Antibodies Raised against Recombinant VP1 and VP2 Expressed in Escherichia coli

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2439
Author(s):  
Song Hee Lee ◽  
Tae-Kyun Oh ◽  
Sung Oh ◽  
Seongdae Kim ◽  
Han Byul Noh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Rapid Detection ◽  
Western Blot Analysis ◽  
Apis Cerana ◽  
Korean Isolate ◽  
Sacbrood Virus ◽  
Positive Reactivity

A Korean isolate of the sacbrood virus infecting Apis cerana (AcSBV-Kor) is the most destructive honeybee virus, causing serious economic damage losses in Korean apiculture. To address this, here, we attempted to develop an assay for the rapid detection of AcSBV-Kor based on immunochromatographic detection of constituent viral proteins. Genes encoding VP1 and VP2 proteins of AcSBV-Kor were cloned into an expression vector (pET-28a) and expressed in Escherichia coli BL21(DE3). During purification, recombinant VP1 (rVP1) and VP2 (rVP2) proteins were found in the insoluble fraction, with a molecular size of 26.7 and 24.9 kDa, respectively. BALB/c mice immunized with the purified rVP1 and rVP2 produced polyclonal antibodies (pAbs) such as pAb-rVP1 and pAb-rVP2. Western blot analysis showed that pAb-rVP1 strongly reacted with the homologous rVP1 but weakly reacted with heterologous rVP2. However, pAb-rVP2 strongly reacted not only with the homologous rVP2 but also with the heterologous rVP1. Spleen cells of the immunized mice fused with SP2/0-Ag14 myeloma cells produced monoclonal antibodies (mAbs) such as mAb-rVP1-1 and mAb-rVP2-13. Western blot analysis indicated that pAb-rVP1, pAb-rVP2, mAb-rVP1-1, and mAb-rVP2-13 reacted with AcSBV-infected honeybees and larvae as well as the corresponding recombinant proteins. These antibodies were then used in the development of a rapid immunochromatography (IC) strip assay kit with colloidal gold coupled to pAb-rVP1 and pAb-rVP2 at the conjugate pad and mAb-rVP1-1 and mAb-rVP2-13 at the test line. One antibody pair, pAb-rVP1/mAb-VP1-1, showed positive reactivity as low as 1.38 × 103 copies, while the other pair, pAb-rVP2/mAb-VP2-13, showed positive reactivity as low as 1.38 × 104 copies. Therefore, the antibody pair pAb-rVP1/mAb-VP1-1 was selected as a final candidate for validation. To validate the detection of AcSBV, the IC strip tests were conducted with 50 positive and 50 negative samples and compared with real-time PCR tests. The results confirm that the developed IC assay is a sufficiently sensitive and specific detection method for user-friendly and rapid detection of AcSBV.

scholarly journals OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Alessandro Bressan ◽  
Francesca Bozzo ◽  
Carlo Alberto Maggi ◽  
Monica Binaschi
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Tandem Repeat ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Ovarian Cancer Cells ◽  
Repeat Region ◽  
Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

scholarly journals A comparison of antigenic peptides in muscle larvae of severalTrichinellaspecies by two-dimensional western-blot analysis with monoclonal antibodies

Parasite ◽  
2001 ◽  
Vol 8 ◽  
pp. S117-S119 ◽  
Author(s):  
M.A. Dea-Ayuela ◽  
F.M. Ubeira ◽  
A. Pitarch ◽  
C. Gil ◽  
A.R. Martínez-Fernández ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Antigenic Peptides ◽  
Two Dimensional ◽  
Muscle Larvae

scholarly journals Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast

1991 ◽  
Vol 276 (1) ◽  
pp. 63-71 ◽  
Author(s):  
J Wang ◽  
J Chao ◽  
L Chao
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Shuttle Vector ◽  
Affinity Column ◽  
Tissue Kallikrein ◽  
E Coli ◽  
Rat Tissue

A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3′ end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence. The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter. Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin. Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa. The recombinant kallikrein from yeast, however, remained mostly inactive. Kallikrein was purified to apparent homogeneity from E. coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA. Both E. coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein. Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties. Our results open the way for the study of kallikrein structure-function relationships through protein engineering.

scholarly journals Monoclonal Antibodies Detecting Regulatory Polypepttdes of the Insect Neuronal Acetylcholine Receptor

1989 ◽  
Vol 147 (1) ◽  
pp. 329-342
Author(s):  
D. BENKE ◽  
I. STAHMER ◽  
H. BREER
Keyword(s):  
Monoclonal Antibodies ◽  
Ligand Binding ◽  
Western Blot ◽  
Acetylcholine Receptor ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Receptor Protein ◽  
Neuronal Membranes ◽  
Neuronal Acetylcholine Receptor

Please address reprint requests to Professor H. Breer, Universität Stuttgart, Hohenheim Farbenstrasse 30, 7000 Stuttgart 70 1. Monoclonal antibodies affecting the ligand binding of the neuronal acetylcholine receptor were prepared. 2. A clone was detected which produced antibodies that increased [125I]-αbungarotoxin binding, but decreased [3H]acetylcholine binding. 3. Western blot analysis established that these antibodies did not recognize the receptor protein, but labelled a 20xl03Mr polypeptide. 4. This putative regulatory polypeptide was purified and was found to inhibit [125I]-α-bungarotoxin binding to pretreated neuronal membranes.

scholarly journals Serological cross-reactivity of environmental Escherichia coli strains with Shigella-specific antisera

2018 ◽  
Vol 33 (1-2) ◽  
pp. 29-33 ◽  
Author(s):  
Nafisa Azmuda ◽  
Rabeya Bilkis ◽  
Humaira Akter ◽  
Anowara Begum ◽  
Sirajul Islam Khan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Surface Antigens ◽  
Biochemical Properties ◽  
Cross Reactivity ◽  
Freshwater Ecosystems ◽  
Shigella Species ◽  
Immunogenic Proteins

Many bacteria of clinical and environmental origin show evidence of sharing common surface antigens. The present study aimed for isolation of Escherichia coli strains that were serologically cross-reactive with Shigella species from freshwater ecosystems in Bangladesh by conventional cultural methods. Among twenty eight isolates, two isolates, termed 12(35) and 6(50) showed cross-reactivity with four polyvalent serogroup-specific Shigella antisera using slide agglutination assay. The isolates were identified and charcterized by cultural and biochemical properties and Western blot analysis. The isolates showed typical Escherichia coli cell morphology and cultural and biochemical properties and were identified as Escherichia coli by API 20E tests. Western blot analysis confirmed the isolates as cross-reactive with all the four group-specific Shigella antisera due to presence of immunogenic proteins and LPS. One of the isolates also showed cross-reactivity with multiple type-specific Shigella boydii antisera (monovalent) because of immunogenic proteins. Both the isolates were identified as nonpathogenic due to absence of virulence marker genes of diarrheagenic E. coli variants.This study revealed that a number of bacteria present in the environment could share important Shigella species surface antigens. Naturally occurring nonpathogenic environmental bacteria expressing surface antigens specific for certain types of Shigella could be a good choice for vaccine candidates against shigellosis. Bangladesh J Microbiol, Volume 33, Number 1-2, June-Dec 2016, pp 29-33

scholarly journals Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

2017 ◽  
Vol 21 (1) ◽  
pp. 1
Author(s):  
Dian Fitria Agustiyanti ◽  
Debbie Sofie Retnoningrum ◽  
Heni Rachmawati ◽  
Asrul Muhamad Fuad
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Soluble Form ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 

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