Akbar Oghalaie; Alireza Shoari; Fatemeh Kazemi-Lomedasht; Fatemeh Rahimi-Jamnani; Fereidoun Mahboudi; Hajarossadat Ghaderi; Mohammad Hosseininejad-Chafi; Reza Moaazami; Arghavan Ashja Ardalan; Somayeh Piri-Gavgani; Delavar Shabazzadeh; Mahdi Behdani
Volume 14, Issue 6 , June 2023, , Pages 323-328
Abstract
Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and ...
Read More
Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.
Azadeh Yektaseresht; Zahra Hemati; Amir Arsalan Khorsand; Shoor Virsingh
Volume 13, Issue 4 , December 2022, , Pages 603-606
Abstract
No diagnostic kits and reagents are available in the market to detect and evaluate camel immune responses to different pathogens. This study aimed to produce sheep anti-camel (Camelus dromedarius) polyclonal antibodies (pAbs) and to determine the specificity with other species immunoglobulin. Immunoglobulins ...
Read More
No diagnostic kits and reagents are available in the market to detect and evaluate camel immune responses to different pathogens. This study aimed to produce sheep anti-camel (Camelus dromedarius) polyclonal antibodies (pAbs) and to determine the specificity with other species immunoglobulin. Immunoglobulins (Igs) from camel serum samples were purified using ammonium sulfate precipitation (40.00% saturated ammonium sulfate). Purity of the camel Igs was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis. PAbs against (Camelus dromedarius) immunoglobulins were generated by immunizing sheep with purified Igs. Anti- camel Ig polyclonal antibodies titer and specificity were determined using ELISA and Western blot techniques. Polyclonal antibodies specific to camel Igs were significantly high in immunized sheep which confirmed the immunization procedure. PAbs reacted specifically with camel serum immunoglobulin and did not react with other species immunoglobulin of horse and chickens. Polyclonal antibodies produced in this study can be regarded as a valuable tool to be used for immune-diagnostic purposes in camel population world-wide.
Immunology
Mojgan Esparvarinha; Hamid Nickho; Leili Aghebati; Jalal Abdolalizadeh; Hadi Nasiri; Zahra Valedkarimi; Jafar Majidi
Volume 10, Issue 3 , September 2019, , Pages 207-211
Abstract
Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal ...
Read More
Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal condition. The purpose of this study was the production and purification of polyclonal immunoglobulin G (IgG) against human kappa light chains. In this study, early human IgG was purified by ion-exchange chromatography, reduced with Dithiothreitol and heavy and light chains were separated with size-exclusion chromatography. Afterward, affinity chromatography with protein L Sepharose at pH 2.00 was displayed to be a dominant condition for the separation and purification of the kappa light chain of immunoglobulins from human serum. Eventually, the rabbit was immunized by human kappa light chains. The rabbit IgG was purified and labeled with horseradish peroxidase (HRP). Direct enzyme-linked immunosorbent assay was planned to determine the titer of HRP conjugated rabbit IgG against the human kappa light chain. The optimum titer of anti-kappa IgG was 1:16000. At the result, purified polyclonal anti-kappa is useful tool in biomedical and biochemical researches and diagnostic kits.
Immunology
Hadi Nasiri; Zahra Valedkarimi; Leili Aghebati-Maleki; Jalal Abdolalizadeh; Tohid Kazemi; Mojghan Esparvarinha; Jafar Majidi
Volume 8, Issue 4 , December 2017, , Pages 307-312
Abstract
Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange ...
Read More
Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits.
