A Genetically Engineered Fusion Protein with Horseradish Peroxidase as a Marker Enzyme for Use in Competitive Immunoassays

2001 ◽  
Vol 73 (6) ◽  
pp. 1134-1139 ◽  
Author(s):  
Vitaly Grigorenko ◽  
Irina Andreeva ◽  
Torsten Börchers ◽  
Friedrich Spener ◽  
Alexey Egorov
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Genetically Engineered ◽  
Marker Enzyme

scholarly journals Easily Prepared Nanobody-horseradish Peroxidase Fusion Protein-based Immunoassay to Detect Antibodies Against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses

2021 ◽  
Author(s):  
Hong Duan ◽  
Xu Chen ◽  
Jiakai Zhao ◽  
Jiahong Zhu ◽  
Guixi Zhang ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Cell Line ◽  
Compliance Rate ◽  
Genetically Engineered ◽  
Respiratory Syndrome Virus ◽  
Small Molecular Weight ◽  
N Protein ◽  
Elisa Kit ◽  
Genotype 2

Abstract Background: Nanobodies are the third generation of genetically-engineered antibodies, possessing advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, a competitive ELISA (cELISA) using nanobody-horseradish peroxidase (HRP) fusion protein was utilized as an ultrasensitive probe for the first time to detect antibodies against genotype 2 porcine reproductive and respiratory syndrome virus (PRRSV). In addition, a platform for easily producing the nanobody-HRP fusion protein against PRRSV was designed and developed. Results: In the present study, three nanobodies against the PRRSV-N protein were screened by Camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-HRP fusion protein against PRRSV-N protein was successfully designed and constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting the antibodies against genotype 2 PRRSV in pig sera, exhibiting a cut‑off value of 23.19% and good sensitivity (100%), specificity, and reproducibility. The compliance rate of cELISA with a commercial IDEXX ELISA kit was 96.4%. By testing the sequential sera from the challenged pigs, the results showed that the sensitivity of cELISA was higher than the commercial IDEXX ELISA kit. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera.Conclusions: By screening the three nanobodies against the genotype 2 PRRSV-N protein, a recombinant HEK293S cell line stably secreting nanobody-HRP fusion protein against PRRSV-N protein was developed (Scheme 1a). Subsequently, a cELISA was developed for detecting the antibodies against genotype 2 PRRSV using the fusion protein and demonstrated high sensitivity, specificity, and reproducibility (Scheme 1b). More importantly, the production of the fusion protein using the developed platform is simple and low cost.

Cytotoxicity of Genetically Engineered Fusion Protein with CD154 Peptide Mimetic (OmpC-CD154p) on B-NHL Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4525-4525
Author(s):  
Bernardo Martinez-Miguel ◽  
Melisa A. Martinez-Paniagua ◽  
Sara Huerta-Yepez ◽  
Rogelio Hernandez-Pando ◽  
Cesar R. Gonzalez-Bonilla ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Fusion Protein ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Genetically Engineered ◽  
B Cell Malignancies ◽  
Peptide Mimetic

Abstract The interaction between CD40, a member of the tumor necrosis factor super family, and its ligand CD154 is essential for the development of humoral and cellular immune responses. Selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically-based diseases and malignancies. CD40 is expressed primarily on dendritic cells, macrophages and B cells. Engagement of CD40-CD154 induces activation and proliferation of B lymphocytes and triggers apoptosis of carcinoma and B lymphoma cells. Agonist CD40 antibodies mimic the signal of CD154-CD40 ligation on the surface of many tumors and mediate a direct cytotoxic effect in the absence of immune accessory molecules. CD40 expression is found on nearly all B cell malignancies. Engagement of CD40 in vivo inhibits B cell lymphoma xenografts in immune compromised mice. Several clinical trials have been reported targeting CD40 in cancer patients using recombinant CD154, mAbs and gene therapy, which were well tolerated and resulted in objective tumor responses. In addition to these therapies, CD54 mimetics have been considered with the objective to augment and potentiate the direct cytotoxic anti-tumor activity and for better accessibility to tumor sites. This approach was developed by us and we hypothesized that the genetic engineering of a fusion protein containing a CD154 peptide mimetic may be advantageous in that it may have a better affinity to CD40 on B cell malignancies and trigger cell death and the partner may be a carrier targeting other surface molecules expressed on the malignant cells. This hypothesis was tested by the development of a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Trp140-Ser149 amino acid strand (Vega et al., Immunology2003; 110: 206–216). This OmpC-CD154p fusion protein binds CD40 and triggers the CD40 expressing B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth and proliferation of the B-NHL cell lines Raji and Ramos. In addition, significant apoptosis was achieved and the extent of apoptosis was a function of the concentration used and time of incubation. The anti-tumor effect was specific as treatment with OmpC alone had no effect. These findings establish the basis of the development of new fusion proteins with dual specificity (targeting the tumor cells directly or targeting the tumor cells and immune cells). The advantages of this approach over conventional CD40-targeted therapies as well as the mechanism of OmpC-CD154p-induced cell signaling and cell death will be presented.

scholarly journals Overcoming rituximab drug-resistance by the genetically engineered anti-CD20-hIFN-α fusion protein: Direct cytotoxicity and synergy with chemotherapy

2015 ◽  
Vol 47 (5) ◽  
pp. 1735-1748 ◽  
Author(s):  
GABRIEL G. VEGA ◽  
LUZ ARELI FRANCO-CEA ◽  
SARA HUERTA-YEPEZ ◽  
HÉCTOR MAYANI ◽  
SHERIE L. MORRISON ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Fusion Protein ◽  
Genetically Engineered ◽  
Direct Cytotoxicity ◽  
Anti Cd20

scholarly journals Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

1984 ◽  
Vol 4 (2) ◽  
pp. 296-301 ◽  
Author(s):  
B Storrie ◽  
M Sachdeva ◽  
V S Viers
Keyword(s):  
Horseradish Peroxidase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Ovary Cell ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Ovary Cells

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

High Expression and Preliminary Purification of Human β-Defensin-2 Fusion Protein

2013 ◽  
Vol 781-784 ◽  
pp. 1076-1079
Author(s):  
Hong Tao Wei ◽  
Zhong Wen Lv ◽  
Xue Mei Han ◽  
Guo Li Zhang
Keyword(s):  
Fusion Protein ◽  
Metal Chelate ◽  
Genetically Engineered ◽  
High Expression ◽  
Soluble Form ◽  
Freezing And Thawing ◽  
Preliminary Purification ◽  
Prokaryotic Expression Vector ◽  
Genetically Engineered Bacteria

This study was undertaken to achieve high expression and preliminary purification of human β-defensin-2 fusion protein to lay a solid foundation for production of human β-defensin-2 using genetic engineering. A prokaryotic expression vector for human β-defensin-2 fusion protein was generated using in vitro gene synthesis before transformation into BL21 (l DE3) plysS TrX-B host bacteria. High expression of TrX-A-HBD-2 fusion protein was induced with IPTG in the bacteria exposed to various expression conditions. The fusion protein then underwent preliminary purification. The protein of interest was released from the genetically engineered bacteria after freezing and thawing. The expression of the target protein accounted for 16.12% of the total bacterial proteins. Fractional precipitation with saturated ammonium sulfate and metal chelate affinity chromatography yielded human β-defensin-2 peptide fusion protein, with a relative purity of 80.53%.Human β-defensin-2 fusion protein could be highly expressed in a soluble form, with a relatively high purity

scholarly journals Graphene Biosensor Programming with Genetically Engineered Fusion Protein Monolayers

2016 ◽  
Vol 8 (12) ◽  
pp. 8257-8264 ◽  
Author(s):  
Miika Soikkeli ◽  
Katri Kurppa ◽  
Markku Kainlauri ◽  
Sanna Arpiainen ◽  
Arja Paananen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Genetically Engineered ◽  
Protein Monolayers

scholarly journals Horseradish Peroxidase-Conjugated-Nanobody-Based cELISA For The Rapid and Sensitive Clinical Detection of African Swine Fever Virus Antibodies

2021 ◽  
Author(s):  
Huijun Zhao ◽  
Jiahui Ren ◽  
Shuya Wu ◽  
Yongkun Du ◽  
Bo Wan ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Fusion Protein ◽  
Expression System ◽  
African Swine Fever ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hemorrhagic Disease ◽  
Serum Samples ◽  
Promising Alternative ◽  
Technical Requirements ◽  
Pig Serum

Abstract Background: African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that affects pigs and has the potential to cause mortality in almost 100% of domestic pigs and wild boars. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, cost-effective detection and strict control and elimination strategies. Traditional molecular and serological testing methods are generally associated with high testing costs, complex operations and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. The application of Nbs in the detection of ASFV antibodies in the serum has not yet been reported, to the best of our knowledge. Results: Using a phage display technology, one specific Nb against the ASFV p54 protein that exhibited high specificity and affinity to the protein, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. The cut-off value for the cELISA was 15.78%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The specificity of the cELISA was 98.97%, and the limit of detection was 1:320 in inactivated ASFV antibody-positive reference serum samples, with the coincidence rate between the two methods being 98.56%. Conclusions: A specific, sensitive and repeatable cELISA was successfully developed based on the unique Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

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