ANS/PLSS 433: Antibodies

1. Antibodies
	A. In higher vertebrates, foreign macromolecules (antigens) 
		stimulate lymphocytes to produce antibodies in the form 
		of immunoglobulins that combine specifically with the 
		antigen to facilitate its destruction and removal from the 
		body.
	B. The area of the antigen that the antibody recognizes is called 
		an epitope.  Antibodies can be made from a number of 
		epitopes on a single antigen.
	C. Types of Antibodies:
		a. Polyclonal--Antibodies that recognize a number of 
		   	epitopes to a single antigen.  Usually these types 
			of antibodies are made by injecting an animal 
			3 times with an antigen, and collecting and 
			purifying the blood serum, called antiserum.
			i.  Animals used:	Rabbit
						Sheep
						Donkey
			ii. Polyclonal Abs can also be made to oligopeptides
				--Make more monoclonal like, recognizes one
				  or two epitopes
				--Need linker molecule for lymphocyte 
				  recognition (Keyhole limpet hemocyanin)
		b. Monoclonal--An antibody that recognizes a single 
			epitope of an antigen--commonly abbreviated MAB.  
			These types of antibodies are made in hybridoma 
			tumors.

2. Production of Monoclonal Antibodies from Hybridomas
	A. MABs are derived by fusing B-lyphocytes from the spleen of 
		immunized animal with Hypoxanthine/Guanine Phophoribosyltransferase
		(HPRT) negative mouse myeloma cells, these fused cells are 
		called hybridomas
		a. HPRT-negative cells selected with 8-azaguanine
		b. Polyethylene glycol (PEG) promotes fusion  
	B. Since only fused cells can survive in HAT medium, cell 
		fusions producing just one antibody can be cloned and 
		grown in cell culture, and kept forever producing the 
		same antibody.
		a. HAT=Hypoxanthine, aminopterin, thymidine
	C. MABs can then be overproduced in cell culture or as ascites
		--a suspension of tumor cells that grow within the 
			peritoneum of an isogenic species. 	

3. Applications of MABs
	A. Diagnostics.
		a. Radioimmunoassay (RIA)
		b. Enzyme-linked Immunosorbant Assay (ELISA)
		c. Western Blot
		d. Immunohistochemistry
	B. Protein Purification.
		a. Affinity Chromatography
	C. Bone Marrow and Organ Transplantation.
		a. MABs will allow for better typing of donors.
		b. MABs can be made against endotoxins that are deadly to 
		  	immunsuppressed marrow and transplant recipients.
		c. MABs can be made against T-lymphocytes that can be used:
			i.	to remove donor T-lyphocytes from bone 
				marrow before transplantation.
			ii. 	to prevent rejection of transplants in the 
				recipient. 
	D. Immunotherapy.
		a. Toxins can be coupled to MABs to treat cancers.
		b. Radioisotopes can be coupled to MABs to treat cancers 
			(RAIT)
		b. Enzymes can be linked to MABs to treat conditions such 
			as blood clots
	E. Diagnostic Imaging.
		a. Radioisotopes are coupled to MABs diagnose certain 
		   	disease states.
	F. Catalytic Antibodies.
		a. Antibody acts like an enzyme or ligand (hormone).

4. Genetically engineered MABS

	A. Genetic engineering has made it possible to manipulate MABs
		a. V (variable), J (join), and D (diversity) domains 
      			in variable region can be manipulated
		b. C (constant) region can also be manipulated somewhat
	B. Humanized MABs
		a. MABs from human source would be advantages to mice
			i.	Prevent cross-reactivity
			ii.	Immunosensitivity--especially with multiple
				treatments
		b. Production of human MABs has been difficult
			i.	Fusion of human lymphocytes with mouse 
				myeloma is unstable and success is 
				very rare.
			ii.	No HPRT negative human myelomas have been 
				discovered
			iii.	Ethical reasons--can't immunize humans 
				against a variety of antigens
		c. Hybrid MABs have been made using genetic engineering
			i.	Fc region made human by gene insertion
			ii.	A chimeric Fab region can be made by 
				inserting human genes
			iii.	PCR has been instrumental in making this 
				possible