3314700304800 Course Code

3314700304800 Course Code: PHRM 407
Course Title: Pharmaceutical Biotechnology
Section: 01
Submitted to:Najneen Ahmed
Senior Lecturer
Department of Pharmacy
East West University
Submitted by:
Md. Marufur Rahman Moni
ID: 2016-1-70-007
Date of Submission: 7th November, 2018
00 Course Code: PHRM 407
Course Title: Pharmaceutical Biotechnology
Section: 01
Submitted to:Najneen Ahmed
Senior Lecturer
Department of Pharmacy
East West University
Submitted by:
Md. Marufur Rahman Moni
ID: 2016-1-70-007
Date of Submission: 7th November, 2018
centercenter
Production of Monoclonal Antibodies Using Hybridoma Technology
9410077300
Production of Monoclonal Antibodies Using Hybridoma Technology

5314950268541522223355667889
022223355667889
-2667001495425 Table of Content
Production of Monoclonal Antibodies Using Hybridoma Technology Introduction ————————————————————————————– Technique involved in hybridoma technology 1. Necessary to initiating a hybridoma project —————————————– 2. Materials and media ——————————————————————— 3. Determination of antigens and immunization plans ——————————– 4. Decision of parent myeloma lines —————————————————- 5. Hybridoma colony and antibody creation ——————————————– 6. Fast essential screening process ——————————————————- 7. Cloning ———————————————————————————— 8. Cryopreservation ————————————————————————- 9. Characterization of monoclonal antibodies —————————————— 10. Production, purification and labeling of monoclonal antibodies —————— Utilization of monoclonal antibody ———————————————————- Conclusion ————————————————————————————— Reference —————————————————————————————–
00 Table of Content
Production of Monoclonal Antibodies Using Hybridoma Technology Introduction ————————————————————————————– Technique involved in hybridoma technology 1. Necessary to initiating a hybridoma project —————————————– 2. Materials and media ——————————————————————— 3. Determination of antigens and immunization plans ——————————– 4. Decision of parent myeloma lines —————————————————- 5. Hybridoma colony and antibody creation ——————————————– 6. Fast essential screening process ——————————————————- 7. Cloning ———————————————————————————— 8. Cryopreservation ————————————————————————- 9. Characterization of monoclonal antibodies —————————————— 10. Production, purification and labeling of monoclonal antibodies —————— Utilization of monoclonal antibody ———————————————————- Conclusion ————————————————————————————— Reference —————————————————————————————–

Production of Monoclonal Antibodies Using Hybridoma Technology
Introduction:
Cells named hybridomas are able to make monoclonal antibodies in a colossal sum which is desirable. The fusion of cells of mice spleen with cells of mice myeloma happen. At that point the secreted antibodies which demonstrate specificity are controlled by parent spleen cell and the characteristics are controlled by myeloma. Two scientist kohler and milstein revealed this innovation in 1975. This innovation manufactures monoclonal antibodies in distinct cells. This antibodies are utilized in hindering, examination and treating infection. They are additionally utilized in sparing organ transplantation where dismissal of organ can happen ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“ISSN”:”0736-2293″,”PMID”:”3038035″,”abstract”:”The technology for producing hybridomas secreting human monoclonal antibodies has not been well established, contrary to the situation for mouse monoclonal antibodies. Therefore, we attempted to find better fusion partners using two EBV-transformed human B cell lines, C5TK1 and TAPC-30 (anti-SRBC IgM and anti-HBs IgG secretors, respectively; provided by Prof. Y. Ono, Nihon Univ. School of Med.). Fifteen partner cell lines available were fused with either of these lines by conventional PEG technique, and their fusion frequencies, cloning efficiencies, and levels and stabilities of specific antibody secretion were examined. Among the fusion partners tested, KR-12, SHM-D33 and 3HL3-6J lines appeared to be the best. Hybridomas with the KR-12 line were easily cloned and produced stable specific antibodies, although the level of Ig secretion was almost the same as the antibody-producing parental B-cell lines. SHM-D33 cells efficiently formed hybrids but the stability of their Ig production was low and cloning was laborious. Although the fusion frequency of 3HL3-6J cells was not so high, some hybrids were good producers of antibodies, and the levels of specific antibody reached levels greater than 10 micrograms/ml. Their cloning was relatively easy. Therefore, the 3HL3-6J line seemed to be the best fusion partner for EBV-transformed B cells. With regard to the low fusion frequency of human cells, we attempted to improve the efficiency using the electrofusion technique. Three to 10 times more hybridomas were obtained by this technique than by the conventional PEG method. Further investigations are under way.”,”author”:{“dropping-particle”:””,”family”:”Tokunaga”,”given”:”T”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Chiba”,”given”:”J”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Ohnishi”,”given”:”K”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”container-title”:”Gan to kagaku ryoho. Cancer & chemotherapy”,”id”:”ITEM-1″,”issue”:”2″,”issued”:{“date-parts”:”2010″},”page”:”2198-2204″,”title”:”Attempts to improve hybridoma technology for the production of human monoclonal antibodies”,”type”:”article-journal”,”volume”:”14″},”uris”:”http://www.mendeley.com/documents/?uuid=28e4e554-5cb0-4e51-8453-ba9a1068d95f”},”mendeley”:{“formattedCitation”:”(Tokunaga, Chiba and Ohnishi, 2010)”,”plainTextFormattedCitation”:”(Tokunaga, Chiba and Ohnishi, 2010)”,”previouslyFormattedCitation”:”(Tokunaga, Chiba and Ohnishi, 2010)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Tokunaga, Chiba and Ohnishi, 2010)
Technique involved in hybridoma technology:
1. Necessary to initiating a hybridoma project:
– Sterility is important in cabinet where hybridoma generation is presented.
– An incubator is required in which temperature is controlled, moistness is kept up and gas concentration is kept up. Fluid nitrogen storage ought to be faciliated as it is basic for hybridomas keeping up in a low temperature store for cryopreservation.
– Ease of animal holding, aseptic careful surgical for mouse dissection, water showers of temperature at 37 and 56 degree Celsius, centrifugation machine, tissue culture product are additionally required ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“DOI”:”10.1109/34.713358″,”ISBN”:”1351091727, 9781351091725″,”author”:{“dropping-particle”:””,”family”:”Hurrell”,”given”:”John G.R.”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”id”:”ITEM-1″,”issued”:{“date-parts”:”2018″},”number-of-pages”:”239″,”title”:”Monoclonal Hybridoma Antibodies: Techniques and Applications – John G.R. Hurrell – Google Libri”,”type”:”book”},”uris”:”http://www.mendeley.com/documents/?uuid=b1d48f2b-6ed6-4f14-914c-4dca3944bc8b”},”mendeley”:{“formattedCitation”:”(Hurrell, 2018)”,”plainTextFormattedCitation”:”(Hurrell, 2018)”,”previouslyFormattedCitation”:”(Hurrell, 2018)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Hurrell, 2018)
2. Materials and media:
– Cell development medium is utilized. RPMI 1640 is utilized. Notwithstanding it, FCS 10%, Glutamine 2mM, Penicillin 100 International Units/ml and streptomycin 100 microgram/ml are utilized.
– HAT particular medium is utilized.
– Polyethylene glycol is used.(Hurrell, 2018)
3. Determination of antigens and immunization plans:
– Highly distinct antibodies against polluted antigens are engaged with purifying the antigens.
– Spleen cells are used in the abtibody producing hybrids.

– BALB/c mice strain are used.(Hurrell, 2018)
4. Decision of parent myeloma lines:
– It`s development ought to be better and high concentrations of immunoglobulin must be discharged from it.
– The fundamental biosynthesis pathway which does nucleic acid synthesis is restrained by an aminopterin. Cell`s duplications are proceeded by utilizing salvage pathway within the sight of hypoxanthine and thymidine. In the event that a compound required in the salvage pathway that is missing in a mutant cell, development of cell is not possible. Myeloma cells lacking hypoxanthine guanine phosphoribosyl transferase catalyst (HGPRT) are applied. (Hurrell, 2018)5. Hybridoma colony and antibody creation:
A particular antigen is infused into mouse. Myeloma, a harmful cell is intermixed with specific plasma cells. This hybrid cell is therefore copied. Numerous specific daughter clones are delivered in immune cells. Antibodies are created from just a single kind of cell. At that point, antibody is created in HAT medium . Here, HGPRT gene is absent. Polyethylene glycol helps in fusion. The mechanism of action relies upon biosynthesis of nucleotides guarantees that just fused hybrids will survive. Tetrahydrofolate is importanr in nucleotide synthesis and this can be obtained by dihydrofolate reductase. Folic acid analouge aminopterin obstructs this catalyst. In the medium,consolidating 6-thioguanine or 8-azaguanine into nucleotides by HGPRT cause cells death. The cells where active HGPRT is missing can survive. Along these lines, just survival partitions are B cell-myeloma hybrids. At that point, incubated medium is thinned into multi well plates are done as such that just a single cell can be available in each well and checking for specific antibody is occurred. (Tokunaga, Chiba and Ohnishi, 2010)(Kulkarni, 2002) Adapted from: Tabll, A. et al. (2015) Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus, World Journal of Hepatology. Baishideng Publishing Group Inc. doi: 10.4254/wjh.v7.i22.2369. (Accessed: 4 November 2018).Figure: Schematic diagram of production of monoclonal antibodies through hybridoma technology 6. Fast essential screening process:
ELISA should be possible. Here, adsorbtion of antigen to the base of 96-well plates, incubation for growth of hybridomas happen. The desired antibody in the samplestays bound to antigen and is recognized by an immune conjugate. This conjugate is comprises of two parts, one immune response is particular for an epitope that the parts are in the consistent space in the first antibodyt. It goes about as hostile to antibody. Second one is basic phosphatase catalyst. This conjugate is retained in the well amid the immobilization at first incubation of antibody. Subsequent to washing dry substrate of compound (ex. p-nitrophenyl phosphate) is changed over to a colored item (ex. p-nitrophenol) by the basic phosphatase. After incubation and the end of enzyme capacity, ELISA reader evaluate the optical density of product.(Kulkarni, 2002) Adapted from: ELISA Guide – Creative Diagnostics (no date). Available at: https://www.creative-diagnostics.com/ELISA-guide.htm (Accessed: 4 November 2018). Figure: Screening process by ELISA (Enzyme-linked immunosorbent assay) 7. Cloning:
In the wake of screening, cloning should be possible by three distinct procedures (cloning by method of limiting dilutions, cloning utilizing semi-solid agars and cloning and choice utilizing the fluorescence-activated cell sorter). The limiting dilutions strategy permits the specification of cells in the culture, thinning and partial adsorption into new wells in which each well is comprise of just a single cell. At that point, cell regeneration process is rehashed to guarantee the presence of monoclonal property. Another technique is soft agar method that permits the expansion of tremendous malignant cells in a low agar content semi-solid medium. The dispersion of culture into single cells and the presence of separated colonies because of such cell concentration guarantee the presence of monoclonal antibody. Both procedures are utilized by consolidating these methods.(Hurrell, 2018) Adapted from: Cloning Method. EuroMAbNet (no date). Available at: https://www.euromabnet.com/protocols/cloning.php (Accessed: 4 November 2018). Figure: Cloning process by technique of limiting dilutions 8. Cryopreservation:
It is important against the loss of useful lines. Hybridoma ought to be frozen down as quickly as to diminish the loss of chromosome.(Hurrell, 2018)9. Characterization of monoclonal antibodies:
Monoclonal antibodies ought to be portrayed as for following parameter:
– Specificity is resolved for particular antigen.
– Titer: By deciding for screening and deciding the highest dilution at which a positive result can be accomplished.
– Affinity of binding can be estimated.
– Storage and stability are resolved to know the impact of manipulations, for example, lyophilization .
– Immunoglobulin class/subclass: For acknowledgment of IgG1, igG2a, IgG2b, IgG3 and IgM , sets of antisera are utilized.
– Monoclonality : Only one subclass of antibody/just a single cell compose ought to be available in hybridoma. (Hurrell, 2018)
10. Production, purification and labeling of monoclonal antibodies:
Monoclonal antibody can be produced by two procedures (tissue culture or by developing hybridoma as a tumor in mice). At that point, ammonium sulfate can precipitate antibody and ion exchanging chromatography procedure and antigen affinity chromatography system can be utilized for higher reach out of antibody purity. Finally, Labeling of desired antibody by radio-isotopic or by fluorescence labeling can be done.(Hurrell, 2018) Adapted from: Affinity Purification – Biologicscorp Blog (no date). Available at: https://www.biologicscorp.com/blog/protein-purification-methods-based-on-bioproperties-affinity/#.W98qUJMzbIU (Accessed: 4 November 2018).Figure: Selective adsorption of the desired antibody is enabled by Affinity chromatography
Utilization of monoclonal antibody:
– Recognition of ABO blood groups
– Diagnosis (ELISA test is utilized for assurance of viruses and imaging)
– Immunopurification that isolates one substance from a blend of very similar to molecule(Tokunaga, Chiba and Ohnishi, 2010)
Conclusion:
A monoclonal antibody can be readied if the arrangement of system can be possible effectively. Immunization of mice, a myeloma cell line for combination and keeping up in culture are the core methodology. The fusion products are permitted to develop in culture where other isolating cells are missing. The developing hybridoma colonies are showed up and after that screened to get the desired antibody. The intriguing hybrids are experienced cloning process for guaranteeing monoclonality. Cloned hybrids that create antibodies of interest are made in huge amounts with the goal that valuable amounts of antibody can be acquired and afterward the cryopreservation of the cells are finished. At last, it is important to characterize, decontaminate and label the monoclonal antibody. (Hurrell, 2018)Reference:ADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY Hurrell, J. G. R. (2018) Monoclonal Hybridoma Antibodies: Techniques and Applications – John G.R. Hurrell – Google Libri. doi: 10.1109/34.713358.

Kulkarni, G. T. (2002) ‘Production of Monoclonal Antibodies’, in Biotechnology and Its Applications in Pharmacy. 1st ed. New Delhi: Jaypee Brothers, Medical Publishers, pp. 4–51.

Tokunaga, T., Chiba, J. and Ohnishi, K. (2010) ‘Attempts to improve hybridoma technology for the production of human monoclonal antibodies’, Gan to kagaku ryoho. Cancer & chemotherapy, 14(2), pp. 2198–2204.