Hybridoma

VP7 is a major group-specific protein of the bluetongue virus (BTV), and is therefore a candidate for use as a diagnostic reagent. In this study, BALB/c mice were immunized with BTV16, and the lymphocyte hybridoma technique and indirect ELISA screening method were employed to obtain two strains of hybridoma cells secreting specific monoclonal antibodies (MAbs) to BTV16. Eukaryotic recombinant plasmids coding for 10 segments of BTV16 separately were transfected into BHK-21 cells, respectively, followed by immunofluorescence, showing that two MAbs only reacted with BTV-VP7...

Adiponectin (AdipoQ) is an adipokine mainly secreted by white fatty tissue, playing a major role in energy homeostasis and insulin sensitivity. For cattle, AdipoQ data are largely limited to mRNA expression; to our knowledge, valid information about the AdipoQ protein in bovine tissues and body fluids is not available. Therefore, we have developed a monoclonal antibody against bovine AdipoQ. This study describes the preparation, application, and characterization of a monoclonal antibody for use in ELISA, Western blot, and histology...

An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Further, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations...

Glypican-3 (GPC3) has been reported as a novel serum and histochemical marker for hepatocellular carcinoma (HCC) by several groups. As an oncofetal protein, it is expressed abundantly in the fetal liver, inactive in the normal adult liver, and frequently reactivated in HCC. Immunology reagents are urgently needed to proceed with mechanism-related research, clinical validation, and application. In this report, monoclonal antibodies (MAbs) against GPC3 were made from hyperimmune BALB/c mice by injecting 100 μg of purified antigen intraperitoneally...

Monoclonal antibody (MAb) against trans-resveratrol (t-RSV) was obtained from hybridoma clones constructed from splenocytes of BALB/c mice immunized with carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]) coupled with synthetic hapten mimicking t-RSV structure. The t-RSV-BSA derivate was more efficient at induction of the immune response than t-RSV-OVA. However, the use of t-RSV-OVA was advantageous during selection of hybridoma clones constructed from splenocytes of t-RSV-BSA-immunized mice...

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA...

Development of a robust immunoassay requires the selection of monoclonal antibodies with desired properties. These properties generally include kinetics parameters such as on-rate and off-rate (i.e., binding affinity), and, often times, the ability to form a sandwich with the analyte of interest. We sought to obtain antibodies suitable for development of an immunoassay capable of detecting human neutrophil gelatinase-associated lipocalin (NGAL), a glycosylated lipocalin of 25 kDa expressed in kidney tubules in response to injury that has been shown to be a urinary biomarker capable of diagnosing acute kidney injury...

Glucosyltransferase-B (GTFB) of Streptococcus mutans is considered a virulence factor because of its activity in the production of insoluble glucan, which is key to the bacterial attachment onto dental surfaces, leading to the formation of dental caries. Local passive immunization with monoclonal antibodies against GTFB is considered to be an effective way to prevent dental caries. Here we amplified a 1.3 kb fragment of the N-terminal half of the gtfB gene (193-1530) of S. mutans by PCR and expressed the truncated protein (GTFBN)...

Prostate-specific membrane antigen (PSMA), a type II integral membrane glycoprotein, is highly overexpressed in all forms of prostate cancer tissues. It has also been demonstrated in a wide range of neovasculature of non-prostatic solid tumors, including bladder, pancreas, lung, kidney, colorectal, and gastric cancers. Given the unique expression of PSMA, it is considered an alluring target for antibody-based imaging and therapy of cancer. In the present study, the production and characterization of camel heavy chain antibodies (HCAbs) specific for the external domain of the PSMA are reported...

B and T lymphocyte attenuator (BTLA) is recently identified as the third co-inhibitory receptor with similarities to CTLA-4 and PD-1. Previous reports have shown that BTLA is associated with autoimmune diseases, viral infections and tumor immune evasion. However, the possibility of the existence and role of a soluble form of human BTLA (sBTLA) has not been revealed. Based on our previously generated mouse anti-BTLA monoclonal antibodies, we intend here to develop a novel enzyme-linked immunosorbent assay (ELISA) for detecting sBTLA...

A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line...

Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis...

Previously we increased the potency of therapeutic antibodies in targeting, induction of apoptosis, and growth inhibition in vitro and in vivo by chemically conjugating a homophilic peptide to the antibody. Here, we describe the construction of a chimeric fusion gene derived from the murine anti-CD20 antibody (1F5) variable region, with an engineered homophilic domain at the C-terminus of the human IgG1 sequence. The construct was expressed in CHO suspension cells and purified. The potency of the homophilic anti-CD20 antibody was compared to a chimeric antibody without the engineered homophilic domain...

Calreticulin (CRT) has various versatile functions. It is one of the family of heat shock proteins (HSPs), and is mainly located in the lumen of the endoplasmic reticulum (ER). In order to elucidate the functional and structural properties of CRT, we expressed and purified CRT protein; we then developed a monoclonal antibody (MAb) against mouse CRT by immunizing BALB/c mice with a specific region of the N and P domains of CRT (dCRT) as antigen, which was expressed in Escherichia coli. A stable hybridoma cell line was established by enzyme linked immunosorbent assay (ELISA) screening...

Renalase is generated mainly by the kidneys, and renalase's expression in chronic kidney disease patients is reduced due to renal dysfunction. In this study, human renalase recombinant protein with prokaryotic expression was used for immunization of male New Zealand rabbits, and polyclonal antibodies against human renalase were obtained. To prepare and verify renalase polyclonal antibody, renalase recombinant protein was used as antigen and male New Zealand rabbits were immunized to obtain anti-serum for identification...

Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells...

Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA)...

The foot and mouth disease virus (FMDV) is an RNA virus composed of single stranded positive sense RNA. FMDV has been known to infect cloven-hoofed animals, including pigs, cattle, and sheep. FMDV is rapidly spreading outward to neighboring regions, often leading to a high mortality rate. Thus, early diagnosis of FMDV is critical to suppress propagation of FMDV and minimize economic losses. In this study, we report the generation and characterization of polyclonal and six monoclonal antibodies against VP1 through immunoblotting and immunofluorescence microscopy analyses...

As a transmembrane enzyme, ATP synthase plays an important role in energy metabolism of organ tissues, as well as in tumors. In this study we generated a monoclonal antibody, 6G11, to the catalytic subunit of F1-F0 ATP synthase (ATP5B). The SDS-PAGE result demonstrated that the hybridoma clone had a molecular weight of 50 and 27 kDa components that could be the heavy and light chains of the monoclonal antibody, respectively. Chromosome analysis of the hybridoma clone proved that they had 98 to 102 chromosomal numbers that were the sum of the SP2/0 and spleen cells...

TonB is known to be a bacterial periplasmic protein that transduces proton from the inner membrane to the outer membrane receptor in complex with the ExbB and ExbD proteins. Actinobacillus pleuropneumoniae TonB2 protein is the second TonB protein that is important for iron acquisition and virulence. The TonB2 protein was verified to be immunogenic and could afford partial protection for animals from lethal infection. In the present study, the recombinant TonB2 (rTonB2) was overexpressed in Escherichia coli BL21(DE3) and purified...