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Antibody Technology and Cell Culture

In your essay you should discuss;

  • The development of the antibody. How was it made, is it a humanized, chimeric or human antibody. As these antibodies should all be monoclonals I would expect a brief overview of how monoclonal antibodies are produced. A brief mention of the company who produce the antibody, are they a big biopharmaceutical company?
  • The uses of the antibody. Is it just used for one particular disease? If not what are the other diseases or conditions it is used for and why is it able to treat more than one condition?
  • What are the mechanisms of action of this antibody? How does it work, what are the pathways that the antibody works on?
  • You should also consider how effective the antibody is as a drug and you could mention the extent of its current usage.

Answer:

The antibody or also known as immunoglobulin is a type of protein produced by the plasma cells of the human body. It is one of the major components of the human immune system. In recent years, antibodies are widely used in the clinical domain for various diseases. Target therapy for cancer treatment is one of the most notable examples of these sorts of therapeutic application (Barouch et al. 2013). Other applications include antibody-drug conjugates that are also used for various purposes. Apart from this, antibodies are used in disease diagnosis, monoclonal antibody therapy, Prenatal treatment, etc. Antibodies are used widely in the treatment of rheumatoid arthritis, psoriasis, multiple sclerosis, colorectal cancer, non-Hodgkin's lymphoma, head and neck cancer and breast cancer. It is also used in many immune deficiency diseases such as hypogammaglobulinemia, X-linked agammaglobulinemia by introducing antibodies into the body (Bernard et al. 2013). One of the significant aspect of monoclonal antibody in recent years is the treatment of Ebola viruses and HIV viruses. The research process is still going on to develop and apply antibody therapy against these viruses as well (Jeong et al. 2012).

Rheumatoid arthritis is a sort of autoimmune disease where a chronic and progressive inflammation occurs in the joints. It is a form of autoimmune disease. In this case, the cytokines produced by human body raise an immune response against its self-component. This reaction causes an inflammatory response in the joint areas of the human body. The result of this disease is deformity and immobility. Fingers, wrists, feet, and


ankles are the most affected areas of rheumatoid arthritis. In the treatment procedure of rheumatoid arthritis, monoclonal antibodies are directed against the pathogenic autoimmune agent cytokine. Monoclonal antibodies are the a kind of monospecific antibodies produced by the immune cells of the human body. In various studies and researches, it is observed that, using mAbs rheumatoid arthritis utilized anti-CD7, anti-CD4 and CAMPATH-1H targets results in varying degrees of efficacy and significant safety concerns (Olinger et al. 2012).

Monoclonal antibodies are also known as mAb. These sets of antibodies are monospecific which are produced by identical immune cells. Until 1975, it was possible to use a mixture of antibodies for therapeutic purposes. But in 1975 Cesar Milstein and George Kohler discovered monoclonal antibodies for the first time. Till then different technologies are used for the production of monoclonal antibodies (Ordas et al. 2012). The production of monoclonal antibodies includes the major steps as Hybridoma cell production, purification of monoclonal antibodies. These monoclonal antibodies are generally made through cell culture techniques. In this method, the primary aspect is to fuse myeloma cells alongside the spleen cells in a mouse that has already been immunized with the desired antigen (Scott, Walchok and old. 2012). In recent studies, rabbit B cells are also used to form rabbit hybridoma. In order to fuse the myeloma cells, polyethylene glycol is used. The selective culture medium employed in the production of monoclonal antibodies is called HAT medium. The name is Hat because it contains hypoxanthine, aminopterin, and thymidine. This medium is a selective medium and only helps to grow the fused hybridoma cells. Unfused myeloma cells cannot grow in this medium as they lack HGPRT. As a result, they cannot replicate their DNA. The mixture of cells is then diluted, and clones of these cells are made of the parent cells (Scott, Walchok and old. 2012). Antibodies that are secreted by the different clones are then assayed according to their ability to the antigen. ELISA method, also known as the Enzyme Linked Immunosorbent Assay or Antigen Microarray or immune dot-blot. After collecting the media or culture sample of hybridomas, it is important to extract the desired monoclonal antibodies. During the purification procedure, at first, it is important to condition the sample. Cells, lipids, cell debris, clotted material are removed (Li et al. 2012). This is done through the use of centrifugation. After centrifugation filtration of the sample is done with a 0.45 µm filter. After this, the sample is condensed. This process is very much important because it will increase the concentration of the monoclonal antibodies in the liquid suspension. After this, most of the charged particles and other impurities like transferring can be removed by respective chromatography techniques (Scott, Walchok and old. 2012).

There are several other techniques used for the production of antibody heterogenicity, recombinant, chimeric antibodies, fully human clonal antibodies, etc. A recombinant aspect of the monoclonal antibodies include properties such as cloning, phage display or yeast display. Recombinant engineering of antibodies involves the uses of viruses and yeasts. This technique mainly depends on the rapid cloning of immunoglobulin gene segments. Through this method library of antibodies are created. Chimeric antibodies are actually partially mouse and partly human monoclonal antibodies. In modern therapeutic approaches, Chimeric antibodies are widely used. Fully human monoclonal antibodies are produced in transgenic mice. Till now, 7 to 9 fully human monoclonal antibodies have been successfully introduced for the therapeutic uses (Scott, Allison, and Wolchok, 2012).

One of the widely used therapeutic antibody used in clinical applications is Infliximab. It is a chimeric form of IgG1 mAb containing human constant regions along with murine variable regions (Birch, 2012). The main problem with this antibody is that it is only available in an intravenous form and should be applied along with the combination of methotrexate (Cheung et al. 2012). The initial dosage of this combination is 3 mg/kg and with respect to certain patient's condition, the dosage can increase to 10 mg/kg with an interval of 4 to 8 weeks. This particular antibody is approved by the FDA/EMA in combination with MTX for moderate and severe rheumatoid arthritis. This drug was first evaluated by Elliott et al. In 1993. The primary cause of the rheumatoid arthritis is a cytokine secreted name TNF α, a kind of tumor necrosis factor. Infliximab neutralizes the activity of this tumor necrosis factor or TNF α through binding. The binding procedure is a property of high affinity. The TNF α becomes a soluble and transmembrane form. This whole process inhibits the binding of TNF α with its respective receptor present in the body. It should be kept in mind that, Infliximab does not neutralize the TNFb or also known as the lymphotoxin a. . It is generally activated by inducing the release of pro-inflammatory cytokines. The event of leukocytes migrating and activating neutrophils are also a concerning event. Neutralization of TNF α reduces the overall inflammatory action caused by rheumatoid arthritis (Kantarjian et al. 2012).

Another drug that is widely used in clinical applications is Golimumab. Golimumab is complex human recombinant IgG1 mAb. It contains no murine components. Golimumab is produced by the phage display technology. The drug form of the antibody was approved by the FDA/EMA in the year of 2009. It is widely used for the moderate and severe level of rheumatoid arthritis. The drug is mainly used as the monotherapy. Sometimes it is also used as a combinational form with some disease-modifying antirheumatic drugs also known as DMARDs (Dinh et al. 2012). The most important aspect of its clinical aspect is that it is found in subcutaneous form as well. The standard dosage for this drug is about 40mg in every two weeks. Golimumab binds to the Tumor Necrosis factor α or TNF α. . As mentioned earlier that Golimumab is a human monoclonal antibody, it binds to the TNF α and inhibits soluble and transmembrane human TNF α. After binding, inhibition of TNF α prevents the binding of it respective receptor (Stanker et al. 2013). This phenomenon prevents the both leukocytes infiltration prevents the cell adhesion proteins such as E-selectin, VCAM-1, and ICAM-1. Another aspect of this drug is that it also prevents the adhesion of pro-inflammatory cytokine secretion such as IL-8, IL-6, G-CSF and GM-CSF in vitro. The patients who are suffering from chronic inflammatory conditions, application of Golimumab reduces the amount of ICAM-1 and IL-6. It also reduces the amount of C-reactive protein also known as CRP., matrix metalloproteinase 3 also known as (MMP-3) and vascular endothelial growth factors known as VEGF (Sandborn et al. 2014).

In modern times. Antibodies are one of the most prosperous and efficient methods for many therapeutic approaches. Monoclonal antibodies are broadly used in cancer therapies. Several other monoclonal antibodies with a combination of other substances are currently in phase 1 and phase 2 of clinical trials. Monoclonal antibody therapy can also be used by antibody fragments (Kolorz et al. 2013). The large size and long half-life of a full-length antibody make them inappropriate component for radioimmunotherapy (Nievves et al. 2012). As a result, Many research processes are carried out to produce some monoclonal antibodies, Diabody and Nanobodies (sdAbs) including scFv, which consist smaller size and consistent long life of the binding activity. Immunotoxins and Immunocytokines are also comprised of toxin and cytokines, which are fused to smaller antibody fragments. In different cancer therapy. Target therapy, monoclonal antibodies are widely used which produces an enormous prospect for future applications (Raison et al. 2013).

References

Barouch, D.H., Whitney, J.B., Moldt, B., Klein, F., Oliveira, T.Y., Liu, J., Stephenson, K.E., Chang, H.W., Shekhar, K., Gupta, S. and Nkolola, J.P., 2013. Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature, 503(7475), pp.224-228.

Bernard, A., Boumsell, L., Dausset, J., Milstein, C. and Schlossman, S.F. eds., 2013. Leucocyte Typing: Human Leucocyte Differentiation Antigens Detected by Monoclonal Antibodies. Specification-Classification-Nomenclature/Typage leucocytaire Antigenes de differenciation leucocytaire humains reveles par lesanticorps monoclonaux: Rapports des etudes com. Springer Science & Business Media.

Birch, J.R., 2012. THE LARGE SCALE CULTIVATION 0F HYBRIDOMA CELLS PRODUCING MONOCLONAL ANTIBODIES. Large-scale mammalian cell culture, 1, p.1.

Cheung, W.C., Beausoleil, S.A., Zhang, X., Sato, S., Schieferl, S.M., Wieler, J.S., Beaudet, J.G., Ramenani, R.K., Popova, L., Comb, M.J. and Rush, J., 2012. A proteomics approach for the identification and cloning of monoclonal antibodies from serum. Nature biotechnology, 30(5), pp.447-452.

Dinh, T.N., Kyaw, T.S., Kanellakis, P., To, K., Tipping, P., Toh, B.H., Bobik, A. and Agrotis, A., 2012. Cytokine Therapy With Interleukin-2/Anti–Interleukin-2 Monoclonal Antibody Complexes Expands CD4+ CD25+ Foxp3+ Regulatory T Cells and Attenuates Development and Progression of Atherosclerosis. Circulation, 126(10), pp.1256-1266.

Jeong, J.S., Jiang, L., Albino, E., Marrero, J., Rho, H.S., Hu, J., Hu, S., Vera, C., Bayron-Poueymiroy, D., Rivera-Pacheco, Z.A. and Ramos, L., 2012. Rapid identification of monospecific monoclonal antibodies using a human proteome microarray. Molecular & Cellular Proteomics, 11(6), pp.O111-016253.

Kantarjian, H., Thomas, D., Wayne, A.S. and O'Brien, S., 2012. Monoclonal antibody-based therapies: a new dawn in the treatment of acute lymphoblastic leukemia. Journal of Clinical Oncology, 30(31), pp.3876-3883.

Kolorz, M., Wroblova, K., Mokranova, J., Bartosova, L., Dite, P., Zbořil, V. and Bartos, M., 2013. Pharmacogenomics of infliximab therapy, impact of TNFRSF1A and TNFRSF1B gene polymorphisms.

Li, H., Ortiz, R., Tran, L., Hall, M., Spahr, C., Walker, K., Laudemann, J., Miller, S., Salimi-Moosavi, H. and Lee, J.W., 2012. General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies. Analytical chemistry, 84(3), pp.1267-1273.

Nieves, A., Ortega, B., Martinez, M., Castejon, O., Lares, M. and Ferrer, E., 2012. Standardization of ELISA technique for the immunological diagnosis of human toxocariasis. Boletín de Malariología y Salud Ambiental, 52(1), pp.21-32.

Olinger, G.G., Pettitt, J., Kim, D., Working, C., Bohorov, O., Bratcher, B., Hiatt, E., Hume, S.D., Johnson, A.K., Morton, J. and Pauly, M., 2012. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences, 109(44), pp.18030-18035.

Ordás, I., Mould, D.R., Feagan, B.G. and Sandborn, W.J., 2012. Anti‐TNF monoclonal antibodies in inflammatory bowel disease: pharmacokinetics‐based dosing paradigms. Clinical Pharmacology & Therapeutics, 91(4), pp.635-646.

Raison, C.L., Rutherford, R.E., Woolwine, B.J., Shuo, C., Schettler, P., Drake, D.F., Haroon, E. and Miller, A.H., 2013. A randomized controlled trial of the tumor necrosis factor antagonist infliximab for treatment-resistant depression: the role of baseline inflammatory biomarkers. JAMA psychiatry,70(1), pp.31-41.

Sandborn, W.J., Feagan, B.G., Marano, C., Zhang, H., Strauss, R., Johanns, J., Adedokun, O.J., Guzzo, C., Colombel, J.F., Reinisch, W. and Gibson, P.R., 2014. Subcutaneous golimumab induces clinical response and remission in patients with moderate-to-severe ulcerative colitis.Gastroenterology, 146(1), pp.85-95.

Sandborn, W.J., Feagan, B.G., Marano, C., Zhang, H., Strauss, R., Johanns, J., Adedokun, O.J., Guzzo, C., Colombel, J.F., Reinisch, W. and Gibson, P.R., 2014. Gastroenterology, 146(1), pp.96-109.

Scott, A.M., Allison, J.P. and Wolchok, J.D., 2012. Monoclonal antibodies in cancer therapy. Cancer Immunity Archive, 12(1), p.14.

Scott, A.M., Wolchok, J.D. and Old, L.J., 2012. Antibody therapy of cancer.Nature Reviews Cancer, 12(4), pp.278-287.

Stanker, L.H., Scotcher, M.C., Cheng, L., Ching, K., McGarvey, J., Hodge, D. and Hnasko, R., 2013. A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in food. Toxins, 5(11), pp.2212-2226.


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