Block A -1

Question 1

what are the Fc regions on antibodies ?

Answer 1

The Fc regions are what communicates with the host cells.

Question 2

binding regions how many and function ?

Answer 2

2 binding regions which can recognise antigens

Question 3

structure of antibodies ?

Answer 3

Antibodies are made up of 4 chains , 2 heavy and 2 light chains with a variable region ( antigen binding site) seen above. The constant region makes up the Fc component. Disulphide bonds are present between the 2 chains.

Question 4

when are antibodies produced ?

Answer 4

Antibodies are produced as part of the immune response , they can eliminate bacterial toxins through neutralisation , bacteria in extracellular space through opsonisation and bacterial in plasma through activation of the complement system. BUT Antibodies also have their uses in research, diagnostics and treatment due to their ability to specifically bind to stuff (antigen!)

Question 5

how are antibodies produced ?

Answer 5

B cells binds virus for example through the viral coat protein - viral particle is internalised and degraded - peptides from the internal proteins of the virus are presented to the T cell which activates the B cell - activated B cell produces antibody against the viral coat proteins.

Question 6

what is antibody production reliant on ?

Answer 6

B cells with T cell help

Question 7

if using an antibody in an immunoassay as an analyte what is needed in terms of describing the antibody ?

Answer 7

If using antibodies to specifically recognise an analyte in an immunoassay, we need it to be as specific as possible and have a high avidity which is strength of binding.

Question 8

how does the occur ?

Answer 8

This occurs naturally due to somatic hypermutation , class switching and gene conversion.

Question 9

what does multiple immunisations do for the antibody ?

Answer 9

Multiple immunisations increases both the amount and affinity of Ab in the serum of immunised animals. Also results in class-switching (more likely to be IgG/IgA).

Question 10

first antibody response then after some time what is the Ab response ?

Answer 10

The first response is to produce IgM and after some time IgG is produced. If there are a number of immunisations then class switching occurs towards IgG.

Question 11

considerations for immunisation ?

Answer 11

Antigen used for antibody production

Adjuvant to increase the effectiveness of the vaccine

Kinetics of when you want to immunise and harvest serum.

Question 12

immunologically primed ?

Answer 12

Once animals are immunologically ‘primed’ (ie. mounted an Ab response), they have high levels of specific Ab in the serum.

Question 13

occur after immunological priming ?

Answer 13

Can therefore collect the blood, separate serum and use this antiserum for future tests, treatments.

Question 14

convalescent serum ?

Answer 14

convalescent serum , is taking the serum with antibodies produced from a patient who has already generated immunity against the infection. There is a limited supply however.

Question 15

can animals only be bleed once and advantages and disadvantages?

Answer 15

Can repeatedly bleed animals, allowing plentiful source of antiserum , . can collect several litres of antiserum from an immunised cow every month.

Relatively cheap and easy approach, allowing generation of polyclonal antibodies.

BUT

Serum contains many other proteins, which may cause problems in sensitive assays (eg. cross-reactivity). Therefore, better to purify Abs for improved storage and higher concentration to avoid cross reactivty in assays.

Question 16

problems with polyclonal antibodies ?

Answer 16

Lots of different classes and isotypes of Ab present

Potentially different specificities against different parts of the molecule

Potentially contaminating antibodies that react with other proteins

Sometimes need reagents that are much more specific & well characterized especially for injecting into patients!

Question 17

how are monoclonal antibodies produced ?

Answer 17

These are identical antibodies that are produced through immunising an animal normally a mouse but can be a rat which fuses the B cells with a tumour cell, so that the B cell is immortal, these cells can then be clonally expanded to be used indefinitely to produce antibodies.

This was first described in 1975 by Kohler , Milsten and Jerne.

Question 18

advantages to monoclonal antibodies ?

Answer 18

They can be produced in large quantities without the need for repeated bleeding of mouse/sheep/cow/etc. Furthermore, they bind very specific epitope of an antigen. They are the exactly same Ig class and isotype and can be highly purified. This allows for in-dept.

Question 19

phage display of monoclonal Ab ?

Answer 19

This is a recent development using bacteriophages to screen and produce monoclonal Antibodies. The first step is isolating a population of genes encoding for the antibody variable regions. Then a construct fusion protein of the V region and a bacteriophage coat protein. There is cloning of a random population of variable regions which gives rise to a mixture of bacteriophage and a phage- display-library. Then a select phage with the desired V region by specific binding to an antigen.

Question 20

advantages of phage screening ?

Answer 20

This allows rapid screening of many more V region-expressing clones against antigen of interest, increasing sensitivity and likelihood of success. Some versions of this method allow production of monoclonal Abs by bacteria (lots of proteins, cheap, but not necessarily correct folding). Others subsequently use genes from positive clones and express into hybridoma (allows production of mammalian/humanised monoclonal Abs).