Detection of Shigella in Milk and Clinical Samples by Magnetic Immunocaptured-Loop-Mediated Isothermal Amplification Assay

Shi

et al. 2019

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Background: Klebsiella pneumoniae is an important human pathogen that causes severe diseases including urinary tract infection, pneumonia, and bacteremia. However, a rapid and sensitive detection method remains to be developed. Objectives: This study aimed to develop a rapid, real-time, and visual detection method for K. pneumoniae. This is a primary screening method to improve the diagnosis of K. pneumoniae infection and save much precious time for clinical practice. Methods: Klebsiella pneumoniae was used as an antigen to produce a monoclonal antibodies (mAb). The mAb 1E6 recognizing outer membrane protein C on the surface of K. pneumoniae was screened by an indirect enzyme-linked immunosorbent assay (ELISA), Western Blot, and mass spectrometry assay, and then conjugated with the protein A/G coated magnetic beads to generate the immune magnetic beads (IMBs). Thereafter, the IMBs were used to capture K. pneumoniae, and the complex (beads-mAb-K. pneumoniae) was used for loop-mediated isothermal amplification (LAMP) assay. Results: We developed a rapid, real-time, and visual detection method employing an immunocapture loop-mediated isothermal amplification (IC-LAMP). The sensitivity of the IC-LAMP was 4 CFU mL-1. The process of K. pneumoniae detection lasted~60 minutes and had no cross-reaction to other microbial strains. Besides, the accuracy of IC-LAMP was further verified by examining 39 clinical isolates. Conclusions: Without the need for enrichment of bacteria and extraction of its genome, the IC-LAMP developed here could be used as a primary screening method supplementary to traditional detection methods to improve the diagnosis of K. pneumoniae infection for clinical practice.

Section: Pneumoniaementioning

confidence: 99%

Visual and Rapid Detection of Klebsiella pneumoniae by Magnetic Immunocapture-Loop-Mediated Isothermal Amplification Assay

Shi

et al. 2019

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“…The titer of the pAb was determined by Enzyme-linked Immunosorbent Assay (ELISA) after the anti-serum was purified (26). Briefly, 1 µg of NS1 protein was coated on each well and maintained overnight at 4ºC.…”

Section: Titer Of Pabmentioning

confidence: 99%

Generation and Characterization of a Polyclonal Antibody Against NS1 Protein for Detection of Zika Virus

et al. 2019

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Background: Zika Virus (ZIKV) is a new type of flaviviruses transmitted by mosquitoes to cause severe diseases including Guillain-Barré syndrome and congenital malformations during recent outbreaks. The early diagnosis of ZIKV infection and efficient vaccines would be effective in controlling epidemics and timely treatment. However, the native nonstructural protein 1 (NS1) mainly acquired from cells infected with ZIKV, which largely limits its application. Objectives: This study aimed to express and purify the recombinant NS1 protein and prepare its antibody that efficiently reacted with the native NS1 protein secreted in the supernatant of ZIKV infected host-cells. Methods: We constructed a prokaryotic expression vector containing the full length of the NS1 gene. Thus, the recombinant NS1 protein was efficiently expressed and purified. The purified NS1 protein was used to prepare an antibody. The Enzyme-linked Immunosorbent Assay (ELISA), Western blot, and Indirect Immunofluorescent Assay (IFA) were used to assess the reactivity and specificity of the antibody. Results: The recombinant NS1 protein from a prokaryotic expression vector had good immunogenicity and the prepared antibody could specifically react with the native NS1 produced in Vero, BHK-21 cells infected with ZIKV. Besides, the ELISA and Western blot showed that the native ZIKV-NS1 protein was secreted extracellularly and could play the role of an early diagnostic biomarker for ZIKV infection. Conclusions: The prepared antibody against the recombinant NS1 protein is a reliable biological tool that enables the rapid and sensitive detection of secreted NS1 from host cells infected with ZIKV. The recombinant NS1 protein and its antibody can contribute to developing vaccines and antibodies targeting the NS1 protein to prevent flavivirus disease progression.

“…In this work, a prokaryotic expression vector was used to express the ZIKV-E protein. ZIKV-E protein was purified and used as an immunogen to inoculate BALB/c mice (19). Western Blot and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react with the native E protein in Vero cells specifically infected with ZIKV.…”

Section: Objectivesmentioning

confidence: 99%

“…The immuno-magnetic beads were conducted as described by Zhang et al (19). A total of 400 µL of mouse anti-ZIKV-E serum (1:100) was transferred into 1.5 mL tubes coated for 0.5 hour at 37°C.…”

Section: Immunoprecipitation Of Zikv Virion In Cell Culture Medium Wimentioning

confidence: 99%

Efficient Capture and Detection of Zika Virion by Polyclonal Antibody Against Prokaryotic Recombinant Envelope Protein

Dai

et al. 2018

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Background: The outbreak of Zika virus (ZIKV) in many countries caused alarming numbers of babies born with microcephalus and severe neurologic disorders, however, the methods for the detection of the ZIKV are limited at present. Objectives: The aim of this study was to produce polyclonal antibody against full length envelop protein of ZIKV (ZIKV-E protein) in prokaryotic system and to evaluate its efficacy to capture ZIKV virion. Methods: The recombinant full length ZIKV-E protein was purified and then used as an immunogen to vaccinate BALB/c mice to produce polyclonal antibody. Protein A/G coated magnetic beads were coated with the polyclonal antibody to capture the ZIKV virion in the culture medium of Vero cells infected with ZIKV. The products of immunoprecipitation were further used for Western Blot, Loop-mediated isothermal amplification (LAMP), and PCR assay. Results: Western Blot and indirect immunofluorescence analysis showed that the mouse polyclonal antibody could react specifically with the native E protein in Vero cells infected with ZIKV. The results of Western Blot, Immunocaptured-LAMP (IC-LAMP), and Immunocaptured-PCR (IC-PCR) showed that polyclonal antibody of ZIKV-E recombinant protein can capture ZIKV virion specifically, however, the polyclonal antibody against the ZIKV-NS1, Shigella, and Salmonella without such a function. Conclusions: These findings may provide the basis for the development of the rapid diagnostic kits such as, IC-PCR, IC-LAMP, and sandwich ELISA. The serum virion capture activity elicited by prokaryotic expressed recombinant ZIKV-E protein may also provide a basis for further preparation of neutralizing antibody and protein subunit vaccine candidate against ZIKV.