Figure 10.10. Basic principles of immunological assays. For details see text.
competitive: The sample is added together with a known amount of labelled antigen to a known amount of antibodies. The labelled antigen binds to the antibody, resulting in a defined signal. If the sample also contains (unlabelled) antigen, this would compete with binding of the labelled antigen molecules, reducing the signal measured in a concentration dependent manner. Competitive assays are often used for the detection of small molecules, like drugs. The problem with that type of assay is to label the antigen without destroying antigen-antibody interaction.
sandwich: Antibodies are fixed on a solid support, then incubated with the antigen. Antigen binding creates binding sites for a second antibody, directed against a different epitope of the antigen, thus the amount of second antibody bound is proportional to the concentration of antigen. This procedure works only with fairly large antigen (with several epitopes), in addition it requires antibody pairs, which are not always available.
direct: The labelled antibody binds to the antigen, which is fixed on a solid support. The amount of antibody bound and hence the signal depends on the amount of antigen present. This type of assay is often used against large antigens like proteins, viruses, bacteria or even whole cells.
indirect: assays work similar to the direct ones, however, the primary antibody is unlabelled. After it has bound to the antigen, a secondary antibody directed against the constant part of the primary antibody bears the label. Since each primary antibody can bind several secondary ones this leads to an increase in sensitivity. Additionally, the complicated labelling procedure needs to be performed only once in the secondary antibody (obtained from large animals like sheep or horse by manufacturers), which can then be used to detect several primary antibodies (obtained in the lab from small animals like rats).
Various different labels may be used to detect the antibody, the most important are radioactive isotopes, fluorescent molecules and enzymes. Note that the assays described above can easily be set up to detect antibodies, rather than the antigens.
Development of immunological assays for drugs, disease marker proteins or for pathogens is a multi-billion € industry, and has revolutionised especially clinical laboratories. In the last couple of years it has been complemented by the detection of nucleic acids, for example by polymerase chain reaction (PCR).
Blood and interstitial fluid contain a group of proteins, that can destroy cell membranes. Collectively, they are known as complement system. The name indicates that these proteins complement and enhance the disease-preventing activity of antibodies and immune cells. There are about 20 different proteins in the complement system, they are produced mostly in the liver.
Obviously, such proteins are very dangerous, and their activity needs to be tightly controlled. The early components of the complement system are highly specific serin-proteases, activation of the complement system is a chain of protein cleavage reactions. Apart from activating another protease, each cleavage results in the production of a small peptide, which acts as inflammatory mediator (anaphylatoxin).
• catalyses the formation of the membrane attack complex from components C5-C9. A complex of C5-C8 catalyses the oligomerisation of C9, which forms pores of about 10 nm diameter in the cell membrane. As a result the affected cell is osmotically lysed.
10.3.1 How is complement activated?
There are three principle sequences of complement activation, known as classical, alternative and lectin pathway respectively.
The classical pathway (see fig. 10.11) is started by IgM or IgG molecules bound to a pathogen.
In the lectin pathway of complement activation the mannan-binding lectin MBL replaces the antibody-antigen complex as starting point. This binds MASP (MBL associated serin protease), which cleaves C4 and C2 just like C1s does in the classical pathway.
The alternative pathway starts with spontaneous cleavage of C3 into C3a and C3b, the latter inserts into membranes. Our own cell membranes have C3b-inactivating components, but some pathogens are lacking those. Thus C3b can hydrolyse B and form the C3bBb-complex, which leads to activation of further C3-molecules. The C3b,Bb complex is the functional homologue of C2b,4b; C2 and B are encoded next to each other in the MHC-locus. Both complexes can bind C3b to form a C5-convertase (C2b,3b,4b or C3b2Bb).
Note that the conversion of complement molecules works as a catalytic cascade quite similar to what is seen in hormone signalling (see fig. 8.1 on page 122). A small number of C1-molecules bound to antigen-antibody-complexes leads to the rapid production of large amounts of C3b and C5b, flooding the surface of the invader.
Cleavage of complement molecules converts a water-soluble protein without enzymatic activity into a membrane-bound Ser-protease and a soluble peptide. This protective mechanism is also observed for example with digestive enzymes.
Once the C5-convertases have been formed, alternative and lectin pathway proceed like the classical. In evolution the alternative pathway probably developed first as part of our innate defense. The lectin- and classical pathway are later additions to direct complement more specifically and with higher activity to foreign material.
10.3.2 What does complement do?
C4a, C3a and C5a are potent activators of inflammation (anaphylatoxins). They induce smooth muscle contraction, increase vascular permeability and recruit granulocytes and macrophages to the site of infection.