L23: Antibodies As Tools Of Discovery

Question 1

Antigen

Answer 1

Foreign substance that elicits an immune response

Question 2

Epitope

Answer 2

Antibodies produced against protein

Example: in mixture of individual antibodies, each one recognising and binding to specific part of protein -> epitopes

Mixture of antibodies produced -> polyclonal

Question 3

Hapten

Answer 3

Small molecule does not result in product of specific antibodies

When covalently attached to large molecule (such as protein) acts as antigenic determinant to elicit antibody production

Some of antibodies produced will bind strongly to hapten when free in solution

Question 4

Antibody

Answer 4

Ig proteins produced by B-lymphocytes in response to invasion by microorganisms

Bund specifically and tightly to epitopes on antigens

Question 5

Classes of immunoglobulin

Answer 5

IgG: most abundant class in serum, slow persistent response

IgA: most abundant in mucosal secretions, 1st defence against microorganism invasion

IgM: first-response Ig produced in serum

IgD: normal biological role not clear

IgE: role not clear

Question 6

IgG

Answer 6

4 polypeptide chains: 2 identical Light chains, 2 identical Heavy

Cleavage of IgG with papain -> 2 Fab fragments still capable of binding antigen and being smaller than whole IgG sometimes used for immunocytochemistry in preference to intact IgGs

Question 7

Polypeptide chain of IgG

Answer 7

Each L chain is bound to one H chain. Stabilised by one interchain S-S bond

H chains bound together in interaction stabilised by 2 interchain -S-S- bonds. Whole structure further stabilised by 12 intrachain -S-S- bonds.

Each L chain: 2 Ig fold domains, one constant domain whose AA sequence does not vary between Igs and on variable domain whose AA sequence is variable

Each H: 4 IgG fold domains with 3 constant domains and one variable domain

Variable domains: arranged at tips of Y of IgG & 2 antigen-binding sites of IgG at tips by variable domains. AA of domains -> HB, electrostatic interactions & hydrophobic interactions with antigen -> bind tightly

Question 8

Diversity and variability

Answer 8

IgG genes for Light and Heavy chains assembled by random combo of coding sequences for Variable and Joining segments of light and heavy chains and diversity segments of heavy chains -> diversity of antibodies which can recognise many millions of antigens

In process, nucleotides randomly lost from V, J & D segments -> more variability in final genes

In assembly of heavy chain genes further variability (random addition of nucleotides during combination)

Total introduced variability -> extremely large diversity in AA sequences of variable parts of chains. Variable regions come together in IgG -> introduces final degree of variability in AA sequence & structure of antigen binding site

Combination occur during maturation of B-lymphocytes bone marrow stem cells -> final mature B-lymphocyte has unique genes for Heavy and light

Constant domain of light has 2 possible AA sequences: lamda and kappa

Question 9

Antibody production- polyclonal

Answer 9

Antibodies produced by injecting animal with antigen

After weeks following booster shots with more antigen -> animal is bled; serum containing antibodies spread out. Serum contains mixture of antibodies against epitopes on antigen (polyclonal)

Question 10

Antibody production- monoclonal

Answer 10

Spleen cells (b-lymphocytes) can be removed; cannot be cultured over extended periods -> die out

Mouse myeloma cells: mutated -> cant grow in HAT medium (aminopterin is inhibitor of de novo purine synthesis-> cells cant synthesise own purines for nucleotides & nucleic acid, have deletion mutation -> one of enzymes for purine salvage pathway is missing -> cell cant use thymidine in medium -> cannot grow)

If one of cells fuse with B-lymphocyte -> b-lymph provides missing enzyme -> fused cells or hybrid can grow. So when spleen cell are mixed with mutant mouse myeloma cells -> hybrids form, leave some B-lymph and some mutant myeloma cell

Hybrids survive and grown on HAT medium; non-fused cells die out

Diluting out cell to point where one cell can be placed in well of culture plat (each cell produces unique antibody -> growth of culture from cells gives rise to cell lines -> produce single type of antibody (monoclonal) against single antigen epitope)

Question 11

ELISA (enzyme-linked immunosorbent assay)

Answer 11

Possible if standard curve used

Detection and quantitative assay of HIV

1. Given: HIV envelope protein
2. Prepare rabbit IgG against HIV protein. Attach horseradish perxidase to goat IgG. Goat IgG (reporter protein) against rabbit-antibody
3. Test for HIV. Polystyrene dish. Remove unbound antibody and reporter protein. Colourless substrate added -> colour product form

Result: amount of coloured product formed in fixed time - proportional to amt of peroxidase present in plate (=amt of HIV envelope protein)

Question 12

Western blotting

Answer 12

Detect and identify particular protein in complex mixture

Electrotransfer. Proteins separated by SDS page

Antibody detection: incubate with Ab1 and wash excess. -> incubate with enzyme linked Ab2 and wash excess -> activate colour reaction

Question 13

Immunocytochemistry: fluorescence microscope

Answer 13

Fixing and staining cells with fluorescent antibodies -> method for specifically detecting and locating small amounts of protein in cell -> cellular architecture and distribution of location of proteins can be studied

Question 14

Drug targetting

Answer 14

Extremely high specificity and tightness of Ab-Ag interaction -> cells displaying certain cell surface can be specifically targeted

Thus, mixture of healthy and cancer cells where cancer cells express specific cell-surface marker proteins, antibodies with cytotoxic reagents or radioactive compounds attached bind and kill cancer cells but not healthy cells