Proteomics

Antibodies

• Campath-1H
• Humanized monoclonal Ab against CD-52 antigen in malignant leukemia cells.
• CD52 unknown function, expressed on >95% B+T cells.
• CD52 not expressed on hematopoietic stem cells so Ab wont attack here - over time new b/t cells can grow healthily from bone marrow.
• Patients are lymphopenic in 2-4 weeks (compromised) - potential use from transplants also.

Epitopes.

• Antibodies only recognize small sites on an antigen called epitopes. These sites bind to membrane receptors of B/T cells/Ab.
• Some large molecules can have multiple epitopes eg. P38 - multiple B-cell clones can be produced to recognize these epitopes - resulting Ab's are heterogenous (polyclonal).

Polyclonal Ab production.

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• Caption: (chromotography) Immunoadsorbent is is solid matrix (agarose gel) & coupled antigen. Antiserum is passed over immunoadsorbent. Abs in the mix specific for antigen and will bind and be retained. Antibodies of other specificities will pass through. Reagent is passed through to remove Abs from the imunnoadsorbent. High salt/low pH usually used to break the noncovalent bond between Ab and An.
• Polyclonal serum and monoclonal serum in sds page (polyacrylamide gel electrophoresis) to determine Ab's present.

Epitope types.

• Discontinuous (conformational) - composed of amino acids from different parts of a polypeptide chain
• Continuous (linear) - composed of amino acids from a single segement of a polypep chain.
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Polyclonal Ab's pt.2

• Ab's generated after immunization are mix of specificities. ~10% of Ab's from immunization will be specific for Ag of interest
• Polyclonal Ab's for Ag has useful properties in biology but disadvantages which relate to its heterogeneity:
• Each antiserum is different even in genetically identical animals using the same method and antigen.
• Limited volumes of polyclonal antisera.
• Even after separation by affinity chromatography Ab's with cross reactions may persist. 2 epitopes could look similar and give a false positive.

Monoclonal Ab's (MAb's)

• To reduce problems & harness full potential of Ab's - method of producing unlimited supply of homogenous structure and known specificity.
• MAb's are from a single B effector cell and grow indefinitely. Early work shows B cells effected by myeloma produced large amounts of homog Ab's ---> fusing immunized spleen cells with myeloma (or cancer) cells.
• These hybrid cells are called hybridomas. Can proliferate indefinitely and produce Ab specific to An used to immunize animal.

MAb production.