scholarly journals Identification and Quantification of Anti-Gp.Mur Antibodies in Human Serum Using an Insect-Cell-Based System

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 966
Author(s):  
Robert John S. Lamis ◽  
Tsong-Shi Chiueh ◽  
Chih-Hsuan Tsai ◽  
Huei-Ru Lo ◽  
Sung-Chan Wei ◽  
...  
Keyword(s):  
Human Serum ◽  
Insect Cell ◽  
Hemagglutination Inhibition ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Blood Group System ◽  
Serum Samples ◽  
Grade Levels ◽  
Asian Populations ◽  
Hospital Patients

Gp.Mur is a clinically relevant antigen of the MNS blood group system that is highly prevalent in several Asian populations. Its corresponding antibody, anti-Gp.Mur, has been implicated in hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Currently, identifying and confirming anti-Gp.Mur antibody presence in sera via agglutination of a panel of red blood cells (RBCs) is inefficient and difficult to quantify. Using a baculovirus expression system to express Gp.Mur antigen on insect cell surfaces, we have developed a quantitative cell-based system to confirm the presence of anti-Gp.Mur antibody in human serum. We obtained 10 serum samples preidentified as having anti-Gp.Mur antibody and another 4 samples containing noncorresponding antibodies from hospital patients. Insect cells displaying Gp.Mur antigen successfully adsorbed anti-Gp.Mur antibody in the sera and inhibited the RBC agglutination mediated by this antibody. By varying the concentration of Gp.Mur-displaying cells, we could grade levels of RBC agglutination by anti-Gp.Mur antibody. Densitometric analysis further enabled quantitative determinations of hemagglutination inhibition by Gp.Mur-displaying cells. We believe that this cell-based hemagglutination inhibition system greatly improves or supplements existing technology and is a convenient means for accurately identifying and quantifying anti-Gp.Mur antibody.

Production of therapeutic proteins with baculovirus expression system in insect cell

2008 ◽  
Vol 38 ◽  
pp. S71-S78 ◽  
Author(s):  
Mi-Hyun AHN ◽  
Mira SONG ◽  
Eun-Yi OH ◽  
Arshad JAMAL ◽  
HyunSoon KIM ◽  
...  
Keyword(s):  
Insect Cell ◽  
Expression System ◽  
Therapeutic Proteins ◽  
Baculovirus Expression System ◽  
Baculovirus Expression

Development of an insect-cell-based assay for detection of kinase inhibition using NF-κB-inducing kinase as a paradigm

2009 ◽  
Vol 419 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Namir J. Hassan ◽  
Sheraz Gul ◽  
Fiona Flett ◽  
Edward Hollingsworth ◽  
Angela A. Dunne ◽  
...  
Keyword(s):  
Insect Cell ◽  
High Throughput Screening ◽  
Expression System ◽  
Scale Up ◽  
Cost Effective ◽  
Baculovirus Expression System ◽  
Nuclear Factor Κb ◽  
Ease Of Use ◽  
Kinase Inhibition ◽  
Whole Cells

Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-κB (nuclear factor κB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IκB (inhibitor of NF-κB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z′ value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-κB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.

scholarly journals Interaction of Gene-Cloned and Insect Cell-Expressed Aminopeptidase N of Spodoptera litura with Insecticidal Crystal Protein Cry1C

2002 ◽  
Vol 68 (9) ◽  
pp. 4583-4592 ◽  
Author(s):  
Neema Agrawal ◽  
Pawan Malhotra ◽  
Raj K. Bhatnagar
Keyword(s):  
Insect Cell ◽  
Spodoptera Litura ◽  
Expression System ◽  
Insect Cell Line ◽  
Baculovirus Expression System ◽  
Aminopeptidase N ◽  
Receptor Protein ◽  
Binding Studies ◽  
Toxin Binding ◽  
Cry Toxin

ABSTRACT Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology

2018 ◽  
Author(s):  
Laura A. Palomares ◽  
Indresh K. Srivastava ◽  
Octavio T. Ramírez ◽  
Manon M. J. Cox
Keyword(s):  
Insect Cell ◽  
Expression System ◽  
Baculovirus Expression System ◽  
System Technology ◽  
Baculovirus Expression
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