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Knowledge in Immunity
IgA - IMMUNOGLOBULIN
IgA is the second most abundant class,constituting about 10-13 per cent of serumimmunoglobulins. The normal serum level is0.6-4.2 mg per ml. It has a half life of 6-8 days. It is the major immunoglobulin in the colostrum, saliva and tears. IgA occurs in two forms. Serum IgA is principally a monomeric 7 S molecule (MW about 160,000). IgA found on mucosal surfaces and in secretions is a dimerformed by two monomer units joined together at their carboxy terminal by a glycopeptide termed the J chain (J for joining). This is called the secretory IgA (SIgA) Dimeric SlgA is synthesised by plasma cells situatednear the mucosal or glandular epithelium. The J chain is also produced in the same cells. J chains are also present in other polymeric immunoglobulin such as IgM.SlgA contains another glycine-rich polypeptidecalled the secretory component or secretory piece. This is not produced by lymphoid cells but by mucosal or glandular epithelial cells. Dimeric IgA binds to a receptor on the surface of the epithelial cells and is endocytosed and transported across the cells to the luminal surface. During this process, a part of thereceptor remains attached to the IgA dimer. This part is known as the secretory component. The secretory piece is believed to protect IgA from denaturation by bacterial proteases in sites such as the intestinal mucosa which have rich and varied bacterial flora.SlgA is a much larger molecule than serum IgA (11 S MW about 400,000).Slag is selectively concentrated in secretions and on mucus surfaces forming an antibody paste and is believed to play an important role in local immunity against respiratory and intestinal pathogens. Secretary IgA is relatively resistant to the digestive enzymes and reducing agents. IgA antibodies may functionby inhibiting the adherence of microorganisms to the surface of mucosal cells by covering the organisms and thereby preventing their entry into body tissues. IgA does not fix complement but can activate the alternative complement pathway. It promotes phagocytosis and intracellular killing of microorganisms.Two IgA subclasses have been described, IgA, and IgA. IgA, lakhs interchain disulphide bonds between the heavy and light chains. Though IgA, is a minor component of serum IgA, it is the dominant form in the secretions.
Yamini Chandru
IgM - IMMUNOGLOBULIN
IgM constitutes 5-8 per cent of serumimmunoglobulins, with a normal level of 0.5-2 mg per ml. It has a half life of about five days. It is a heavy molecule (19 S; MW 900,000 to 1000000 hence called 'the millionaire molecule). IgM molecules are polymers of five four-peptide subunits, each bearing an extra CH domain (Fig. 12.5). As with IgA, polymerisation of the subunits depends upon the presence of the chain. Though the theoretical valency is ten, this is observed only with small haptens.With larger antigens, the effective valency falls to five, probably due to steric hindrance. Most IgM (80 per cent) is intravascular in distribution. Phylogenetically IgM is the oldest immunoglobulin class. It is alsothe earliest immunoglobulin to be synthesised by the fetus, beginning by about 20 weeks of age. As it is not transported across the placenta, the presence of IgM in the fetus or newborn indicates intrauterine infectionand its detection is useful in the diagnosis of congenital infections such as syphilis, rubella, HIV infection and toxoplasmosis. igM antibodies are relatively short lived, disappearing earlier than IgG. Hence, theirdemonstration in serum indicates recent infection.Treatment of serum with 0.12 M 2-mercaptoethanol selectively destroys IgM without affecting IgG antibodies. This is a simple method for the differential estimation of IgG and IgM antibodies. The isohemagglutinins (anti-A, anti-B) and many other natural antibodies to microorganisms are usually IgM, as also antibodies to typhoid 'O' antigen(endotoxin) and reagin antibodies in syphilis.The unique structural features of IgM appearparticularly suited to the biological role of providing protection against microorganisms and other large antigens that have repeating antigenic determinants on their surface. A single molecule of IgM can bring aboutimmune hemolysis, whereas 1000 IgG molecules are required for the same effect. IgM is also 5001000 times more effective than IgG in opsonisation, 100 times more effective in bactericidal action and about 20 times in bacterial agglutination. In the neutralisationof toxins and viruses, however, it is less active than IgG. Being largely confined to the intravascular space, IgM is believed to be responsible for protection againstblood invasion by microorganisms. IgM deficiency is often associated with septicemia.Monomeric IgM is the major antibody receptor on the surface of B lymphocytes for antigen recognition.
IgD - IMMUNOGLOBULIN
IgD resembles IgG structurally. It is present inconcentration of about 3 mg per 100 ml of serum and is mostly intravascular. It has a half life of about three days. IgD and IgM occur on the surface of unstimulated B lymphocytes and serve as recognition receptors forantigens. Combination of cell membrane-bound IgD or IgM with the corresponding antigen leads to specific stimulation of the B cell either activation and cloning to produce antibody, or suppression.
IgE - IMMUNOGLOBULIN
This immunoglobulin was discovered in 1966by Ishizaka during the investigation of atopic reagin antibodies. It is an 8 S molecule (MW about 190,000), with a half life of about two days. It resembles IgG structurally. It exhibits unique properties such as heat lability (inactivated at 56 °C in one hour) and affinityfor the surface of tissue cells. (particularly mast cells) of the same species (homocytotropism) It mediates the Prausnitz-Kustner reaction It is susceptible to mercaptoethanol. It does not pass the placental barrier or fix complement. It is mostly extravascular in distribution Normal serum contains only traces (a few nanograms per ml) but greatly elevated levels are seen in atopic (type 1 allergic) conditions such as asthma,hay fever and eczema. Children living in insanitary conditions, with a high load of intestinal parasites, havehigh serum levels of IgE.IgE is chiefly produced in the lining of the respiratory and intestinal tract. IgE deficiency has been associated with IgA deficiency in individuals with impaired immunity who present undue susceptibility to infection.IgE is responsible for the anaphylactic type ofhypersensitivity. The physiological role of IgE appears to be protection against pathogens by mast cell degranulation and release of inflammatory mediators. It is also believed to have a special role in defenceagainst helminthic infections.In general, IgG protects the body fluids, IgA thebody surfaces and IgM the bloodstream, while IgE mediates reaginic hypersensitivity. IgD is a recognition molecule on the surface of B lymphocytes.
ABNORMAL IMMUNOGLOBULIN
Apart from antibodies, other structurally similar proteins are seen in serum in many pathological processes, and sometimes even in healthy persons. The earliest description of an abnormal immunoglobulin was the discovery by Bence Jones (1847) of the protein thatbears his name. The Bence Jones protein is typically found in multiple myeloma. It can be identified in urine by its characteristic property of coagulation when heated to 50 °C but redissolving at 70 °C(Bence Jonesproteins are the light chains of immunoglobulins and so may occur as the kappa or lambda forms. But in anyone patient, the chain is either kappa or lambda only and never both, being uniform in all other respects.This is because myeloma is a plasma cell dyscrasia in which there is unchecked proliferation of one clone of plasma cells, resulting in excessive production of the particular immunoglobulin synthesised by theclone. Such immunoglobulins are, therefore, called Monotonal)Multiple myeloma may affect plasma cellssynthesising IgG, IgA, IgD or Ige. Similar involvementof IgM producing cells is known as Waldenstrom macroglobulinemia In this condition, there is excessive production of the respective myeloma proteins M proteins) and of their light Chains (Bence Jones proteins). A different disorder is found in 'heavy chaindisease', which is a lymphoid neoplasia characterised by the overproduction of the Fc parts of the immunoglobulin heavy chains.Cryoglobulinemia is a condition in which a gelor precipitate is formed on cooling the serum,which dissolves on warming. It may not always be associated with disease but is often found in myelomas macroglobulinemias and autoimmune conditions such as systemic lupus erythematosus. Most cryoglobulinsconsist of IgG, IgM or their mixed precipitates.Because of the monoclonal nature of Bence Jones and other M proteins, they have been valuable models for the understanding of immunoglobulin structureand function.
IMMUNOGLOBULIN SPECIFICITY
The immunoglobulin specificity of the greatestbiological importance is idiotypic specificity pertaining to the nature of the antigen binding sites (paratopes).The specific antigenic determinants on the paratope are called idiotopes. The sun total of idiotopes on an Ig molecule constitutes its idiotype. By immunisation with Fab fragments, anti-idiotypic antibodies can be produced. These resemble the epitopes of the original antigen. Used as a vaccine, theseshow protection against the original antigen (pathogen or tumour) in experimental animals. Sequential anti- idiotypic antibody formation is the basis of Jerne's network hypothesis of immune regulation.Immunoglobulins exhibit other geneticallydetermined specificities based on their antigenic structure. The antigenic specificities which distinguish between the different classes and subclasses of immunoglobulins present in all normal individuals of a given species are termed isotypic specificities. Antigenicspecificities which distinguish immunoglobulins of the same class, between different groups of individuals inthe same species, are called allotypic specificities. Immunoglobulin allotypes have been studied in detail in the rabbit and guinea pig by using type-specific immunesera. Such deliberate immunisation is not possible in human beings, but anti isotype-specific antibodies may develop following blood transfusion or passage of maternalIgG into the fetus. Anti Allotypic antibodies are also found in sera containing rheumatoid arthritis factor'Two allotypic systems are known in humans: the Gm system (for gamma marker) and the InV system (abbreviation of patient's name). Gm is associated with the Fc portion of the IgG heavy chain. More than 25 Gm types have been identified so far. The InV system is associated with the kappa light chain and so has been renamed Km. Three Km allotypes have been identified.Genetic markers associated with IgA are called 'Am. To date, in the human system no allotypic markers have been found for lambda light chains or u, dor e heavy chains.
SEROLOGICAL REACTIONS - PRECIPITATION REACTION
When a soluble antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature and pH, the antigen-antibody complex forms an insoluble precipitate. When, instead of sedimenting, the precipitate remains suspended as floccules, the reaction is known as flocculation. Precipitation can take place in liquid media or in gels such as agar agarose or polyacrylamide.The amount of precipitate formed is greatlyinfluenced by the relative proportions of antigens and (antibodies. If increasing quantities of antigens are added to the same amount of antiserum in different tubes, precipitation will be found to occur most rapidly and abundantly in one of the middle tubes in which the antigen and antibody are present in optimal or equivalent proportions. In the preceding tubes in which the antibody is in excess and in the later tubes in whichthe antigen is in excess, the precipitation will be weak or even absent. For a given antigen-antibody system, the optimal or equivalent ratio will be constant, irrespective of the quantity of the reactants. If the amounts of precipitate in the different tubes are plotted on agraph, the resulting curve will have three phases: an ascending part (prozone or zone of antibody excess), a peak (zone of equivalenče) and a descending part (postzone or zone of antigen excess).This is called the zone phenomenon. Zoning occurs in agglutination and some other serological reactions. The prozone is of importance in clinical serologyas sera rich in antibody may sometimes give a false negative precipitation or agglutination result, unless several dilutions are tested.
MECHANISM OF PRECIPITATION REACTION
Marrack (1934) proposed the lattice hypothesis to explain the mechanism of precipitation. According to this concept, which is supported by considerable experimental evidence and is now widely accepted, multivalent antigens combine with bivalent antibodies in varying proportions, depending on the antigen-antibody ratio in the reacting mixture. Precipitation results when a large lattice is formed consisting of alternating antigen and antibody molecules. This is possible only in the zone of equivalence. In the zones ofantigen or antibody excess, the lattice does not enlarge, as the valencies of the antibody and the antigen, respectively, are fully satisfied. The lattice hypothesis holds good for agglutination also.
APPLICATIONS OF PRECIPITATION REACTION
The precipitation test may be carried out as aqualitative or a quantitative test. It is very sensitive in the detection of antigens and as little as 1 ug of protein can be detected. It therefore finds forensic application in the identification of blood and seminal stains, and in testing for food adulterants. Precipitation is relatively less sensitive for the detection of antibodies.The following types of precipitation and flocculation tests are in common use:•Ring test: This, the simplest type of precipitation test, consists of layering the antigen solution over a column of antiserum in a narrow tube. A precipitate forms at the junction of the two liquids. Ring tests have onlya few clinical applications now. Examples are Ascoli's thermoprecipitin test and the grouping of streptococci by the Lancefield technique.•Slide test: When a drop each of the antigen and the antiserum are placed on a slide and mixed by shaking, floccules appear. The VDRL test for syphilis is an example of slide flocculation.•Tube test: The Kahn test for syphilis is an example of a tube flocculation test. A quantitative tube flocculation test is used for the standardisation of toxins and toxoids.Serial dilutions of the toxin/toxoid are added to the tubes containing a fixed quantity of the antitoxin. The amount of toxin or toxoid that flocculates optimally with one unit of the antitoxin is defined as an Lf dose.
ELECTROIMMUNODIFFUSION
The development of precipitin lines can be speeded up by electrically driving the antigen and antibody. Various methods have been described combining electrophoresiswith diffusion. Of these. one-dimensional double electroimmunodiffusion(counterimmuno electrophoresis) and one-dimensional single electroimmunodiffusion (rocket electrophoresis) are used frequently in the clinical laboratory.1. Counterimmunoelectrophoresis (CIE, counter-current immunoelectrophoresis): This involves simultaneous electrophoresis of the antigen and the antibody in gel in opposite directions resulting in precipitation at a point between them. This method produces visible precipitation lines within thirty minutes and isten times more sensitive than the standard double diffusion techniques. The clinical applications are for detecting various antigens such as alpha fetoprotein in serum and specific antigens of cryptococcus and meningococcus in the cerebrospinal fluid.2. One-dimensional single electroimmunodiffusion (rocket electrophoresis): The main application of thistechnique is for quantitative estimation of antigens.The antiserum to the antigen to be quantitated is incorporated in agarose and gelled on the glass slide. The antigen, in increasing concentrations, is placed in wells punched in the set gel. The antigen is thenelectrophoresed into the antibody containing agarose. The pattern of immunoprecipitationresembles a rocket and hence the name.A variant of this is Laurel's two-dimensionalelectrophoresis. In this technique, the antigen mixtureis first electrophoretically separated in a directionperpendicular to that of the final rocket stage. By thismethod one can quantitate each of several antigens in a mixture.
NEUTRALISATION TEST
Virus neutralisation test: Neutralisation of viruses by their antibodies can be demonstrated in various systems. Neutralisation of bacteriophages can bedemonstrated by the plaque inhibition test. When bacteriophages are seeded in appropriate dilution on lawn cultures of susceptible bacteria, plaques oflysis are produced. Specific antiphage serum inhibits plaque formation. Neutralisation of animal viruses can be demonstrated in three systems: animal, egg and tissue culture.Toxin neutralisation: Bacterial exotoxins are good antigens and induce the formation of neutralising antibodies (antitoxins) which are important clinically, in protection against and recovery from diseases such as diphtheria and tetanus. The toxicity of endotoxins isnot neutralised by antisera.Toxin neutralisation can be tested in vivo or in vitro.Neutralisation test in animals consist of injecting toxin- antitoxin mixtures and estimating the least amount of antitoxin that prevents death or disease in the animals.With the diphtheria toxin, which in small doses causes a cutaneous reaction, neutralisation tests can be done on rabbit skin. The Schick test is based on the ability of circulating antitoxin to neutralise' the diphtheriatoxin given intradermally, and indicates immunity or susceptibility to the disease. Toxin neutralisation in vitro depends on the inhibition of some demonstrable toxic effect. An example is the antistreptolysin O test, in which anti toxin present in patient sera neutralises the hemolytic activity of the streptococcal O hemolysin.
OPSONISATION
The name 'opsonin' was originally given by Wright (1903) to a heat labile substance present in fresh normal sera, which facilitated phagocytosis. This factor was subsequently identified as a complement.A heat stable serum factor with similar activity was called "bacteriotropin'. This appears to be a specific antibody. The term opsonin is now generally used to refer to both these factors.Wright used the 'opsonic index' to study the progress of resistance during the course of diseases. The opsonic index was defined as the ratio of the phagocytic activityof the patient's blood for a given bacterium, to the phagocytic activity of blood from a normal individual.It was measured by incubating fresh citrated blood with the bacterial suspension at 37 °C for 15 minutes and estimating the average number of phagocytosed bacteria per polymorphonuclear leukocytes (phagocytic index) from stained blood films.