Amplification methods

At low levels of Ab and Ag, no visible precipitation  may be formed. In modem methods  of immunological analysis, therefore,  use is made of amplification methods which enable the  Ab/Ag reactions to  be visualized and quantitated.  Amplification  methods  will  be  discussed  here  in  more-or-less historical order. the enzyme and immunogold methods  being more recent.

1 . Complement fixation

Complement is  a name given to a group of serum proteins which bind Ag/Ab complexes and cause lysis of cells displaying  surface  antigens. Complement thus functions in vivo  as part  of the  immune response, aimed at the  lysis of foreign cells.  This activity  is exploited in the complement fixation assay, which is a sensitive  means of detecting Ag/Ab complexes.  The  complement  fixation  assay  is  about  100-fold more sensitive than the precipitin reaction.

Figure 1. The complement fixation assay.

The complement fixation assay depends on the fact that  complement is consumed (fixed) by Ag/Ab complexes,  making less complement available. The amount of complement left (i.e. not fixed by the Ag/Ab complexes) can be measured by its ability to lyse red blood cells sensitized by antibodies binding to  surface  antigens.  The  amount  of  lysis  can  be quantitated by measuring the  haemoglobin released into  the  supernatant, by its absorbance at 541  nm.

If all  of the  complement  was  bound  by  Ag/Ab  complexes,  none  would be available to  bind to  the  red blood cells and no  lysis, and consequently no release of haemoglobin, would occur.  On the other  hand,  if no  Ag/Ab complexes were formed, no complement would be fixed, so all of it  would be available to  lyse  red blood cells and the  absorbance of the  released haemoglobin would be at a maximum.

In order to measure the amount of a specific Ag present  in a complex mixture, it is necessary to first establish a standard curve by measuring the complement fixed when different amounts of Ag are added to  a fixed amount of complement and Ab.  The standard curve (Fig. 2) has a shape similar to that of an immunoprecipitation  curve.

Because of the shape of the standard curve, two different Ag concentrations can give the same degree  of complement  fixation  (dotted lines in Fig.  2).  In  measuring  an  unknown, therefore,  it  is  necessary  to test several dilutions of the Ag to establish on which side of the  curve  the Ag concentration falls.  The  [Ag]  can then be  read off from  the  standard curve.

Figure 2.  Standard curve for complement fixation.

The method has two principal disadvantages:-

•  Certain crude mixtures  cause haemolysis by mechanisms  unrelated  to complement, and,

•  Some crude  mixtures  inactivate  complement  in  the  absence  of  the appropriate Ag/Ab complex.

2.  Radioimmunoassay (RIA)

Radioimmunoassays (RIAs) combine the  sensitivity  of  radioisotope detection with the selectivity of immunoassays.  RIAs can be used to detect molecules that  do not  fix complement  when combined with a specific antibody,  for  example  haptens,  and RIAs are mostly used for the assay of small molecules.  Examples  of compounds  that  can be assayed by RIA are peptide hormones,  steroids (such as testosterone), cyclic AMP,  morphine,  digitalis  etc.

The principle of the  RIA is the  same  as  that  of the  chemical  assay technique known as an  isotope dilution assay.  In an isotope dilution assay a known amount of a radioisotope,  of known  specific activity  (i.e radioactivity per unit mass),  is added to a sample and a pure  sample of the element of interest  is subsequently extracted  and its specific activity determined. The decrease in the specific activity of the isolated element is due to  the  presence  of the  non-radioactive isotope which dilutes the radioactive isotope.  From  the  extent  of  this  dilution,  the  concentration of the  endogenous,  non-radioactive isotope can be determined.

Figure 3. Radioimmunoassay.

A RIA  is illustrated  in  Fig.  3.  In  a  RIA,  a  radio-labelled antigen  is used to dilute an unknown amount of an unlabelled antigen, present in the sample. A non-saturating amount of,  say, rabbit antibody to  the  antigen is added (i.e. the  antigen  must  be in  excess).  The  labelled and unlabelled antigens will bind to the antibodies in the same ratio as they are present in the  sample.  The  Ab/Ag  complexes  can  then  be  precipitated,  for example by addition of a goat anti-rabbit IgG antibody.  The radioactivity in the precipitate will be inversely related to the amount of the antigen originally present in the  sample.  The  concentration  of the  unknown antigen in the sample can thus be measured by reference to a straight line standard curve in which the  % inhibition  is plotted  against log[non-radioactive Ag].

Radioimmunoassay has the following disadvantages:

•  A radioactively  labelled Ag  may  not  be  available, especially  for  an antigen which has not been extensively studied.

•  Associated with the  radioimmunoassay  are  all of  the  hazards of radioisotopes, which means that specially equipped laboratories and special licenses are necessary.

3 .Enzyme amplification

The advantage of enzyme methods over radioisotope methods of amplification is that, because enzymes are safe and biodegradable, no special licences or safety facilities are required, and disposal after use is no problem.

3.1 Enzyme linked immunosorbent assay (ELISA)

The ELISA method was introduced in its modern form by Engvall and Perlmann and van Weemen and  Schuurs. The principle of an ELISA is that an enzyme, linked to an immunoreactive molecule (an antibody  or protein A) can be used to detect the presence of an antigen with great sensitivity, due to the  amplification  achieved by the enzyme catalyzed reaction. The method can also be turned  about and used to  detect Ab. Several different  formats  of ELISA are  possible.  Only  some  concepts pertaining to ELISAs are discussed here.  For details on how to  conduct an ELISA, a specialist text should be consulted.

The competitive ELISA for measurement of Ag  is conceptually  similar to a RIA,  in that  a labelled Ag competes  with an unlabelled Ag for binding to  the  antibody,  but in  this  case  the  label  is  an enzyme (Fig.  4).

Figure 4. A competitive ELISA for measuring [Ag]. Unknown [Ag] is proportional to the absorbance difference between the wells A and B.

Antibody is immobilized by adsorption  onto  the  walls of a plastic microtitre plate.  Excess antibody is washed off and the  enzyme-linked Ag is added to  one  set of wells while the  unknown  sample,  mixed  with enzyme-linked Ag is added to another set.  The  unknown  sample Ag thus serves to  dilute the  amount of labelled Ag which  reacts  with  the immobilized Ab  After  incubation,  excess  antigen  is  washed  off  and  a solution of the enzyme substrate is added.  The enzyme catalyzed reaction generates a colour which can be measured.  The  intensity  of this colour is  inversely  proportional  to  the  amount  of Ag  originally  present. The microtitre plate contains 96 wells in an 8 x  12  array,  which permits the  exploration  of a number of different  combinations  of coating  and Ab/Ag concentrations, permitting optimization of the reaction.

In another format,  related to  immunoblotting  ,  the Ag (usually a solution  containing  a mixture  of proteins,  including the protein of interest) may be  adsorbed onto  the  walls of a microliter  plate and the Ag of interest subsequently  detected  with  an  enzyme-labelled Ab. For increased sensitivity,  further  amplification  may  be obtained  by using two antibodies,  an  Ag-specific primary  Ab and an enzyme-labeled secondary Ab that  is specific for the type  of primary Ab.  This has the advantage that  the  secondary enzyme-labeled secondary Ab can be a universal reagent, targeting primary Abs of the  same type  but with different specificities.

3.2 Immunoblotting

In one ELISA format the principle of amplified detection  of  an immobilized Ag using an enzyme labeled Ab is used.  The Ag, in  this  case, may be coated onto  the walls of a microtitre  plate.  A similar principle applies to  immunoblotting,  whereby Ag immobilised on  for  e.g.  a nitrocellulose filter, can be detected using an enzyme-linked antibody system (Fig.  5).  An  example  is dot blotting, in which a small volume of the  antigen  of  interest  is  dotted  onto  a  nitrocellulose  filter. The surrounding protein-binding sites may be blocked with milk proteins, which do not tend to bind proteins non-specifically. The dot can then be probed with a primary  antibody,  followed by an  enzyme-labelled secondary antibody, as in an ELISA.  The  only difference  is that  in  an ELISA, the  enzyme  product  is  soluble,  whereas  in  an  immunoblotting  an insoluble product is necessary.  A  commonly  used  combination  is horseradish peroxidase as the  enzyme  label, with  4-chloro- l-naphthol as the substrate.  The blue/grey product is insoluble in water. Alternatively, a gold-labelled Ab or protein A may be used.

Figure 5.  A schematic sketch of immunoblotting.

A related technique is western blotting. The curious name  of this technique is due to  a pun.  A technique  for the  blotting of DNA fragments, and probing these with labelled RNA, was named Southern blotting after  its originator,  E. M. Southern.  The  reverse process:

blotting RNA and probing this with DNA fragments,  was then  named northern blotting,  to indicate that it was an opposite process.  The blotting of proteins was subsequently called “western blotting” to indicate a similar process but with different molecules, i.e. in a different direction. In western blotting, proteins are first separated  by SDS-PAGE. The separated proteins are then drawn out of the gel, and blotted onto nitrocellulose, by transverse electrophoresis,  forming a replica of the gel separation (Fig.  6). In this process the nitrocellulose sheet must be on the anodic side of the gel, as protein/SDS complexes are negatively charged and have an anodic migration.

Within the gel the proteins are not accessible to probing by antibodies, but they  become accessible after electroblotting  onto  the surface of a nitrocellulose sheet.  Antibodies bound to the blotted proteins can be detected with an enzyme-labelled secondary antibody, as in a dot blot.

Figure 6.  Electroblotting.

Treatment with SDS tends to denature proteins, although the effects can be minimized if the  sample is not boiled in SDS. The denatured protein might not be recognized by the antibodies used to probe the  blot.  In this case a renaturing  blot  system, can be used to advantage.  In this technique the transfer buffer does not contain SDS  and the SDS  may  be  removed  from  the  gel  before  the  transfer  step.  The nitrocellulose sheet must be placed on the  appropriate  side of the gel, depending on the direction of migration  of the  protein  at the  pH of the transfer buffer.  Normally  a high pH  is used, giving an  anodic  migration, but this is not appropriate if the protein is not stable at a high pH.

In a gel of constant  composition,  proteins  separate  by virtue  of their differential rates of migration,  smaller proteins  migrating faster than large ones.  This same differential will apply to the migration  of proteins during the  transfer  step,  i.e.  smaller  proteins  will  transfer  to  the nitrocellulose more rapidly.  If insufficient time  is allowed for the transfer, the blot will be biased in favour of smaller proteins.  This  effect can be overcome  by running the  first electrophoresis  in a gradient gel.  In  this  case  the  proteins  will  each  reach  a  point  in  the gradient where they  are  about equally impeded by the  gel and during the lateral transfer step they will all migrate out of the  gel at  about the  same rate.

Western blotting is useful for determining the presence or absence of a specific Ag in a complex mixture.  It is also useful for testing  the specificity of an Ab, before this is used in immunocytochemistry,  for example.  Blotting (not immunoblotting) is also used as a step in the sequencing of a protein  band purified by gel electrophoresis.  For  this purpose the protein  is electroblotted  onto  a polyvinylidene  difluoride (PVDF) membrane, the blot excised and transferred into the sequenator.

4 .Immunogold labeling with silver amplification

Colloidal gold particles, ranging  in  diameter  from  1  to  30  nm,  can  be prepared by reducing dissolved gold chlorides with various  reducing agents . Proteins bind readily to such particles and stabilize the colloids against a salt challenge.  Colloidal gold particles can thus be used  as labels, attached to either antibodies or protein A, to form immunogold probes. Such immunogold probes  find their  greatest  use  in  electron  microscopy immunocytochemistry, whereby the subcellular distribution of an Ag of interest may be determined.  Colloidal gold particles  are very  electron dense and show up readily in electron micrographs.

With silver amplification, immunogold probes may  also be used at the light microscopy level and for staining immunoblots. In the immunogold-with-silver-amplification (IGSS) technique, the colloidal gold label serves as a nucleation  center  for the  deposition  of metallic  silver. This yields a black stain which marks the position of the Ag of interest.

5 . Colloid agglutination

As mentioned  above  ,  proteins  can  stabilize colloids. The proteins bind to the colloidal particles and similar charge repulsion between the  bound proteins  keeps the  colloidal  particles  apart,  thus preventing flocculaton.  Latex  beads are  commonly  used as colloidal suspensions for analysis.  A natural system, which is virtually  colloidal,  is blood, in which the red cells are prevented  from  aggregating by virtue  of their similar surface charges.

Antibodies are divalent and are thus able to simultaneously bind to two similar antigens on two different colloidal particles.  Such cross-linking of the colloidal particles causes them to flocculate out of suspension, and this provides a very sensitive method for the  detection  of Abs specific for the  colloid-bound Ag.  The  method  is particularly  useful for medical diagnosis in the field.  Since flocculation can easily be detected by eye,  no sophisticated instrumentation is required.  The  method  gives a simple yes-or-no answer, but  it can be made semi-quantitative by  dilution  of the Ab, until the “definite yes” becomes a “maybe”. The dilution at which this happens is inversely related to the initial antibody concentration.

An elegant diagnostic method uses agglutination of endogenous  red blood cells as the reporter system. Monoclonal Abs are raised against glycophorin, a glycoprotein present on the surface of all red blood cells. These Abs are species specific, i.e. they only recognize glycophorin  from a particular species.  From  these  monoclonal  Abs, F(ab’),  fragments  can be made by proteolysis with pepsin.  F(ab’)2  fragments consist of the  two Fab arms of the Ab, bound together by disulfide  bridges.  The  presence  of the disulfide bridges is useful as these can be reduced and  subsequently used to conjugate a peptide epitope to the free -SH groups of the  two separate Fab fragments.  This  generates  a  specific  diagnostic  reagent  (Fig.  7).

Addition of this reagent to  a drop of blood will cause haemagglutination, if Abs targeting the peptide epitope  are present.  For example, a person  infected with the  AIDS virus will, in the  early stages, have anti-AIDS virus Abs present in their blood.  These  Abs will target specific epitopes  on the  AIDS virus proteins.  These  epitopes  can  be identified and corresponding peptides can be  synthesized.  Conjugation  of one such peptide to an anti-human glycophorin monoclonal  Ab half-F(ab’)2  fragment will generate  a specific diagnostic  reagent.  Addition of an appropriate  dilution of this  reagent to  a drop  of a patient’s  blood will give a yes/no indication of the  presence  of anti-AIDS virus Abs in the patients blood.  A positive answer is given by  the  agglutination  of the red blood cells.  Such diagnostic  analyses are  useful  for  screening  in  the field but they  should be followed by confirmatory  laboratory-based tests, such as ELISA

Figure 7.  Generation of a reagent for detecting the presence of specific antibodies, using endogenous red blood cells as the reporter system.

References

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