Establishment of an antigen-capture enzyme-linked immunosorbent assay for detecting avian reticuloendotheliosis virus

In this study, an antigen-capturing enzyme-linked immunosorbent assay (AC-ELISA) was established for the detection of avian reticuloendotheliosis virus (REV) using monoclonal and polyclonal antibodies against gp90. New Zealand white rabbits were immunized with recombinant REV-gp90 protein, and polyclonal antibodies were obtained after purification and used as the capture antibody. Mice monoclonal antibody 1A12D against REV-gp90 protein previously prepared in our laboratory was used as the detection antibody. The specificity of the AC-ELISA was confirmed with REV, avian leukosis virus subgroup J, Marek’s disease virus, avian hepatitis E virus and Fowl adenovirus serotype 4. The results showed that the AC-ELISA had specific binding reaction with REV, and did not react with other viruses.
The detection limit of this assay was 195 TCID50 units of joplink Rabbit antibodies for IHC and WB REV. Furthermore, commercial vaccine artificially contaminated with REV was detected by three methods: AC-ELISA, the TaqMan probe fluorescence real-time quantitative RT-PCR (RT-qPCR) and indirect immunofluorescence assay (IFA). The results showed that the positive coincidence rate of RT-qPCR and AC-ELISA was 90.63 %, and the positive coincidence rate of RT-qPCR and IFA was 96.88 %, indicating that the AC-ELISA established in this study was effective and feasible. This method simplified the detection process for REV contamination in poultry attenuated vaccines, and provide necessary technical tools for high-throughput detection of REV.

Prokaryotic expression and polyclonal antibody preparation of human adenovirus type 7 DNA binding protein

Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody.

Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA).
In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV.

Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1024000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.

Prokaryotic expression and identification of PPE15 protein from Mycobacterium tuberculosis and preparation of rabbit polyclonal antibodies

Objective To clone, express and purify PPE15 recombinant protein from Mycobacterium tuberculosis (H37Rv), as well as prepare and characterize its rabbit polyclonal antibody. Methods By The PPE15 gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR, and the His-tagged prokaryotic PPE15 prokaryotic expression plasmid pET28a-PPE15 was constructed by homologous recombination cloning technique, and transformed into E. coli BL21 (DE3). PPE15 expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG).
Recombinant PPE15 was identified by SDS-PAGE, and further purified by affinity chromatography with a Ni-NTA column. The renaturation purified PPE15 protein was used to immunize New-Zealand rabbit to prepare polyclonal antibodies. The antibody specificity was analyzed by Western blot analysis, and antibody titer was determined by indirect ELISA. Results Recombinant prokaryotic PPE15 protein was successfully expressed and purified with a molecular weight of 38 kDa.
The purified PPE15 protein exhibited positive reaction with the serum of TB patients and the PPE15 protein, the titer of the polyclonal antibodies reaches more than 1:1 300 480. Conclusion The recombinant protein PPE15 was successfully expressed and purified, and high titer rabbit-derived polyclonal antibody was prepared which provided an experimental basis for further functional studies of PPE15 protein.

GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus

A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli-derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice. Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line.
In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples. The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital.
The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.

Molecular characterization and antibacterial immunity functional analysis of the antimicrobial peptide hepcidin from Coregonus ussuriensis berg

Antimicrobial peptides are immune system molecules existing in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine rich antimicrobial peptides, which plays an important role in fish response to a variety of pathogens. In this study, we cloned and identified Hepcidin from the Coregonus ussuriensis Berg, and its functions in vivo and in vitro was investigated. Our results showed that, CuHepc contains a 267 bp coding sequence (CDS) region that encodes 88 putative amino acids with a molecular weight of 9.77 kD. Hepcidin transcripts were most abundant in the liver of healthy C. ussuriensis Berg.
The synthesized Hepcidin peptide exhibited a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria in vitro, and the results of in vivo bacterial attack assays showed that the CuHepc gene was differentially up-regulated in the six tissues investigated after infection with Aeromonas hydrophila. To analyze the changes in protein levels in C. ussuriensis, we generated Hepc polyclonal antibodies in rabbits and verified that the protein expression was increased after bacterial infection with Western blot assay.
MIC assay results showed a geometric mean value of 5.513 μM for CuHepc peptide. In the in vivo experiment, immune-related genes IL-10, NF-κB, TLR3 were up-regulated post-infection CuHepc peptide in liver and intestine. Finally, CuHepc peptide reduced the tissues microbial load compared to infection with Aeromonas hydrophila. The above results indicate that Hepc plays a role in the immune response of C. ussuriensis to exogenous disturbances, indicate that CuHepc might act a candidate for modulation of the innate immune system in C. ussuriensis.

Blocking Buffer for ICC and IHC

10 ml 128 EUR

Permeabilization Buffer for ICC and IHC

10 ml 122 EUR

Antibody Dilution Buffer for ICC and IHC

10 ml 122 EUR

Cell Lysis Buffer for WB and IP

100 ml 217 EUR

HiQ Block for IHC

100ml 134 EUR

HiQ Block for IHC

500ml 263 EUR

Alk.Phos IHC Enhancing Wash Buffer for rinsing IHC and Fluorescence, 4 Liter Ready-To-Use

4 Liters 427 EUR

WB Developing and Fixing Kit

  • 189.00 EUR
  • 230.00 EUR
  • 100 ml
  • 500 ml

WB Developing and Fixing Kit

500 ml 189 EUR

Rabbit Kappa light chain antibody (Prediluted for IHC)

7 ml 187 EUR