Antibody production

The immune system  is  designed  to  ward  off  “foreign”  invaders. Injection of a foreign molecule, usually a protein, into an animal elicits an immune response, which includes the  production of antibodies which react with the foreign protein and target it for removal  from  the  system. The foreign protein in this context used to be called an antigen, from antibody generator, but the terminology has changed and now the molecule which elicits antibody production  is called an immunogen  and the molecule with which the antibody reacts is called an  antigen  (Fig. 1).

The terminology changed when it was realized that  although the immunogen and the antigen are often the same molecule, sometimes they are not.  Also, some molecules are antigenic (i.e. they react with antibodies) but are not immunogenic (i.e. they will not elicit antibody production when injected into an  animal).  The  current  hypothesis  takes account of the fact that the immunogen and the antigen may or may not be the same molecule.

Figure 1.  The relationship between immunogens, antibodies and antigen.

Many small molecules will not by themselves elicit antibody production but when conjugated to a larger molecule they will elicit  antibodies,  which will react with the unconjugated small molecule.  Such small molecules are known as haptens. The cut-off in  size is not  absolute and experience with peptide  antibodies (antibodies elicited by peptide immunogens) suggests that  it  may be that smaller molecules simply take  longer to elicit antibodies.  For example,  free  peptides  are  often  able to  elicit antibodies, but the antibody titre takes longer to rise than when a peptide conjugated to a carrier protein is used as the immunogen.

A single injection of an  immunogen  is not  optimally  effective  in eliciting antibody production.  In  a  natural  infection,  molecules  from  the infecting agent will leak from the site of infection  to  become exposed to the immune system in small amounts, but over an extended time.  To elicit antibodies, this  natural  process  must be mimicked.  One way would be to  inject a small amount  of immunogen  at a time  and  to  repeat  the  injection many  times over a time period.  This  for exposure to the immune  system  in  small would work, but it would subject the  animal  to  unnecessary  trauma,  which  is  ethically unacceptable. Another way would be to make an emulsion of the aqueous antibody solution with an adjuvant oil and inject this subcutaneously or intramuscularly. The emulsion is made by a process known as trituration. The injected emulsion would exist at a focal site, mimicking a natural infection, and would break down slowly over time, thereby slowly releasing the  immunogen  and exposing  it to  the  immune  system  over  a period. This is the principle of Freund’s  incomplete  adjuvant.  However, the immune system is particularly geared to combating microbial infection and its response is stimulated by the presence of components  of the bacterial cell wall.  Freunds  complete adjuvant thus contains  bacterial cell wall components, in addition to an emulsifying oil. The first injection of an immunogen gives a relatively  small response and the  antibodies produced are of the  high molecular  weight IgM type (IgM antibodies are comprised of five subunits, each equivalent to a single IgG molecule, joined  together).  This  is  known  as  the  primary  response. Further inoculations  with the  same  immunogen  gives a much greater secondary response, in which mainly IgG-type antibodies are produced (Fig. 2).

Antibodies arc made by a class of white blood cells (leukocytes) known as B-cells. In any one animal there are a large number of different B-cells,  each  capable of making  a single type  of antibody  molecule.  Each B-cell carries an “advertisement” γ-globulin on its surface  and interaction of this  molecule  with  an  antigen  molecule  causes  that  B-cell to divide into a clone of similar B-cells, a process known as clonal expansion.  Some of these B-cells mature  into  antibody-producing plasma cells  and some remain as memory cells.  The existence  of an  expanded  clone  of memory cells accounts for the faster and more extensive secondary response.

Figure 2. Time-course of the immune response. Arrows indicate inoculations with immunogen.

Making  an antiserum

An antiserum, or an antibody isolated from  it,  constitutes  a useful reagent in protein biochemistry.  However, it is a reagent  which is specific to  the  immunogen/antigen  couple  and often  it must be prepared in-house, especially when a novel  protein  is under investigation Preparation of an antiserum  starts  with the  immunogen, which is usually a protein  isolated by one  or more  of the  methods  described in the previous chapters.  The  more  pure  the  immunogen,  the  more  specific  will be the antibodies which it elicits.  For  most  purposes,  therefore,  it  is best to use as pure an immunogen as possible.

Alternatively, for the production of so-called peptide  antibodies,  the immunogen might be a synthetic peptide of ten or more  residues.  The peptide is chosen from the amino acid sequence of the Ag  of interest,  i.e. the complete protein which it is hoped the  Abs will recognize.  For peptide antibodies to recognize the whole protein, it is necessary that  the peptide sequence chosen be accessible, i.e. it must be on the surface of the protein. This can be readily determined if the 3-D structure of the protein is known.  If the  3-D structure is not  known,  then  the  peptide can be chosen by analysis of the  amino  acid sequence of the  Ag of interest for hydrophobicity (since hydrophilic residues will tend  to  be on the surface) or mobility (since residues on the  surface, and especially at the N- or C-terminus are likely to be more mobile).

For inoculation  into  an animal, the  immunogen must be emulsified with Freunds complete  adjuvant and this  can  conveniently  be done  by trituration in an apparatus such as shown in Fig. 3.  In this device, the solutions are  emulsified by passage back and forth  between two  syringes, through a fine stainless steel mesh.

Figure 3.  A device for emulsifying antibody solution with adjuvant oil.

The triturated immunogen/adjuvant emulsion may be injected subcutaneously in a rabbit, or into the  breast muscle of a laying hen. Animals have an idiosyncratic response to  immunogens and so it is best to use at least two animals, in case the one is a poor responder   this is provided sufficient immunogen  is available, of course.  A typical inoculation schedule would involve  injection of 50-----100 µg of protein per time, first  in  Freund’s  complete  adjuvant,  followed at  one,  two,  four and six weeks thereafter  by further inoculations  in  Freunds  incomplete adjuvant. If necessary, further booster inoculations can be given at monthly intervals.  In the case of rabbits, blood samples are taken immediately before each inoculation, so that the  increase in antibody titre with time  can be followed.  An  illustrative  example  of the  increase in antibody titre with time is shown in Fig. 4.

Figure 4.  A typical ELISA of the progress of an immunization. The open triangles represent preimmune  serum and the other three  symbols represent serum at 2,4 and 6  weeks.

An advantage of using hens for antibody production is that  it  is not necessary to bleed them  in order to harvest the  antibodies.  It  is, of course, necessary to bleed rabbits and this may be done by warming one of their ears with  a hot,  wet towel,  in  order to  dilate the  blood vessels,  and nicking the  peripheral  ear vein with a sharp  scalpel blade.  About 25  ml can be collected from  a rabbit at one time.  The  blood is best collected into a clean,  dry 25  ml conical  flask as this  has an almost  optimal  ratio of volume to surface area and the  exposed area is also minimal.  With  a high volume to surface area ratio, the clot can more  easily contract  away from the vessel walls, thereby obviating tearing of the clot and lysis of the red blood cells.  Optimal clot  formation  is promoted  by incubation overnight at 4C.  Ideally, no haemolysis should occur and the  serum should be  a  pale  straw  colour.  It  may  be  harvested  by  careful  aspiration with a Pasteur pipette.

An IgG preparation  may be isolated from the  antiserum,  or IgY isolated from egg yolks, by precipitation with polyethylene  glyco .

References 

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .