Limiting dilutions and subcloning while ensuring Monoclonal antibodie - PDF document

Limiting dilutions and sub-cloning while ensuring Monoclonal antibodies (Mabs) are antibodies Hybridomas, cell culture, monoclonal in primary culture, presence or absence of growth factorsImmediately after fusions of genetically compatiblemay be achieved in soft agar, by limiting dilution or by Sukanya Shyama Sundar , Kaavya Krishna Kumar , Deepa Parvathi.V Solomon F. D. Paul On days 4 and 7, post fusion, 100 µl Dr. M. RAVI Department of Human Genetics seen under the microscope or upon a change in cultureactively growing cultures on two or three occasions. (in culture flasks). expansion is done byAccustoming the hybridoma cells to serum free (SF)entirely with

SF medium without any adaptation phase; byreducing the serum content slowly over a number of passages-clone in culture. about 0.5 ml volumes, ultra concentrators with a molecularslides. Wells were punched in the gel and scooped. 10 µl day of limitingfinal concentrated supernatants of the 13 clones. As thedata stored.A 50% antigen was prepared as 1:1 v/v ratio of CHO mitoticThe plate was decanted and washed thrice with aspirated after gentle mixing, and limiting dilution wasdone on 96 well plates containing feeder cells (Figure 1)in HAT medium. For the expansions, 13 actively growinghybridomas were aspirated from 96 well plates, and addedinto 2ml HAT in 24 we

ll plates. One ml of supernatantfrom the wells containing actively expanding hybridomaswere collected into clean eppendorf tubes periodically andscreened. The wells were re fed with one ml hybridoma SFmedium (with 10% FBS) and the plates incubated at 37ºC; 5 % CO2. The supernatants collected were concentratedby sucrose dialysis (for larger supernatant volumes) or usingultra concentrators with cut-off of 50K and screened forantibody by Ouchterlony double diffusion (ODD) andELISA.Figure2:Atypical young hybridoma clone stabilizing in cul-ture in the presence of feeder cell.The spherical cells are thehybridomas whereas the cells with extendedmorphology are the feed

er cells. (Inverted Phase Contrast;Magnification 40 X) One clone 20E5 (Figure 2) was chosen for furtherexpansions into T-25 flasks and the supernatant wasconcentrated followed by fractionation of antibodies andfinally to obtain affinity purified Mabs. 8 ml of supernatantfrom actively expanding Hybridoma was collected intodialysis tubing and was placed on sucrose powder in apertidish. The tubing was covered completely with sucrosepowder and left undisturbed at room temperature. The moistOriginal Article in substrate buffer)4 and 7 post fusion as guided by the rate of cell growth.secreted. (Inverted Phase Contrast; Magnification 40 X) color of the medium where t

here is no apparent cellular and regularly observed for the 36Sri Ramachandra Journal of Medicine Nov. 2007 S.No123456789101112131.Supernatant 10 D510 F920 E510 C815 E510 G610 F320 C720 C820 D910 G1015 D410 F52.OD value0.1030.1170.1110.1220.1210.1060.110.1190.1170.1160.1210.1140.115 volume of one ml. The concentrated samples were collectedthe supernatant and anti mouse whole serum indicating thepresence of Mabs of the IgG isotype. The ELISA resultswithin close range. In denotes the supernatant from the wells that containhave been exposed to 10µl of antigen. Figure 7: A semi confluent culture of the Hybridoma clone Original Article situations. Monoclonal anti

bodies (Mabs) on the other immunizations of murine splenocytes whichcheck for a generated immune response in the experimentalreduction in time required for stimulation of eretentatefor the presence of antibodies was done using Ouchterlonyfractionated using ammonium sulphate precipitation methodas cell-lines in culture was optimized by expansions fromoffs that gave us culture supernatants with detectable1.Kohler G, Milstein C. Continuous culture of fused cells2.Edwards PAW. Some properties and applications of3.Payne WJ, Marshall DL, Shockley RK, Martins WJ. 1988;1:313-329.4.McGregor MC. Monoclonal antibodies: production and5.Peters JH, Baumgarten H. Monoclonal

antibodies.6.Hudson L, Hay FC. Practical immunology. 3 ed.7.Howard GC, Bethell DR. Basic methods in antibody 51-53 8.Talwar GP. A Handbook of practical and clinical ed. India: CBS publishers and9.Murakami H, Masui H, Sato G, Sueoka N, Chows T,10.Wilson K, Walker J, editors. Practical biochemistry: ed. Cambridge: Cambridge11.Maddaly Ravi, Sukanya Shyama Sundar, Kaavya Krishna Immunizations of12.Boer MD, Ossendorp F, Duijin GV, Voorde T, Tager under serum free13.Takahashi M, Fuller A, and Hurell GR. Production of14.Borrebaeck CAK. Human mAbs produced by primary immunization. Immunol today 1988;9:15.Borrebaeck CAK. Strategy for the production of human activated B