Serology is the study of serum (singular) or sera (plural). Serum, the fluid obtained when blood clots are removed contains antigens and antibodies. Serology may be used in detection, identification and classification of microorganism. The reactions in vitro between antigens and antibodies (antisera) form the basis of serological tests (techniques). A range of methods are available to make serological reactions visible, such direct tests may be in liquid or in solid media.
Serological tests can distinguish between closely related microorganisms (viruses, bacteria, ‘fungi) by detecting differences in their cell surface antigens, for example serological tests have been used to distinguish thousands of different strains of Salmonella based primarily on the slight chemical differences in their 0-antigens (lipopolysaccharides – protein antigen) and H-antigens (flagella antigens).
Visible serological reactions are of two main types.
- Agglutination in which particulate antigen such as ‘microbial cells are caused to gather together in clumps.
- Precipitation in which a soluble antigen is precipitated from solution.
There are other antigen – antibody reactions which are not directly visible and are demonstrated by-various other methods.
Immunochemical techniques are very useful in serology and employ the use of the antibodies involved in humoral immunity. The basic principle involved in most immunochemical techniques is that a specific antigen will combine with its specific antibody to give an antigen antibody complex. Since antigens .and antibodies are both multivalent the antigen-antibody complex is usually insoluble and may be seen with the naked eye. Serological tests can be used in two ways, a known antibody can be used to detect and measure an unknown antigen or a known antigen can be used to detect and measure an unknown antibody.
1. THE PRECIPITIN REACTIONS (In free solution) Principle
When increasing amounts of an antigen are mixed with suitable antibody solution a precipitate forms. The antigen-antibody precipitate may be isolated from the supernatant by centrifugation and may be estimated with a suitable protein assay.
Qualitative analysis of antigen
It is possible to ascertain the presence of any specific protein or antigen, in the presence of many related compounds by adding a range of different concentrations of the suspected antigen to a fixed amount of monospecific antiserum. Note: False negative data may be obtained if the ratio of antigen to antibody do not fall within zone of equivalence.
Quantitative Analysis
When the zone of equivalence can be determined, the precipitin reaction provides an accurate method for quantitative assay of antigens. It is particularly useful during the quantitative assay of a protein synthesised in vitro under direction of an isolated or chemically synthesised RNA.
Usually the total reaction volume is not more than 100mm3 and the precipitation is allowed to occur overnight at 4°C. The antigen antibody complex is then washed and its amount determined by any sensitive protein or nitrogen assay. Precipitation tests may be carried out in narrow glass tubes for viruses where clarified virus suspension and antiserum are mixed at various dilutions.
2. THE PRECIPITIN REACTION IN GELS (Immunodiffusion)
Precipitation may also take place in solid media such as agar. One of the techniques involves the diffusion of antigen from a solution into a gel containing antibody (Radial or single diffusion). Both reactants move and form a visible band where they meet in optimal proportions asin § the Ouchterlony technique. These gel diffusion tests were originally in tubes but are now mostly in petri dishes.
DOUBLE IMMUNODIFFUSION IN TWO DIMENSION:
Ouchterlony Technique
This technique involves the diffusion of both antigenand antibody towards each other. It can be used for detecting which sera or cell fractions contain a particular antigen and whether or not two antigens are identical, different or share antigenic determinants.
IMMUNOELECTROPHORESIS (IE)
Antigenic mixtures may be analysed by immuno electrophoresis (sero-electrophoresis). In this technique the components are first separated in one direction by an electric – current, and thereafter antiserum is put into a long trough at one side of the well parallel to the direction of the current. Antigenic components and antibodies then diffuse together to produce bands. Strains of microorganisms which are closely related are then distinguished.
QUALITATIVE IMMUNOELECTROPHORESIS
This technique combines the specificity of immunoprecipitin reactions with separation of molecules in a molecular sieving, medium using electrophoresis.
QUANTITATIVE IMMUNOELECTROPHORESIS (Laurell rocket immunoelectrophoresis)
In this technique the antigen sample is placed in wells cut in agar containing specific antiserum to the antigen to be assayed. On application of a direct electric current, most antigens migrate towards the anode and the IgG antibodies migrate towards the cathode. When all the antigen has migrated into the gel, equivalence is reached and antigenantibody complexes precipitate in rocket shaped figures. The area under the rocket shape is directly propotional to the antigen concentration when the rocket height is plotted against the concentration, a linear curve is obtained, thus when standards are used with the preparation of a calibration curve, the concentration of antigen solutions may be determined.
RADIOIMMUNOASSAY (RIA)
This method uses radioisotopes to detect and quantify the antigen – antibody interaction. It combines the specificity of the immune reaction with the sensitivity of radioisotope techniques. This technique is based on the competition between unlabelled antigen and a finite amount of the corresponding radiolabelled antigen happen for a limited number of antibody binding sites in a fixed amount of antiserum. The quantity of antigen in a test solution can be determined when a known amount of radiolabelled antigen and a fixed amount of antibody is known and a calibration curve can be constructed.
IMMUNO RADIOMETRIC ASSAY (IRMA)
This technique makes use of purified radiolabelled antibody to the antigen to be estimated rather than antigen for direct measurement of the amount of antigen in a sample. Immunoradiometric assay is a direct binding assay in which the radiolabelled antibody reagent is in excess and the amount bound to the antigen is measured.
ENZYME IMMUNOASSAY ENZYME LINKED IMMUNOSORBENT ASSAY
Enzyme immunoassay techniques combine the specificity of antibodies with the _ sensitivity of spectrophotometric enzyme assays by using either an antibody or an antigen linked to an enzyme at a site which does not affect the activity of either compound. The enzyme is easily assayed by adding a substrate that yield’s a coloured product. ELISA is cheap to operate and lacks the radiological hazards associated with RIA. ELISA is suitable for use-in small laboratories that lack radioactive counting facilities, it is more sensitive and uses more stable reagents. Automation is straight forward because it uses disposable microtitre plate and an optical scanner.
DOUBLE ANTIBODY SANDWICH ELISA
This technique detects specific antigens involving a complex made up of three components, an antigen, an antibody linked to a solid support and a second antibody which is linked to an enzyme. Some of solid phase or supports used in ELISA are polyacrylamide beads, crosslinked dextran filter paper discs made of “Cellulose and polypropylene tubes. The appropriate antigen or antibody May be attached to the solid phase by passive adsorption or Covalent coupling with cyanogen bromide.
INDIRECT ELISA
This technique is used to measure antibody levels with a specific antigen attached to a solid support. When the specific antibody is added, its molecules bind to the antigen and will not be washed away while all other materials are washed away during rinsing. The bound antibody i detected by the use of an enzyme-linked anti-immunoglobulin.
DOT IMMUNOBINDING ASSAY
In this technique antigens can be applied directly to nitrocellulose filter paper (widely used as a support for solid phase immunoassays) as a dot to give a simple and reliable assay. The dot permits the colour reaction to be viewed against a white background.
IMMUNOFLUORESCENCE MICROSCOPY
Several modifications of this technique have been described. The procedures to be used does not depend so much on the bacterium to be tested but more on the scientist. Normal slides can be used in these tests but special IF-slides should be preferred. The special IF-slides have six to eight holes so that several tests can be performed on each slide.
FLUORESCENCE IMMUNO ASSAY (FIA)
This technique combines the specificity of antibodies with the sensitivity of fluorimetric assays. In this technique antibodies are combined with fluorescent dyes, so that the antigens they have reacted with will be recognised by their fluorescence when illuminated with UV light under a microscope, with this technique the exact location of infecting organisms in tissues can be found.
OTHER IMMUNOCHEMICAL TECHNIQUES
1. Immunoferritin probes:
In this technique antibodies are labelled with feritin and are used for locating antigens using electron microscopy.
2. Immunoradioisotope probes:
Antibodies are labelled with radioisotopes and used for locating and detecting antigens by autoradiography.
3. Particle counting immunoasays:
This technique is based on the principle that the number of free particles coated with antigen will decrease during agglutination. Also the angle at which a beam of light is scattered is a function of particle size.
AGGLUTINATION TESTS
Agglutination tests also employ precipitation when a suspension of a particular antigen (which may be viral or bacterial cells’or even latex particles) interact with antibodies, immune complexes are formed resulting in visible clumping known as agglutination. The agglutination test can be done either on a slide (slide agglutination) or ina tube (tube agglutination).
THE COMPLEMENT FIXATION TEST
The test employs the capacity of the antibody-antigen reaction to fix complement, a labile component of fresh unheated rabbit antiserum and the need for this complement for lysis in a mixture of sheep red blood cells and their complement free antiserum. The reaction of complement with antigen – antibody complexes is termed complement fixation.
Two important properties of complement are made use of in this test
1. A mixture of antigen and antibody that does not react will not fix complement
2. Haemolysin, an antibody specific for red blood cells will only cause lysis of ted blood cells in the presence of complement.
SPECIFIC AGGLUTINATION TESTS
Agglutination tests for Salmonella (widal reaction) Salmonella Species possess both flagellar and somatic antigens. In addition to diphasic flagella variation strains may show other variations in their antigenic components. This agglutination test is for detection of antibodies which appear in the course of enteric fever and other salmonella infections. A range of suspensions is available for use and include Salmonella typhi (H and.O), S. paratyphi A(H), S. paratyphi B(H and OQ), a non specific salmonella suspension which contains several H phase 2 antigens, S. typhi (Vi), S. typhimurium (H phase 1) S. paratyphi A(o) and S. paratyphi c(H and O). Other salmonella and Brucella arbotus suspensions can be used when required.
QUELLUNG (swelling) reaction, or the specific capsula reaction
Quellung is a German word meaning to swell. When a specific antibody combines with an antigen which is of the same composition as the capsules of an organism the capsules appears to swell. This is because the antibodies in the antiserum bind to the capsule giving it a swollen appearance. This specific capsular reaction may be used in typing bacteria with capsules by using specific antisera raised against particular capsular antigens.
DEMONSTRATION OF ANTIBODIES AS EVIDENCE OF PATHOGENICITY
When a microorganism normally regarded as harmless, appears to be causing a localised or generalised infection, part of the evidence which must be accumulated before its causal role can be established or confirmed is the demonstration of specific antibodies to the organism in the serum of the patient.
The type of test selected for the demonstration of the antibodies depends on certain characteristics of the organism such as its growth pattern. Several methods of detecting antibodies have been described and can be used. The complement fixation test has the advantage that it does not depend on the physical state of the bacteria in the antigen.