Monoclonal antibodies and their applications

Various diagnostic tools now a days relied on the Hybriodoma technology and monoclonal antibodies,so this presentation will give some basic information about mAb and Hybridoma technology.

1. Monoclonal antibodies . Hybridoma technology Asst.prof . Dnyaneshwari Joshi. Department of Biotechnology and winetechnology

2. First immortal monoclonal Ab’s produced by George Kohler and Cesar Milstein in 1975 They shared Nobel prize for this discovery in 1984 The term Hybridoma was coined by Leonard Herzenberg in 1975. HISTORY..

3. ANTIBODY: It is a soluble globular protein ( Glycoprotein),Which is produced in response to a specific antigen and binds via non-specific interactions . The term Immunoglobulin is used to describe any Antibody regardless of its specificity , whereas Antibody describes ANTIGEN-SPECIFIC immunoglobulin. MONOCLONAL ANTIBODY( MAb ) : Single type of Antibody which is directed against specific type of antigenic determinant (epitope) is called as monoclonal antibody.

4. Monoclonal antibodies…. Antibodies produced from a single clone of B cells. Produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture. Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same epitope with the same affinity. They are all of the same antibody class ( isotype ).

5. IMMUNOGLOBULIN STRUCTURE.

6. Murine antibody. 1.Whole of the antibody is of murine origin Major problems associated with murine antibodies include  reduced stimulation of cytotoxicity  Formation of complexes after repeated administration  allergic reactions. 2. Chimeric antibody. 1.Chimeric antibodies are composed of murine variable regions fused onto human constant regions. 2.Antibodies are approximately 65% human. 3.Humanized antibody. 1.Humanized antibodies are produced by grafting murine hyper variable amino acid domains into human antibodies. 2.This results in a molecule of approximately 95% human origin

7. Hybridoma technology A hybridoma is a hybrid cell obtained by fusion of B lymphocyte with usually a tumor cell of antibody forming system or B lymphocyte (these are called myelomas)

8. The hybrid cell has the capacity of antibody production derived from B cells At the same time it can divide continuously by the quality derived from Myeloma cells By combining the desired qualities of both the cells, the technology ensures large scale Antibody production of single specificity Specific hybridomas are either cultured in vitro or passed through mouse peritoneal cavity to obtain monoclonal antibodies, this is called as hybridoma technology PRINCIPLE

9. 1.Isolation of B cells -Mice , 2-4 weeks old are immunized with the antigen against which monoclonal antibodies are to be raised by subcutaneous injection -Later B cells are isolated from the spleen of an immunized mouse 2.Isolation of myeloma cells -Myeloma cells are isolated from bone marrow -The myeloma cells used are HGPRT(Hypoxanthine-guanine phosphoribosyl transferase ) mutant cells ( raised by mutations using 8-azaguanine ) STEPWISE PROCEDURE

10. This Cell Line Is Deficient In HGPRT ( hypoxantine guanine phosphoribosyl transferase ) Alternatively TK (thymidine kinase deficient) Cell Line Cannot Survive In Selection Medium Aminopterin Inhibits “ De novo Pathway”, “Salvage Pathway” Is Not Possibe Due To HGPRT or TK Deficiency It Is Also Ig Deficient It can not secret any immunoglobulins Aminopterin (folic acid antagonist) Blocks De novo Pathway P3.653 cells die in the presence of aminopterin They cannot utilize the “salvage pathway” because they are HGPRT deficient P3.653 Myeloma

11. Somatic cell fusion - Electrofusion : cells are allowed to fuse with the application of an electric field -Done by using PEG medium -PEG stands for Poly Ethylene Glycol The New Hybrid Cell Exhibits Properties Of Both Cell Types Unlimited growth Secretes monoclonal antibody Or Secretes cytokines

12. Selection of hybrid cells -HAT medium is used for the selection of hybrid cells -HAT stands for Hypoxanthine Aminopterine Thymidine Nucleotide synthesis is essential for cell survival In HAT medium, aminopterine blocks the cellular synthesis of purines and pyramidines from simple sugars ( denovo pathway) But cells can succeed by using hypoxanthine and thymidine present in the medium by salvage pathway using the enzyme HGPRT

13. -B cells are HGPRT+ and can survive in the HAT medium, but they undergo normal cell death after some division - In hybridoma technology, the myeloma cells used are HGPRT deficient -So these cells can’t survive in HAT medium as Aminopterine blocks the Denovo pathway Hybrid cells has HGPRT enzyme from the B cell as well as they have the ability to multiply repeatedly as myeloma cells So only hybrid cells can survive in HAT medium How HAT medium works in the selection of hybrid cells?

14. Identification and isolation of the hybridoma cells • The first screening technique used is ELISA -Done by incubating the hybridoma culture supernatant, secondary enzyme labeled conjugate and chromogenic substrate -Formation of a coloured product indicates a positive hybridoma

15. DIAGNOSTIC APPLICATIONS A. Biochemical Analysis B.Diagnositic imaging ELISA Eg . HIV test Generally used Radioisotopes are Iodine 131, technetium 99, Indium chloride 111. 1. Radiolabel the specific monoclonal antibody. 2. Inject them intravenously into the patients. 3. Detection by using SPECT(Single Photon Emission computed tomography). Used in detection of cancer, Bacterial infections , Heart attack etc.

16. Metastatic medullary Thyroid carcinoma . Patient was injected with Anti-CEA bispecific antibody with indium111, and immunoscintigaphy was performed after 4 days. Liver , Bone and Brain shows good uptake of labelled Antibody which shows metastatic tumour .

17. Therapeutics 1.Direct 2.Targeting agents Eg . As immunosupresent in organ transplants. OKT3 monoclonal antibody specifically directed against CD3 antigen of T lymphocyte. The toxins can be coupled with MAb’s to form immunotoxins .Anti –IL2-R conjugated with exotoxin of Pseudomonas sp. Can be used to destroy malignant T cells of T cell leukemia Some drugs , radioisotopes can be also attached to MAb’s and carried out towards the specific tissue for efficient actions

18. Thank you