Antigen-Antibody is a bimolecular interaction like enzyme substract interaction but it is an irreversible interaction.
The strength of antigen-antibody interaction depends on the bonding between them. The involved in the interaction are hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions as these bondings are very weak so large number of such bondings are required. Furthermore, each of these noncovalent interactions operates over a very short distance, generally about 1× 10-7 mm (1 angstrom, Å); consequently, a strong Ag-Ab interaction depends on a very close fit between the antigen and antibody. The strength of antigen-antibody interaction is expressed in terms of avidity and affinity. Avidity is expressed as valancy of an antibody. For example, IgM has 5 valancy or it is a pentamer. Higher avidity can compensate the low affinity (strength of an antibody to bind with the antigen). IgM has low affinity than IgG but pentameric nature of IgM has increased the affinity of IgM than IgG.
It is the interaction of antibody with an antigen with unrelated epitope.
Condition for cross reactivity:
· If two different antigen share an identical epitope.
· Unrelated antigen possessing epitope with similar chemical properties.
However such antibody interaction usually has less affinity than that for the original epitope.
Example for cross reactivity:
The ABO blood group differs in the terminal sugar residues. Cross-reactivity is induced by the exposure of microbial antigen of one individual to the other which lack these antigen in their blood. In some cases these microbial antigens have been shown to elicit antibody that cross-react with the host-cell components, resulting in a tissue damaging autoimmune reaction. The bacterium Streptococcus pyrogens, for example express cell wall proteins called M antigens have been shown to cross react with several myocardial and implicated in heart and kidney damage following Streptococcus infections.
Importance of cross-reactivity:
Vaccinia virus, which caused cowpox, expresses cross-reaction epitope with variola virus, the causative agent of small-pox. This property had been used by the Jenner to produce vaccine against the small-pox.
The interaction between an antibody and a soluble antigen in aqueous solution form a lattice that eventually develops into a visible precipitate. Antibodies that aggregate soluble antigen are called precipitins.
Condition for the precipitation reaction:
· Antibody must be at least divalent, precipitation will not form with monovalent Fab fragments.
· Antigen must be either divalent or polyvalent; that is it must have at least two copies of the same apitopes.
Precipitation reaction in fluid:
Precipitation of antigen and antibody in visualized in fluid when paratope is equal to the epitope. The region where precipitation reaction is visualized is known as zone of equilibrium (Ag=Ab).
Precipitation reaction in gel:
The reaction is based on immunodiffusion i.e. diffusion of antigen or antibody on the semisolid surface. Precipitation is seen in the region of antigen-antibody equivalent region. Two very common immunodiffusion techniques are:
· Radial immunodiffusion (Mancini Method).
· Double immunodiffusion (Oucterlony Method).
The relative concentration of an antigen can be determined by a simple quantative assay in which an antigen sample is placec in a well and allowed to diffuse in a semisolid medium containing uniformly distributed antibody. Precipitation is seen in Ag-Ab equivalent region. The area of the precipitin ring is proportional to the concentration of antigen. By comparing the are of the precipitin ring with a standard curve, the concentration of the antigen sample can be determined.
Fig: Radial Immunodiffusion.
The technique establishes the effective quanlitative nature of antigens and numbers of different antigen-antibody system present.
Fig: Double Diffusion
Three types of patterns are produced by this reaction:
a. Identity: It occurs when two antigens share identical epitopes.
b. Nonidentity: It occurs when two antigens are unrelated and share no common epitopes.
c. Partial identity: It occurs when two antigen share one common epitopes but have one or more different unique epitope.
Fig: a. Identity; b. Partial identity; c. Nonidentity
It is a technique which is applied to separate mixture of antigens and its identification. This technique is generally used in clinical laboratories to detect the presence or absence of particular protein in the serum.
· Useful in determination whether protein produces abnormally low amount of one or more isotypes, characteristicsof certain immunodeficiency disease.
· This technique is use to detect the protein overproduced such as albumin, transferin etc.
Immunoelectrophoresis is strictly quantative technique. The quantitative analysis is done by rocket electrophoresis. In rocket electrophoresis, a negatively charged Ag is electrophoresed in a gel containing Ab. The precipitin formed between Ag and Ab has shape of rocket, the heoght if which is proportional to the concentration of the Ag in the well.
Limitation of Rocket Electrophoresis:
Only negatively charged antigen is used.
Fig: Rocket Electrophoresis.
The interaction between Ab and a particulate Ag results in visible clumping called agglutination. Antibodies that produce such reaction are called agglutinins. The reaction is based on polyvalent linkage.
Fig: An example of Agglutination reaction.
Excess of Ag and Ab inhibit agglutination reaction. This is known as prozone effect.
Mechanism causing prozone effect:
· High Ab concentration, exceeds the number if epitopes.
· Incomplete Ab lacks agglutinin activity, this may be due ti restriction flexibility in the hinge region making it difficult for the Ab to assume the required angle for optimal cross-linking of the epitope on two or more particulate antigen.
· Location of epitope in the deep pocket f Ag making it difficult for the Ab for cross reactivity.
It is the process of extention of agglutination reaction. In this technique, Ag-coated RBCs are prepared for mixing a soluble Ab with RBCs that have been treated with tannic acid or chromium chloride, both of which promote absorption of the Ag to the surface of the cells.
A modification of the agglutination reaction called agglutination inhibition provides a highly sensitive assay for small quantities of an antigen. Its applications are:
· Pregnancy test kit: This technique is use to detect HCG (Human chirionic gonadotrophin) which is released in the urine during the early pregnancy period.
· Use for the detection for the use of illegal drugs, such as cocaine or heroin. Though major disadvantage of this technique is that some legal drugs may illicit same chemical structure to that of illegal drugs and may cross-react with Ab to give false results.
· Clinical uses: Use in determination of whether an individual has been exposed to certain types of viruses that caused agglutination of RBCs. This technique is commonly used in premarital testing to determine the immune status of women with respect to rubella virus. The reciprocal of the last serum dilution to show inhibition of rubella hemagglutination is the titer of the serum. A titer greater than 10 (1:10 dilution) indicates that a woman is immune to rubella,whereas a titer of less than 10 is indicative of a lack of immunity and the need for immunization with the rubella vaccine.
The technique was developed by endocrinologist, S.A. Berson and Rasalyn Yallow in 1960 to determine the level of insulin-antiinsuline complex in diabetics.
The Principle of RIA involves competitive binding of radiolabeled Ag and unlabeled Ag to a high affinity Ag. The Ag is generally labelled with γ-emitting isotype such as 125I, but β-emitting isotypes such as 3H are also routinely used as labels. The Ab doesn’t distinguish labaled and unlabeled Ag, so the two kinds of Ag compete for available binding sites of Ab. With increasing concentration of unlabled Ag, more lebled Ag will be displaced for the binding sites. The decrease in Ab and levelled Ag coples is proportional to the amount of unlabeled Ag. A standard curve is obtained by adding increasing concertration of unlabeled HBs Ag to a fixed quantity of I125 HBs Ag and specific Ab. From the plot of the perdentage of labelled Ag bound versus the concentration of unlabeled antigen, the concentration of Abs Ag inunknown serum can be determined by using the linear part of the curve.
Enzyme Linked Immunosorbent Assay (ELISA)
The principle is similar to RIA but instead of radioactive labelling, enzyme conjugated Ab is used. The enzyme reacts with colousless substrate to give coloured product. This is known as chromogenic substrate. Enzyme involved in ELISA are alkaline phosphate, horsederadish peroxidise and β-galactosidase. Five types of variation has been introduced in ELISA which allow quantitative determination and quantitative analysis of either Ag or Ab. These are:
· Indirect ELISA.
· Sandwitch ELISA.
· Compititive ELISA.
· ELIPSOT Assay.
It is an modified ELISAwhich allows the quantitative determination of the number of cells in a population that are producing Abs specific for a given Ag or an Ag which one has a apecific Ab.
The immunoprecipitation technique has the advantage of allowing the isolation of the antigen of interest for further analysis. It also provides a sensitive assay for the presence of a particular antigen in a given cell or tissue type. If the antigen concentration is low (often the case in cell and tissue extracts), the assembly of antigen-antibody complexes into precipitates can take hours, even days, and it is difficult to isolate the small amount of immunoprecipitate that forms. Fortunately, there are a number of ways to avoid these limitations:
· One is to attach the antibody to a solid support, such as a synthetic bead, which allows the antigen-antibody complex to be collected by centrifugation.
· Another is to add a secondary antibody specific for the primary antibody to bind the antigen-antibody complexes. If the secondary antibody is attached to a bead, the immune complexes can be collected by centrifugation. A particularly ingenious version of this procedure involves the coupling of the secondary antibody to magnetic beads. After the secondary antibody binds to the primary antibody, immunoprecipitates are collected by placing a magnet against the side of the tube
· Radiolabeling are also used for the isolation. Radiolabeling of proteins synthesized by cells of interest can be done by growing the cells in cell-culture medium containing one or more radiolabeled amino acids.