NEET MDS Lessons
General Microbiology
Test 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.
CHEMICAL AGENTS
Chlorine and iodine are most useful disinfectant Iodine as a skin disinfectant and chlorine as a water disinfectant have given consistently magnificent results. Their activity is almost exclusively bactericidal and they are effective against sporulating organisms also.
Mixtures of various surface acting agents with iodine are known as iodophores and these are used for the sterilization of dairy products.
Apart from chlorine, hypochlorite, inorganic chioramines are all good disinfectants but they act by liberating chlorine.
Hydrogen peroxide in a 3% solution is a harmless but very weak disinfectant whose primary use is in the cleansing of the wound.
Potassium permanganate is another oxidising agent which is used in the treatment of urethntzs.
Formaldehyde — is one of the least selective agent acting on proteins. It is a gas that is usually employed as its 37% solution, formalin.
When used in sufficiently high concentration it destroys the bacteria and their spores.
Classification of chemical sterilizing agents
Chemical disinfectant
Interfere with membrane functions
• Surface acting agents : Quaternary ammonium, Compounds, Soaps and fatty acids
• Phenols : Phenol, cresol, Hexylresorcinol
• Organic solvent : Chloroform, Alcohol
Denatures proteins
• Acids and alkalies : Organic acids, Hydrochloric acid , Sulphuric acid
Destroy functional groups of proteins
• Heavy metals : Copper, silver , Mercury
• Oxidizing agents: Iodine, chlorine, Hydrogen peroxide
• Dyes : Acridine orange, Acriflavine
• Alkylating agents : Formaldehyde, Ethylene oxide
Applications and in-use dilution of chemical disinfectants
Alcohols : Skin antiseptic Surface disinfectant, Dilution used 70%
Mercurials : Skin antiseptic Surface disinfectant Dilution Used 0.1 %
Silver nitrate : Antiseptic (eyes and burns) Dilution Used 1 %
Phenolic compound : Antiseptic skin washes Dilution Used .5 -5 %
Iodine : Disinfects inanimate object, Skin antiseptic Dilution used 2%
Chlorine compounds : Water treatment Disinfect inanimate objects , Dillution used 5 %
Quaternary ammonium Compounds : Skin antiseptic , Disinfects inanimate object, Dilution Used < 1 %
Glutaraldehyde: Heat sensitve instruments, Dilution used 1-2 %
Cold sterilization can be achieved by dipping the precleaned instrument in 2% solution of gluteraldehyde for 15-20 minutes. This time is sufficient to kill the vegetative form as well as spores ofthe organisms that are commonly encountered in the dentistry.
Ethylene oxide is an a agent extensively used in gaseous sterilization. It is active against all kinds of bacteria and their spores. but its greatest utility is in sterilizing those objects which are damaged by heat (e.g. heart lung machine). It is also used to sterlise fragile, heat sensitive equipment, powders as well as components of space crafts.
Evaluation of Disinfectants
Two methods which are widely employed are:
Phenol coefficient test, Kelsey -Sykes test
These tests determine the capacity of disinfectant as well as their ability to retain their activity.
Measurement of Bacterial of Growth
A convenient method is to determine turbidity by photoelectric colorimeter or spectrophotometer.
The cell number can be counted as total cell number as well as viable count. Viable Count Viable number of bacteria can be counted by inoculating the suspension onto solid growth medium and counting the number of colonies. Since each colony is the end product of one viable bacterium, their count gives the number of viable bacteria in the suspension.
Total number of bacteria can be ascertained in specially designed chambers such as Coulter counter.
ANTIGENS
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).
Complement Fixation Test (CFT)
This test is based upon two properties of the complement viz:
a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.
Test system
It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.
Indicator system
To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.
NUTRITION OF BACTERIA
Nutrients
Chemoheterotrophs: nutrient source is organic material
Bacteria also requires a source of minerals.
Oxygen
On this basis bacteria have been divided into four groups.
Obligate Anaerobes: These grow only under conditions of high reducing intensity. These bacteria catalase peroxidase, superoxide dismutase and cytochrome systems
Clostridium and Bacteroides are important examples.
Facultalive Anaerobes. These can grow under both aerobic and anaerobic conditions and include members of family enterobacteriaceae and many other bacteria.
Obligatory Aerobes. These cannot grow unless oxygen is present in the medium. Pseudomonas belong to this group.
Microaerophillic. These organisms can grow under conditions with low oxygen tension. Clostridium tetani is an important example.
The strict anaerobes are unable to grow unless Eh is as low as 0.2 volt
Temperature
• On the basis of temperature requirements, three groups of bacteria are recognised.
• Psychrophilic : Growth in the range of —5 to 30°C with an optimum of 10-20
• Mesophillic : bacteria grow best at 20-40°C with a range of 10-45°C.
• Medically important bacteria belong to this group
• Myco. leprae is one such important example and it can grow only at reduced temperature such as footpad of mouse
• Thermophillic organisms prefer high temperature (25-80°C) for growth and yield maximum growth at 50-60°C
pH : Most pathogenic bacteria require a pH of 7.2-7.6 for their own optimal growth.
Immunology:
The branch of life science which deals with immune reaction is known as immunology.
Components of Immune System:
The immune system consists of a network of diverse organs and tissue which vary structurally as well as functionally from each other. These organs remain spreaded throughout the body. Basically, immune system is a complex network of lymphoid organs, tissues and cells.
These lymphoid organs can be categorized under three types depending upon their functional aspects:
i. Primary lymphoid organ.
ii. Secondary lymphoid organ.
iii.Tertiary lymphoid organ.
White blood cells or leukocytes are the basic cell types which help to give rise to different types of cells which participate in the development of immune response . WBC are classified into granulocytes and agranulocytes depending on the presence or absence of granules in the cytoplasm.
Agranular leukocytes are of two types, viz., lymphocytes and monocytes. Lymphocytes play pivotal role in producing defensive molecules of immune system. Out of all leukocytes, only lymphocytes possess the quality of diversity, specificity, memory and self-non self recognition as various important aspects of immune response.
Other cell types remain as accessory one; help to activate lymphocytes, to generate various immune effector cells, to increase the rate of antigen clearance
All cells of the immune system have their origin in the bone marrow
myeloid (neutrophils, basophils, eosinpophils, macrophages and dendritic cells)
lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells .
The myeloid progenitor (stem) cell in the bone marrow gives rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T cells and B cells.
For T cell development the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD4+ T helper cell and the CD8+ pre-cytotoxic T cell.
Two types of T helper cells are produced in the thymus the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic T cells, and TH2 cells, which help B cells, differentiate into plasma cells, which secrete antibodies.
Function of the immune system is self/non-self discrimination.
This ability to distinguish between self and non-self is necessary to protect the organism from invading pathogens and to eliminate modified or altered cells (e.g. malignant cells).
Since pathogens may replicate intracellularly (viruses and some bacteria and parasites) or extracellularly (most bacteria, fungi and parasites), different components of the immune system have evolved to protect against these different types of pathogens.