📖 General Microbiology
Precipitation Reaction
General MicrobiologyPrecipitation Reaction
This reaction takes place only when antigen is in soluble form. Such an antigen when
comes in contact with specific antibody in a suitable medium results into formation of an insoluble complex which precipitates. This precipitate usually settles down at the bottom of the tube. If it fails to sediment and remains suspended as floccules the reaction is known as flocculation. Precipitation also requires optimal concentration of NaCl, suitable temperature and appropriate pH.
Zone Phenomenon
Precipitation occurs most rapidly and abundantly when antigen and antibody are in optimal proportions or equivalent ratio. This is also known as zone of equivalence. When antibody is in great excess, lot of antibody remains uncombined. This is called zone of antibody excess or prozone. Similarly a zone of antigen excess occurs in which all antibody has combined with antigen and additional uncombined antigen is present.
Applications of Precipitation Reactions
Both qualitative determination as well as quantitative estimation of antigen and antibody can be performed with precipitation tests. Detection of antigens has been found to be more sensitive.
Agglutination
In agglutination reaction the antigen is a part of the surface of some particulate material such as erythrocyte, bacterium or an inorganic particle e.g. polystyrene latex which has been coated with antigen. Antibody added to a suspension of such particles combines with the surface antigen and links them together to form clearly visible aggregate which is called as agglutination.
Application of precipitation reactions
Precipitation reaction Example
Ring test Typing of streptococci, Typing of pneumococci
Slide test (flocculation) VDRL test
Tube test (flocculation) Kahn test
Immunodiffusion Eleks test
Immunoelectrophoresis Detection Of HBsAg, Cryptococcal antigen in CSF
ANTIGENS
General MicrobiologyANTIGENS
Immunogen
A substance that induces a specific immune response.
Antigen (Ag)
A substance that reacts with the products of a specific immune response.
Hapten
A substance that is non-immunogenic but which can react with the products of a specific immune response. Haptens are small molecules which could never induce an immune response when administered by themselves but which can when coupled to a carrier molecule. Free haptens, however, can react with products of the immune response after such products have been elicited. Haptens have the property of antigenicity but not immunogenicity.
Epitope or Antigenic Determinant
That portion of an antigen that combines with the products of a specific immune response.
Antibody (Ab)
A specific protein which is produced in response to an immunogen and which reacts with an antigen.
FACTORS INFLUENCING IMMUNOGENICITY
- Larger the molecule the more immunogenic it is likely to be.
- More complex the substance is chemically the more immunogenic it will be.
- Particulate antigens are more immunogenic than soluble ones and denatured antigens more immunogenic than the native form.
- Antigens that are easily phagocytosed are generally more immunogenic. This is because for most antigens (T-dependant antigens, see below) the development of an immune response requires that the antigen be phagocytosed, processed and presented to helper T cells by an antigen presenting cell (APC).
- Some substances are immunogenic in one species but not in another. Similarly, some substances are immunogenic in one individual but not in others (i.e. responders and non-responders). The species or individuals may lack or have altered genes that code for the receptors for antigen on B cells and T cells or they may not have the appropriate genes needed for the APC to present antigen to the helper T cells.
Method of Administration
1. Dose
The dose of administration of an immunogen can influence its immunogenicity. There is a dose of antigen above or below which the immune response will not be optimal.
2. Route
Generally the subcutaneous route is better than the intravenous or intragastric routes. The route of antigen administration can also alter the nature of the response
3. Adjuvants
Substances that can enhance the immune response to an immunogen are called adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as fever and inflammation.
TYPES OF ANTIGENS
T-independent Antigens
T-independent antigens are antigens which can directly stimulate the B cells to produce antibody without the requirement for T cell help In general, polysaccharides are T-independent antigens. The responses to these antigens differ from the responses to other antigens.
Properties of T-independent antigens
1. Polymeric structure
These antigens are characterized by the same antigenic determinant .
2. Polyclonal activation of B cells
Many of these antigens can activate B cell clones specific for other antigens (polyclonal activation). T-independent antigens can be subdivided into Type 1 and Type 2 based on their ability to polyclonally activate B cells. Type 1 T-independent antigens are polyclonal activators while Type 2 are not.
3. Resistance to degradation
T-independent antigens are generally more resistant to degradation and thus they persist for longer periods of time and continue to stimulate the immune system.
T-dependent Antigens
T-dependent antigens are those that do not directly stimulate the production of antibody without the help of T cells. Proteins are T-dependent antigens. Structurally these antigens are characterized by a few copies of many different antigenic determinants as illustrated in the Figure 2.
HAPTEN-CARRIER CONJUGATES
Hapten-carrier conjugates are immunogenic molecules to which haptens have been covalently attached. The immunogenic molecule is called the carrier.
Structure
Structurally these conjugates are characterized by having native antigenic determinants of the carrier as well as new determinants created by the hapten (haptenic determinants). The actual determinant created by the hapten consists of the hapten and a few of the adjacent residues, although the antibody produced to the determinant will also react with free hapten. In such conjugates the type of carrier determines whether the response will be T-independent or T-dependent.
SUPERANTIGENS
When the immune system encounters a conventional T-dependent antigen, only a small fraction (1 in 104 -105) of the T cell population is able to recognize the antigen and become activated (monoclonal/oligoclonal response). However, there are some antigens which polyclonally activate a large fraction of the T cells (up to 25%). These antigens are called superantigens .
Examples of superantigens include: Staphylococcal enterotoxins (food poisoning), Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal exfoliating toxins (scalded skin syndrome) and Streptococcal pyrogenic exotoxins (shock).
STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES
General MicrobiologySTRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES
A. IgG
1. Structure
All IgG’s are monomers (7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region.
2. Properties
IgG is the most versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.
a) IgG is the major Ig in serum – 75% of serum Ig is IgG
b) IgG is the major Ig in extra vascular spaces
c) Placental transfer – IgG is the only class of Ig that crosses the placenta. Transfer is mediated by a receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally well; IgG2 does not cross well.
d) Fixes complement – Not all subclasses fix equally well; IgG4 does not fix complement
e) Binding to cells – Macrophages, monocytes and neutrophils and some lymphocytes have Fc receptors for the Fc region of IgG. A consequence of binding to the Fc receptors on such cells is that the cells can now internalize the antigen better. The antibody prepares the antigen for killing by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. (Coating of the surface of pathogen by antibody is called opsonization).IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.
IgM
1. Structure
IgM normally exists as a pentamer (19S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S-S bond called the J chain. This chain functions in polymerization of the molecule into a pentamer.
2. Properties
a) IgM is the third most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.
c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig
Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminus to anchor it into the membrane . Cell surface IgM functions as a receptor for antigen on B cells.
IgA
1. Structure
Serum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure 10. When IgA exits as a dimer, a J chain is associated with it.
When IgA is found in secretions is also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the secretions . The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.
2. Properties
a) IgA is the 2nd most common serum Ig.
b) IgA is the major class of Ig in secretions – tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in local (mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells – PMN’s and some lymphocytes.
IgD
1. Structure
IgD exists only as a monomer.
2. Properties
a) IgD is found in low levels in serum; its role in serum is uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen.
c) IgD does not bind complement.
E. IgE
1. Structure
IgE exists as a monomer and has an extra domain in the constant region.
2. Properties
a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.
b) Involved in allergic reactions – As a consequence of its binding to basophils and mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms.
c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.
Test for Antigen - Antibody Reactions
General MicrobiologyTest for Antigen - Antibody Reactions
Antigens are those substance that stimulates the production of antibodies which, when enter into the body it reacts specifically in a manner that are clearly visible.
Some antigens may not induce antibody production, but instead creates immunological tolerance.
An antigen introduced into the body produces only specific antibodies and will react with only those specific antigens.
These antibodies appear in the serum and tissue fluids. All antibodies are considered as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE.
Antigen- antibody reactions are known as serological reactions and are used as serological diagnostic tests for the identification of infectious diseases.
The reaction occurs mainly in three stages;
1. The initial interaction between the antigen and antibody, which produces no visible effects. It is a reversible and rapid reaction.
2. The secondary stage leads to the demonstration proceedings, such as precipitation, agglutination, etc.
3. The tertiary reaction follows the neutralization or destruction of injurious antigens. These results in clinical allergy and other immunological diseases.
There are certain characteristics for antigen-antibody reactions;
1. Reaction is specific.
2. The whole molecules participate in the reaction, and not just a part of it.
3. No denaturation of antigen or antibody occurs during the reaction.
4. The combination usually occurs at the surface.
5. The combination is firm, but reversible
6. Agglutinins formed after agglutination usually are formed by both antigen and antibody together.
7. They can combine in varying proportions.
Measurement of antigen and antibody are made in terms of mass or as units or titre.
Serological reactions include;
1. Precipitation reaction
a soluble antigen combining with the specific antibody in the presence of electrolytes at a suitable temperature and pH forming insoluble precipitins. Commonly used tests are ring test, slide test, tube test, immunodiffusion, etc.
Radial Immunodiffusion
In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed .
This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.
Immunoelectrophoresis
In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs .
This tests is used for the qualitative analysis of complex mixtures of antigens
This test can also be used to evaluate purity of isolated serum proteins.
Countercurrent electrophoresis
In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line.
2. Agglutination reaction
when a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. When the antigen is an erythrocyte the term hemagglutination is used.
Applications of agglutination tests
i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.
Passive hemagglutination
The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination test for antibody to the soluble antigen . This is called passive hemagglutination.
The test is performed just like the agglutination test.
Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.
Coomb's Test (Antiglobulin Test)
DIRECT ANTIGLOBULIN TEST (DAT)
The DAT is used to detect IgG or C3 bound to the surface of the red cell. In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.
A positive DAT can occur without hemolysis
Immune causes of hemolysis including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions are characterized by a positive DAT.
INDIRECT ANTIGLOBULIN TEST (IAT)
The IAT (antibody screen) is performed by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination. If the antibody screen is positive, additional testing is required to determine the specificity of the antibody.
The IAT is used to detect red cell antibodies in patient serum. Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.
3. Complement fixation test (CFT)
the ability of antigen antibody complexes to fix complement is made use in this test. Complement is something which takes part in any immunological reaction and absorbed during the combining of antigen with its specific antibody.
The best example of CFT is the Wassermann reaction done for the detection of Syphilis.
4. Neutralization test
different types of these are available. Virus neutralization, toxin neutralization, etc. are some of its kind.
5. Opsonization
this makes use of the determination of opsonic index, which is the ratio of the phagocytic activity of patient’s blood to the phagocytic activity of the normal patient’s for a given bacterium.
6. Immunfluorescence
the method of labeling the antibodies with fluorescent dyes and using them for the detection of antigens in tissues.
7. Radioimmunoassay (RIA)
is a competitive binding radioisotopes and enzymes are used as labels to conjugate with antigens or antibodies.
8. Enzyme Immuno Assay (EIA)
the assays based on the measurement of enzyme labeled antigen or antibody. The most common example is ELISA used to detect HIV.
9. Immunoelectroblot
it uses the sensitivity of Enzyme immunoassay with a greater specificity. Example is Western blot done for the serodiagnosis of HIV infection.