Monoclonal Antibody Production using Animal Cell Culture

Monoclonal Antibody Production using Animal Cell Culture Introduction Monoclonal antibodies, in biomedical research, are used as reagents in diagnosis and treatment of diseases like cancer and infections [1]. It has been almost century their introduction, mAbs are still produced from splenocytes fused to myeloma cells [2]. The antibodies are produced by obtaining cell lines from animals immunized with substance to be studied. To produce the cell lines, B cells obtained from immunized mice are fused with myeloma (immortalized) cells [1][3]. For production of desired monoclonal antibodies, the cells should grow in one of the two ways: injecting the peritoneal cavity of mouse (known as in vivo method or mice ascites method) or by in vitro method (Tissue culture method). Further mouse ascites fluid or supernatant of tissue culture is processed and monoclonal antibody of desired concentration and purity is obtained (figure1) [1]. Mice ascites method is preferred as it is familiar, properly understood and extensively used in laboratories in comparison to tis sue culture method which is time consuming, expensive and laborious and ails to give required amount of antibodies[1][3]. Presently, twenty two monoclonal antibodies for transplantation, oncology, infectious, cardiovascular and chronic inflammatory disease have been approved by FDA [3]. Strict guidelines has been setup by IACUC for use of animal for mAb production which includes (i) use of animal is scientifically justified (ii) methods to be used which gives minimum pain to the animal[1]. Monoclonal antibody production (Past to Present) Mouse mAbs This technology was introduced in 1975, which works on generation of mouse hybridomas by fusion of B cells, obtained from immunized mice, and myeloma cells. But mAbs produced by this method have many limitations and is not preferred due to high immunogenicity in humans and due to production of human anti-mouse antibody which leads to their rapid clearance from patient’s body [3]. Chimeric mAbs These are produced by gene manipulation method in which constant regions of mouse Abs are replaced by human Abs. Like mouse mAbs, chimeric mAbs also leads to formation of human anti-mouse antibodies and leads to various immunogenicity in patients thus to make it potent in therapeutics further better understanding is required in their structure and function [3]. Humanized mAbs In this method, complementary determining regions (CDRs) are transferred to human IgG from mouse mAb. There is only 5-10% non-human content in humanized mAbs in comparison to 30% in chimeric mAbs [3]. Generation of mAbs Immunization of mice Screening of sera Spleen cell isolation Myeloma cells prep. Cell fusion (Tissue culture) Hybridoma screening Selecting cultures for cloning Mouse Feeder cells Cloning (limiting dilution) Clone isolation and expansion Cell freezing and recovery Supernatant production (from tissue culture media) mAbs purification and testing Figure1. Flowchart showing steps for production of monoclonal antibodies by tissue culture method [1]. Monoclonal Antibody Production Against various Diseases 5C3 mAb against Tumor Growth and Angiogenesis In this method, S100A4 was used for immunizing female Balb/cAnNHscl mice and mAbs were obtained from fused myeloma and spleen cells using PEG-1500. Hybridomas were selected on HAT medium and further screened for it reaction with S100A4 by ELISA. Clones were selected which were corresponding to 5C3 mAb. Cell culture was scaled up in humid conditions (air 94% and 6% CO2) at 37 °C temperature [4]. Supernatant (serum free) from hybidomas was obtained and purified on column containing protein A with the help of AKTA purifier FPLC system and elutions containing 5C3 mAbs were concentrated and filtered in PBS centrifuge Amicon Ultra-15 which has low binding Ultracel membrane and then quantifying mAbs at 280nm [5][6]. 2-4F mAb against Oxytetracycline in shrimps Oxytetracycline is used as medication feed in aquaculture [7], its overuse can lead to its accumulation in aquaculture food and its consumption then leads to serious health problems in sea food lovers. To prevent consumers from its harmful effects mAb 2-4F, highly sensitive and specific, were produced for detection of OTC in aquaculture food animals by ELISA. Hybridomas were obtained by standard protocol, by immunizing the female BABL/c mice with OTC-BSA, hybridomas were cultured and supernatants from culture were screened for antibodies using iELISA and antibodies were cloned by limiting dilution method to obtain monoclones then in serum free media these moloclones were cultured in 500 ml spinner flask [6][7]. Further mAbs were purified from this culture using protein G by affinity chromatography. The elute fractions were collected and its protein concentration was determined at 280nm spectometrically and mAb was filtered using cellulose acetate membrane (0.2  µm) and kept at -20à ‚ °C until used [8]. Human anti-human IL-21 monoclonal antibody. Interleukin-21 is a type I cytokine with four helical bundles that exerts effect on hematopoietic cells like NK cells, T and B lymphocytes. CD4+ T and NK T cells produce interleukin-2 cytokine, over expression of IL-2 lead to variety of autoimmune disorders. Genetically modified Kirin-Medarex mice were immunized with rhIL-21, immunogens were emulsified with P-adjuvant and CpG and recombinant mouse GM-CSF. Hybridomas obtained were cultured in IMDM containing 1x GlutMax, 1x Penicillin, 10% fetal clone serum and 10% Hybridoma Cloning Factor. Hybridomas were selected with IMDM in conjugation with HAT medium and cloning was carried out with 1x HT and distributed in 96 well Elisa plate and wells were examined microscopically for monoclonality and screened with phosphorylated-STAT3. Wells with positive results were distributed in 24 well cultures to obtained density 6105 cells/ml and then supernatant was collected and cells cryopreserved. Further media with human IgG was obtained and filter ed through 0.2 µm membrane and from this filtered media antibody protein was purified by combing Protein G Sepharose Affinity Chromatography Size Exclusion Chromatography and absorbance was taken at 280nm and further its quality was accessed by size exclusion HPLC [9]. mAbs L317, L363, L386 ÃŽ ±-galactosylceramide:CD1d complex The ÃŽ ±-galactosylceramide also known as KRN7000 is best studied ligand that binds to protein CD1d. KRN7000:mCD1d complex is easily recognized by iNKT cells and leads to number of proinflammatory and immunoregulatory functions. To understand the mechanism of antigen presentation to CD1d by iNKT cell three monoclonal antibodies L317, L3363, and L386 were produced. Primary immunogen was prepared with protein obtained from strain H37Ra of Mycobacterium tuberculosis (PPD) and it was conjugated with the complex KRN7000:CD1d. The complex KRN7000:mCD1d:PPD was studied by SDS-PAGE. Mice were first vaccinated with Mycobacterium bovis (BCG) then after 23 days mice were immunized with 5 µg KRN7000:CD1d:PPD complex in 1:1 PBS and Imject alum. At day 61 booster dose was given to mice, of the complex, with 7106 cells. Mice were then sacrificed and spleens dispersed PBS, cells were obtained and further washed with PBS and erythrocytes were lysed and cells were suspended in FBS/HEPES free DMEM [10][11]. The preparation was then mixed with myeloma cells and centrifuged and tubes with pellet were placed in water bath set at 40 °C and into this heated PEG was added followed by FBS/HEPES- free DMEM and then cells again centrifuged and re-suspended in DMEM. Hybridomas along with MRC-5 fibroblast feeder blast cells were plated in 96 well tissue culture plates. Supernatant from culture was screened and cloning of hybridomas carried out by limit dilution. Then 108 cells were inoculated in 2 liters roller bottles containing 500ml medium and OptiMAb supplement was added. MAbs were obtained by filtering of supernatant through protein G column chromatography [12]. Stx2f-1, Stx2f-3, Stx2f-4 mAb against Shiga toxin, a gastrointestinal disease Shiga Toxin 2 also designated as Stx2 is virulence causes gastrointestinal disease in humans’ world by food poisoning. It subtype Stx2f cannot be easily detected by immunological methods and thus three monoclonal antibodies specific to it were produced. Complete hybridoma media contains Iscove’s modified DMM with NaHCO3 and 1 Glutamax, containing fetal calf serum (heat inactivated) [13]. Female Balb/cJ mice were immunized with His-tagged Stx2f and hybridomas were obtained and screened for antibodies against Stx2f by ELISA and were further transferred to MPCM/HT/cHM media and diluted 500cells/ml and then the cells were grown in cHM media. Media containing antibody (400ml) was filtered through protein G column and elution were obtained in 0.1M glycine giving 5mg of purified antibody Stx2f [14][15]. Monoclonal antibody from EB66 Cell lines with enhanced ADCC activity EB66 cell lines are derived from embryonic stem cells of duck which can be genetically engineered and production of mAbs can be increased above 1g/L when grown in serum free media. EB66 have various other characteristic features like short doubling time, high cell density and unique metabolic profile with low accumulation of ammonium and lactate and low consumption of glutamine [16]. Further, EB66 cell lines used for production of mAbs has reduced fucose content with enhanced ADCC activity. EB66 cell lines produce chimeric IgG1 anti-cancer mAb against antigen anti-X by nucleofection. EB66 clones when grown in Erlenmeyer flask with standard fed batch culture produces 1.28g/L of IgG1 of cell density with 36 millions cells/ml. Further by accumulation of monoclonal antibodies in supernatant culture no degradation was observed in antibody production assessed by HPLC, SDS-PAGE and western blot. When the supernatant was purified with Protein-A HPLC showed 98% mAbs as monomers. Glycosylation profile of monoclonal antibodies was analyzed by MALDI-TOF-MS, enhanced activation of the monoclonal antibodies obtained from EB66 cell lines was analyzed by flow cytometry[16][17]. FDA Approved mAbs in market [18][19] Infliximab Remicade ® TNF Rituximab Rituxan ®, MabThera ® CD20 Trastazumab Herceptin ® HER2 Bevacizumab Avastin ® VEGF Adalimumab Humira ® TNF Cetuximab Erbitux ® EGFR Ranibizumab Lucentis ® VEGF Palivizumab Synagis ® RSV Tositumomab Bexxar ® CD20 Alemtuzumab Campath ® CD52 Certolizumab pegol Cimiza ® TNF Gemtuzumab ozogamicin Mylotarg ® CD33 Muromonab-CD3 Orthoclone Okt3 ® CD3 Efalizumab Raptciva ® CD11a Abciximab ReoPro ® GP IIb/IIIa Basiliximab Simulect ® CD25 Eculizumab Soliris ® C5 Natalizumab Tysabri ® a-4 integrin Panitumumab Vectibix ® EGFR Omalizumab Xolair ® IgE Daclizumab Zenapax ® CD25 Ibritumomab tiuxetan Zevalin ® CD20 Recent advances in mAbs production Engineered Monoclonal antibodies Advancement in mAb engenrreing has lead to transformation in this field which has lead to production of new drugs which as many useful characteristics like decreased immunogenicity, improved specifity along with stability and potency [18]. The replacements of murine as well as chimeric mAbs with full human mAbs are boon of this novel technology for example adalimumab, ranibizumab and cetrolizumab pegol. Adalimumab, the human mAb, is created by using phage display technology and now it is the top selling drug in the market. Cetrolizumab pegol has been engineered to increase its half-life by making changes in its Fab fragments [19]. Ranibizumab which is derived from bevacizumab wet AMD (age-related macular degeneration) and is considered as care indication standard. These new engineered mAbs have potential to compete with the drugs already in market and have bright future ahead [19][20]. Biosimalar Monoclonal antibodies Biosimilars are the copies of drugs whose patient has expired and now these drugs can be produ- -ced and manufactured by any company. But due to complex molecule used and then its approval from U.S makes it a complex process therefore most of the biotechnology companies are not in favor of production of biosimilars. Dr. Reddy in India has launched Reditux ® which is anti-CD20 monoclonal antibody and it is claimed, as the first biosimilar monoclonal antibody, by the company. In spite of approval of Reditux ® in India, it is thought that it would not have sufficient data that can fulfill the set standards of developed countries in terms of strict safety, efficacy and manufacturing standards[18][19][20]. Conclusion Monoclonal antibodies are expanding rapidly in pharmaceutical industries with already hundreds of candidates are under development and trials. Both cytotoxic and radiology methods are emerging to increase efficacy of the present therapeutic molecules. Moreover, advances have also been made to use mAbs in treatment of bacterial and viral infection. Biosimilars and bio-superiors are the next generation drugs which can be produced as most of the blockbuster monoclonal antibody are at verge to their patent expiry. The future of the monoclonal antibodies in therapeutics is bright and continued discovery, research and development in this field can take it to the heights that have not been achieved before. Abstract Monoclonal antibodies today have gained a breakthrough and are used in treatment of numbers of disease. Over 30% of the Engineered Monoclonal antibodies are under clinical trials. Moreover, different methods to generate human monoclonal antibodies are present today like generation of humanized and chimeric antibodies from genetic engineering of mouse antibodies, phage display method and transgenic mice development. Monoclonal antibodies are in great demand today and FDA has approved almost 22 mAbs till date and all these are commercially available in market. Biosilimars are also taking up the pace as most of the blockbuster mAbs are at verge of their patient expiry and Reditux ® developed by Dr. Reddy claimed as first biosimilar in India and is half the cost of Rituximab ®.

The Role of the Inspector in An Inspector Calls Essay examples -- An I

The Role of the Inspector in 'An Inspector Calls' An Inspector Calls is a play with many social and political messages. J. B. Priestley believed a great deal in socialism and he used several of his plays to try and influence people to be Socialist as well. It was written in a time when Britain was ruled by a Labour government and socialist policies were seen as the way forward. It was a popular way of thinking at that time so Priestley's aim for the play was probably to teach the unconvinced. The Inspector in J. B. Priestley's 'An Inspector Calls' is one of the most thought-provoking and mysterious characters that modern day literature has yet produced. It is this mysterious element that contributes greatly to making him a very interesting character and one that may be perceived in many ways. The audience does not find a great deal out about the Inspector and nothing is explicitly told to us; we are given hints and clues from the way he acts and what he says and are forced to piece these together to form our own ideas about his identity and his intentions. In this way, Priestley has asked his audience to act as a judge and to reach personal conclusions about him. The role of the Inspector is one of many levels. In terms of how he is used in the basic structure of the play, he is there to move the play along in that he encourages the characters to tell their stories. If there was not the revelation that he was not a real Police Inspector, he would only be considered as a narrator and not play a big part in the play. Because it transpired that he was an impostor of sorts, further questions are asked by the audience and different insights have become likely and it is clear that the Inspector is in the play for many reasons. T... ...e unpunished. One must conclude that the Inspector's main purpose is to teach. In the context of the play, he told the characters what had happened to a particular girl because they had each been guilty of selfishness. In regards to the whole of society, he voiced Priestley's opinions that we cannot make any progress if we do not work together. In my opinion, those watching or reading the play today would not gain as much from the story in regards to the moral teachings because most have now accepted the advantages of Socialism over Capitalism and so do not have as much to learn on the arguments of this issue as the audiences of 1947. In regards to the question of what the Inspector actually was, I personally feel that there is not enough evidence given for even a strong, fact-supported theory to be produced to answer the question, let alone an infallible answer.

Commodore Perry’s Journey to Japan

After the conclusion of the War of 1812 and prior to the Civil War, the United States Navy entered into a peacetime role. Initially, this role was to protect commerce trading in both inland and international waterways. However, that role was soon expanded upon with Commodore Matthew Calbraith Perry’s journey to Japan. The journey had its immediate impact, including the signing of a comprehensive treaty that established trade relations with Japan and provided protection for sailors and their ships. Perry’s expedition also had the impact of serving as a precursor for the change in what the Navy’s responsibilities encompassed, which even carry on to the present day Navy. Commodore Perry left for Japan with the objectives of opening up Japanese ports to trade and ensuring American presence and protection in East Asia. These terms were outlined in â€Å"detailed instructions from the Secretary of the Navy John P. Kennedy, diplomatic instructions from the State Depart ment, and a letter from President Millard Fillmore to the Emperor of Japan†2 that Perry carried with him on his voyage.From beginning to end Perry’s voyage spanned nine months and was filled with trials and tribulations. The Japanese were initially turned off to the idea of Americans entering their country, and would not even let them step on land. Only twice did Perry and his squadron come ashore in the nine months prior to the signing of the official treaty. Most of the negotiations took place upon various ships in Perry’s control and the meetings were often difficult to coordinate.Based on notes from Perry’s personal journal, these complications often lead to frustration and Perry was constantly considering employing â€Å"whole force† that he was granted to use if he deemed it necessary to achieve his goals. 3 However, this was ultimately unnecessary, and Perry did well to remind himself that his voyage was diplomatic and pacific in nature. The negotiations were an arduous process and Perry even left Japan returning later with twice as many ships, anticipating a struggle. This was unnecessary as the Japanese agreed to Perry’s desires and the â€Å"black ships† saw no combat.With the agreement of the Japanese the Treaty of Kanagawa was drafted and subsequently signed on 31 March 1854. This treaty allowed for a U. S. consul to be created at Shimoda, and allowed access to the ports of Hakodate and Shimoda for the purpose of obtaining â€Å"wood, water, provisions, and coal, and other articles their necessities may require. † The treaty also required that â€Å"whenever ships of the United States are thrown or wrecked on the coast of Japan, the Japanese vessels will assist them, and carry their crews to Shimoda. Thirdly, men staying in Shimoda and Hakodate, or any seamen shipwrecked shall be free and â€Å"shall not be subject to†¦restrictions and confinement. †4Although there was not a formal agreement on trade in these open ports, Perry assumed correctly that with an American presence in port, trade would come naturally. 5 The initial impact of Perry’s expedition and the treaty with Japan gave the United States Navy many new roles and an international presence on the high seas. Japan had been a country focused on isolationism for centuries. This isolationism is mainly connected to the zeal of early missionaries who traveled to Japan.The United States was able to avert this conflict in values by Commodore Perry’s outright statement to the Japanese leadership that the United States government â€Å"does not interfere with the religion of its own people, much less with that of other nations. †6 Several attempts were made to open Japan to American trade, but all had failed. One such failure was that of Commodore James Biddle, which proved to be a complete embarrassment for the United States, as he made several mistakes in his conduct and on top of it a ll needed to be towed out of port by a Japanese ship. The fact that Commodore Perry was successful in his mission changed the status quo in regards to what the United States Navy could and could not do. Perry proved that the United States was capable of having a forward presence in foreign lands and was able to establish international trade in East Asia. The establishment of commercial relations with Japan furthered the Navy’s responsibility in protecting trade. Perry’s exploits also showed that diplomacy was a possible way for the United States to establish influence in other countries.Thirdly, Perry and his â€Å"black ships† were the first sign of American deterrence. The fact that American ships were off the coast of Japan ready to attack an underprepared country made it very difficult for the Japanese to negotiate anything in their favor or make any tactical or strategic decisions to remove the threat of Perry’s force. The roles of the Navy that Comm odore Perry established in the mid-nineteenth century are still prevalent in the present day.The idea of the Navy as a protector of commerce (although established before Perry, he was instrumental in expanding the Navy’s prevalence in ensuring safe trade) continues into the present day. An example of this would be ships stationed in the Mediterranean Sea. This area, specifically around the Strait of Hormuz is crucial to trade in the Middle East. The presence of the United States Navy maintains a safe trading environment between the United States and its allies, and other countries in the region.Commodore Perry also introduced the idea of deterrence, which is crucial in the operations of the Navy in today’s world. One example of American deterrence is the use of submarines, equipped with nuclear war heads and ballistic missiles, which are virtually invisible to our enemies. Perry also proved that diplomacy was a very potent way to establish influence in foreign countrie s and maintain a presence without force. This is also seen in the United States establishment of embassies in foreign countries and the use of diplomats to negotiate with foreign countries.Commodore Perry’s expedition to Japan had a tremendous impact on the United States at the time it occurred, but it also had an everlasting impact on how the Navy operates and what roles and responsibilities it chooses to take on. Notes 1. Walworth, Arthur. Black ships off Japan; the story of Commodore Perry's expedition 242. New York: A. A. Knopf, 1946. 2. Bradford, James C. Quarterdeck and bridge: two centuries of American naval leaders 115. Annapolis, MD: Naval Institute Press, 1997. 3. Perry, Matthew Calbraith, and Roger Pineau.The Japan Expedition, 1852-1854; the personal journal of Commodore Matthew C. Perry 157. Washington: Smithsonian Institution Press, 1968. 4. Barrows, Edward Morley. The great commodore; the exploits of Matthew Calbraith Perry 365. Indianapolis: The Bobbs-Merrill C o, 1935. 5. Anderson, David. â€Å"Perry, Matthew Calbraith. â€Å"American National Biography Online Feb. 2000 (accessed October 2, 2012). 6. Walworth, Arthur. Black ships off Japan. 243. 7. Bradford, James C. Quarterdeck and Bridge. 113. Bibliography Anderson, David. â€Å"Perry, Matthew Calbraith. â€Å"American National Biography Online Feb. 000 (accessed October 2, 2012). Barrows, Edward Morley. The great commodore; the exploits of Matthew Calbraith Perry 365. Indianapolis: The Bobbs-Merrill Co, 1935. Bradford, James C. Quarterdeck and bridge: two centuries of American naval leaders. Annapolis, MD: Naval Institute Press, 1997. Perry, Matthew Calbraith, and Roger Pineau. The Japan Expedition, 1852-1854; the personal journal of Commodore Matthew C. Perry 157. Washington: Smithsonian Institution Press, 1968. Walworth, Arthur. Black ships off Japan; the story of Commodore Perry's expedition. New York: A. A. Knopf, 1946.

Edit and Display Boolean Fields using a CheckBox in Delphi

Tip submitted by Rene van der Heijden A series of articles titled Adding components to a DBGrid discusses placing just about any Delphi control (visual component) into a cell of a DGBrid. The idea is to create visually more attractive user interfaces for editing fields inside a DBGrid: a ComboBox for drop down lists; a DateTimePicker (calendar) for date values; a check box for boolean fields. CheckBox for Boolean Fields CheckBox inside a DBGrid As noticed by Rene van der Heijden the solution is rather lengthy, and it doesnt work, at least not when using the mouse to click on the checkboxes. Rene suggest an easier approach needing only two even handlers: OnCellClick and OnCustomDrawCell for your DBGrid control: //OnCellClik event of a DBGrid1 procedure TForm.DBGrid1CellClick(Column: TColumn) ; begin   Ã‚  if (Column.Field.DataTypeftBoolean) then   Ã‚  begin   Ã‚  Ã‚  Ã‚  {toggle True and False}   Ã‚  Ã‚  Ã‚  Column.Grid.DataSource.DataSet.Edit;   Ã‚  Ã‚  Ã‚  Column.Field.Value: not Column.Field.AsBoolean;   Ã‚  Ã‚  {immediate post - see for yourself whether you want this}   Ã‚  Ã‚  Ã‚  Column.Grid.DataSource.DataSet.Post;   Ã‚  Ã‚  Ã‚  {you may add additional functionality here,   Ã‚  Ã‚  to be processed after the change was made}   Ã‚  end; end; //OnDrawColumnCell event of a DBGrid1 procedure TForm.DBGrid1DrawColumnCell(   Ã‚  Sender: TObject;   Ã‚  const Rect: TRect;   Ã‚  DataCol: Integer;   Ã‚  Column: TColumn;   Ã‚  State: TGridDrawState) ; const   Ã‚  CtrlState: array[Boolean] of integer (DFCS_BUTTONCHECK, DFCS_BUTTONCHECK or DFCS_CHECKED) ; begin   Ã‚  if (Column.Field.DataTypeftBoolean) then   Ã‚  begin   Ã‚  Ã‚  Ã‚  DBGrid1.Canvas.FillRect(Rect) ;   Ã‚  Ã‚  Ã‚  if VarIsNull(Column.Field.Value) then   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  DrawFrameControl(DBGrid1.Canvas.Handle,Rect, DFC_BUTTON, DFCS_BUTTONCHECK or DFCS_INACTIVE) {grayed}   Ã‚  Ã‚  Ã‚  else   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  DrawFrameControl(DBGrid1.Canvas.Handle,Rect, DFC_BUTTON, CtrlState[Column.Field.AsBoolean]) ; {checked or unchecked}   Ã‚  end; end; Delphi tips navigator: » Remove Duplicate Items in Delphis TStringList « 5 Facts you Did Not Know about Delphi and Classes and the VCL and Inheritance and Custom Controls and...