Human and primate monoclonal antibodies for in vivo therapy.

1988 ◽  
Vol 34 (9) ◽  
pp. 1681-1688 ◽  
Author(s):  
P H Ehrlich ◽  
Z A Moustafa ◽  
J C Justice ◽  
K E Harfeldt ◽  
I K Gadi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Protein A ◽  
Human Chromosomes ◽  
Human Monoclonal Antibodies ◽  
Western Blot Assay ◽  
Human Antibodies ◽  
Low Levels ◽  
High Concentrations

Abstract Human monoclonal antibodies, owing to their decreased immunogenicity, are expected to be an improvement over mouse monoclonal antibodies for in vivo therapy. Human and primate monoclonal antibodies are best produced with a human x mouse heteromyeloma. Several human chromosomes are stable in the human x (human x mouse) hybrids. Chimpanzee anti-digoxin monoclonal antibodies were prepared and characterized. Because they are structurally very similar to human antibodies, they should be well tolerated in humans. The anti-digoxin antibodies can be used for therapy of extreme overdoses or as an in vivo diagnostic tool for slight overdoses. Because the advantage of using human monoclonal antibodies is their lack of immunogenicity, preparation of the antibody must be scrupulous so as not to introduce extraneous immunogens. Analysis to ensure the purity of the preparation can be complicated by the presence of high concentrations of the antibody and the low levels of contamination that must be detected. We describe a Western blot assay for Protein A that is sensitive even in the presence of human IgG.

The Anti-tumor Activity and Mechanisms of rLj-RGD3 on Human Laryngeal Squamous Carcinoma Hep2 Cells

2020 ◽  
Vol 19 (17) ◽  
pp. 2108-2119
Author(s):  
Yang Jin ◽  
Li Lv ◽  
Shu-Xiang Ning ◽  
Ji-Hong Wang ◽  
Rong Xiao
Keyword(s):  
Wound Healing ◽  
Western Blot ◽  
Morphological Changes ◽  
In Vivo Studies ◽  
Migration And Invasion ◽  
Tunel Staining ◽  
Dna Ladder ◽  
Western Blot Assay

Background: Laryngeal Squamous Cell Carcinoma (LSCC) is a malignant epithelial tumor with poor prognosis and its incidence rate increased recently. rLj-RGD3, a recombinant protein cloned from the buccal gland of Lampetra japonica, contains three RGD motifs that could bind to integrins on the tumor cells. Methods: MTT assay was used to detect the inhibitory rate of viability. Giemsa’s staining assay was used to observe the morphological changes of cells. Hoechst 33258 and TUNEL staining assay, DNA ladder assay were used to examine the apoptotic. Western blot assay was applied to detect the change of the integrin signal pathway. Wound-healing assay, migration, and invasion assay were used to detect the mobility of Hep2 cells. H&E staining assay was used to show the arrangement of the Hep2 cells in the solid tumor tissues. Results: In the present study, rLj-RGD3 was shown to inhibit the viability of LSCC Hep2 cells in vitro by inducing apoptosis with an IC50 of 1.23µM. Western blot showed that the apoptosis of Hep2 cells induced by rLj- RGD3 was dependent on the integrin-FAK-Akt pathway. Wound healing, transwells, and western blot assays in vitro showed that rLj-RGD3 suppressed the migration and invasion of Hep2 cells by integrin-FAKpaxillin/ PLC pathway which could also affect the cytoskeleton arrangement in Hep2 cells. In in vivo studies, rLj-RGD3 inhibited the growth, tumor volume, and weight, as well as disturbed the tissue structure of the solid tumors in xenograft models of BALB/c nude mice without reducing their body weights. Conclusion: hese results suggested that rLj-RGD3 is an effective and safe suppressor on the growth and metastasis of LSCC Hep2 cells from both in vitro and in vivo experiments. rLj-RGD3 might be expected to become a novel anti-tumor drug to treat LSCC patients in the near future.

Ginkgolic acid induces apoptosis and autophagy of endometrial carcinoma cells via inhibiting PI3K/Akt/mTOR pathway in vivo and in vitro

2021 ◽  
pp. 096032712110237
Author(s):  
L Zhou ◽  
S Li ◽  
J Sun
Keyword(s):  
Endometrial Cancer ◽  
B Cells ◽  
Western Blot ◽  
Signal Pathway ◽  
Mtor Pathway ◽  
Related Protein ◽  
Western Blot Assay ◽  
Ginkgolic Acid

Endometrial cancer (EC) is the fourth most common malignancy in women in developed countries. The prognosis of EC is extremely poor, and it is an important factor that contributes to the death of patients. Therefore, studying EC pathogenesis and therapeutic targets, and exploring effective drugs are the primary tasks to improve the prognosis of EC. In the present study, we aimed to explore the function of ginkgolic acid (GA) in EC cell apoptosis and autophagy through PI3K/Akt/mTOR signal pathway in vitro and in vivo. Firstly, MTT assay and clone formation assay were employed to analyze the Ishikawa and HEC-1-B cell viabilities and proliferation after treatment with GA. The results showed that GA inhibited endometrial cancer cell survival. Flow cytometry assay and western blot assay were applied to examine the apoptosis and apoptosis related protein Bcl-2, Bax, Cleaved caspase-3 expression levels of Ishikawa and HEC-1-B cells after treatment with GA. Next, we applied western blot assay to analyze the autophagy associated proteins LC3I, LC3II, p62 and Beclin-1 in GA treated Ishikawa and HEC-1-B cells. We found that GA promoted apoptosis and induced autophagy of endometrial cancer cells. Meanwhile, western blot assay was also used to determine the expression levels of the PI3K/Akt/mTOR signal pathway related protein and the results revealed that GA inhibited the activity of PI3K/Akt/mTOR pathway. Finally, we found that GA inhibited tumor growth in vivo through immunohistochemistry assay. In conclusion, GA induces apoptosis and autophagy of EC cells via inhibiting PI3K/Akt/mTOR pathway in vivo and vitro.

scholarly journals A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 37 ◽  
Author(s):  
Jennifer Linden ◽  
Kiel Telesford ◽  
Samantha Shetty ◽  
Paige Winokour ◽  
Sylvia Haigh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Clostridium Perfringens ◽  
Specificity And Sensitivity ◽  
Epsilon Toxin ◽  
Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

scholarly journals Novel human monoclonal antibodies specific to the alternatively spliced domain D of Tenascin C efficiently target tumors in vivo

mAbs ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 1836713
Author(s):  
Lisa Nadal ◽  
Riccardo Corbellari ◽  
Alessandra Villa ◽  
Tobias Weiss ◽  
Michael Weller ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Monoclonal Antibodies ◽  
Tenascin C ◽  
Alternatively Spliced

scholarly journals Circ_0015756 promotes the progression of ovarian cancer by regulating miR-942-5p/CUL4B pathway

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenhua Du ◽  
Lei Wang ◽  
Yu Xia
Keyword(s):  
Ovarian Cancer ◽  
Tumor Growth ◽  
Western Blot ◽  
Gynecologic Cancer ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Western Blot Assay

Abstract Background Ovarian cancer (OC) is the gynecologic cancer with the highest mortality. Circular RNAs (circRNAs) play a vital role in the development and progression of cancer. This study aimed to explore the potential role of circ_0015756 in OC and its molecular mechanism. Methods The levels of circ_0015756, microRNA-942-5p (miR-942-5p) and Cullin 4B (CUL4B) were determined by quantitative real-time PCR (qRT-PCR) or Western blot assay. Cell proliferation, apoptosis, migration and invasion were assessed by Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry and transwell assay. The levels of proliferation-related and metastasis-related proteins were measured by Western blot assay. The relationship between miR-942-5p and circ_0015756 or CUL4B was verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Xenograft assay was used to analyze tumor growth in vivo. Results Circ_0015756 and CUL4B levels were increased, while miR-942-5p level was decreased in OC tissues and cells. Depletion of circ_0015756 suppressed proliferation, migration and invasion and promoted apoptosis in OC cells. Down-regulation of circ_0015756 hindered OC cell progression via modulating miR-942-5p. Also, up-regulation of miR-942-5p impeded OC cell development by targeting CUL4B. Mechanistically, circ_0015756 up-regulated CUL4B via sponging miR-942-5p. Moreover, circ_0015756 silencing inhibited tumor growth in vivo. Conclusion Knockdown of circ_0015756 suppressed OC progression via regulating miR-942-5p/CUL4B axis, suggesting that circ_0015756 might be a potential therapeutic target for ovarian cancer.

scholarly journals Aurora-a confers radioresistance in human hepatocellular carcinoma by activating NF-κB signaling pathway

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ze-Tian Shen ◽  
Ying Chen ◽  
Gui-Chun Huang ◽  
Xi-Xu Zhu ◽  
Rui Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Aurora A ◽  
Western Blot Assay ◽  
Induced Apoptosis ◽  
Mtt Assays ◽  
Hcc Cells

Abstract Background Radiotherapy failure is a significant clinical challenge due to the development of resistance in the course of treatment. Therefore, it is necessary to further study the radiation resistance mechanism of HCC. In our early study, we have showed that the expression of Aurora-A mRNA was upregulated in HCC tissue samples or cells, and Aurora-A promoted the malignant phenotype of HCC cells. However, the effect of Aurora-A on the development of HCC radioresistance is not well known. Methods In this study, colony formation assay, MTT assays, flow cytometry assays, RT-PCR assays, Western blot, and tumor xenografts experiments were used to identify Aurora-A promotes the radioresistance of HCC cells by decreasing IR-induced apoptosis in vitro and in vivo. Dual-luciferase reporter assay, MTT assays, flow cytometry assays, and Western blot assay were performed to show the interactions of Aurora-A and NF-κB. Results We established radioresistance HCC cell lines (HepG2-R) and found that Aurora-A was significantly upregulated in those radioresistant HCC cells in comparison with their parental HCC cells. Knockdown of Aurora-A increased radiosensitivity of radioresistant HCC cells both in vivo and in vitro by enhancing irradiation-induced apoptosis, while upregulation of Aurora-A decreased radiosensitivity by reducing irradiation-induced apoptosis of parental cells. In addition, we have showed that Aurora-A could promote the expression of nuclear IkappaB-alpha (IκBα) protein while enhancing the activity of NF-kappaB (κB), thereby promoted expression of NF-κB pathway downstream effectors, including proteins (Mcl-1, Bcl-2, PARP, and caspase-3), all of which are associated with apoptosis. Conclusions Aurora-A reduces radiotherapy-induced apoptosis by activating NF-κB signaling, thereby contributing to HCC radioresistance. Our results provided the first evidence that Aurora-A was essential for radioresistance in HCC and targeting this molecular would be a potential strategy for radiosensitization in HCC.

scholarly journals Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Xuyao Jiao ◽  
Sarah Smith ◽  
Gabrielle Stack ◽  
Qi Liang ◽  
Allan Bradley ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Typhoid Fever ◽  
Severe Disease ◽  
Genetically Engineered ◽  
Human Monoclonal Antibodies ◽  
Genetically Engineered Mice ◽  
Typhoid Toxin

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

scholarly journals Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

1982 ◽  
Vol 156 (6) ◽  
pp. 1739-1754 ◽  
Author(s):  
M E Medof ◽  
K Iida ◽  
C Mold ◽  
V Nussenzweig
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Complexes ◽  
Solid Phase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alpha Chain ◽  
Sds Page ◽  
High Concentrations ◽  
Polypeptide Chains

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

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