MONOCLONAL ANTIBODIES What are they? How are they produced? What potential uses?

MONOCLONAL ANTIBODIES : Uses Medical diagnostic tests Cancer treatment : targeted delivery of cytotoxic drugs Transplant surgery: knock out specific T cells that would cause rejection if left active Immunoassay : Drug / Pregnancy testing Separation of a chemical from a mixture by labeling it

Text pages Copy figure 1 and label the antibody molecule

Epitopes Often, antigens have more than one binding site. This means that several antibodies can be raised against it. Also, membranes generally have several different antigens on their surface so a range of antibodies will be produced. How can you target ONE particular antigen? Pathogen A Pathogen B Antigen

A membrane will have more than one single antigen to which T helper cell surface proteins can bind. Alternatively, some antigens may more than one binding sites (‘epitopes’)

T2 T1 T3 A membrane will have more than one single antigen to which T helper cell surface proteins can bind

T2 T1 T3 All of the bound T-helper cells become ‘ACTIVATED’ Animation of monoclonal antibody production Activated Look at these great animations

Therefore, several DIFFERENT antibodies will be produced against each pathogen (= POLYCLONAL) Animation of monoclonal antibody production TO TARGET AN ANTIGEN ACCURATELY, we need ONE SPECIFIC antibody. We need to produce what is called a “MONOCLONAL” antibody. Antibody from activated B1 Antibody from activated B2 Antibody from activated B3

Diagram of sequence for monoclonal antibody production

Preparing monoclonal antibodies SEQUENCE THE FOLLOWING IN THE CORRECT ORDER Mouse immune system stimulated to produce antibodies against antigens on the pathogen’s membrane (there will be several different antibodies at this stage = polyclonal) Each hybridoma cell line produces the antibody from the B cell it fused with Inject pathogen against which antibody is to be raised into a mouse Antiserum (blood serum rich in antibodies) extracted from mouse. Hybridoma colonies are screened to identify those which produce the desired antibody. Clone selected and cultured to produce the “Monoclonal Antibody” Mouse B cells fused with tumor B cells to create a immortal cell lines for producing antibodies (called an ‘hybridoma’ or ‘myeloma’). Therefore some colonies will produce the desired antibody. Hybridoma cells allowed to multiply in-vitro on nutrient medium. A B C D E F G H I

Preparing monoclonal antibodies 1.Inject pathogen against which antibody is to be raised into a mouse (C) 2.Mouse immune system stimulated to produce antibodies against antigens on the pathogen’s membrane (there will be several different antibodies at this stage = polyclonal) (A) 3.Antiserum (blood serum rich in antibodies) extracted from mouse.(D) 4.Mouse B cells fused with tumor B cells to create a immortal cell lines for producing antibodies (called an ‘hybridoma’ or ‘myeloma’). (G) 5.Hybridoma cells allowed to multiply in-vitro on nutrient medium. (I) 6.Each hybridoma cell line produces the antibody from the B cell it fused with (B) 7.Therefore some colonies will produce the desired antibody. (H) 8.Hybridoma colonies are screened to identify those which produce the desired antibody. (E) 9.Clone selected and cultured to produce the “Monoclonal Antibody” (F)

Monoclonal antibodies for cancer treatment One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cells specific antigen and induce immunological response on the target cancer cell (naked antibodies). mAb can be modificated for delivery of [[toxin], radioisotope, cytokine or other active conjugatecancerantigen

ELISA (Enzyme Linked ImmunoSorbant Assay) For diagnosis of diseases (test for presence of antigens in blood samples) Mechanism: The basic mechanism involved in these test utilizes absorption of antigen to a solid substrate which is placed in contact with a dilution of serum. The reaction which detects and quantifies the binding of antibody uses an antibody labelled with an enzyme followed by the addition of an appropriate substrate on which the enzyme can act to produce a colour reaction.

Why is it ESSENTIAL to rinse the well with distilled water between steps 2 and 3? (both mAb-1 and mAb-2 have been raised against the different epitopes (binding regions) on this antigen for the pathogen that is being tested for) Using the key, complete the flow diagram

Why is it ESSENTIAL to rinse the well with distilled water at the times stated? Using the key, complete the flow diagram Add serum to all wells Rinse all wells with distilled water (both mAb-1 and mAb-2 have been raised against the different epitopes (binding regions) on this antigen for the pathogen that is being tested for) If the sample contains the antigen to which mAb-1 has been raised is added, it will bind to mAB-1 Rinse wells again BEFORE adding enzyme substrate Add mAb-2 that has been raised against the antigen. mAb-2 has a reporter enzyme attached to it Positive result is generally a colour change of the well with the desired antigen

Antigen Monoclonal Antibody Anti-mouse Antibody with Marker, tag or enzyme attached Monoclonal antibody uses : Use page 110 and the Fact file to list medical applications of monoclonal antibodies

Text pages List the four main applications of monoclonal antibodies Produce a table highlighting the benefits and disadvantages of this technology. Include as many ‘ethical’ issues as possible. Answer exam style Q 3 on page 117.

Answer to exam style Q3 p116 – 3 (a) injection of antigens; –stimulates the formation of memory cells; (antigen from) attenuated microorganism/non-virulent microorganisms/dead microorganisms/isolated from microorganism; 2 max The first two mark points are sufficient. You do not need to know the different types of vaccines described. –(b) (i) antibodies are specific to mumps antigen; secondary antibodies specific to mumps antibody;1 max The important idea is the specificity of the antibody to the antigen. –(ii) removes unbound secondary antibodies; otherwise enzyme may be present/may get colour change anyway/false positive;2 –(iii) no antibodies to bind (to antigen); –therefore secondary antibody (with the enzyme attached) will not bind; no enzyme/enzyme-carrying antibody present (after washing in step 4); 2 max Ensure you are very precise in your answer, particularly when referring to antibody and secondary antibody. 3a 3b(i) 3b(ii) 3b(iii)

Resources for animations on monoclonal antibody production nimations/content/monoclonalantibodies.ht mlhttp:// nimations/content/monoclonalantibodies.ht ml