Autoantibodies

Anti-nuclear antibodies (ANA)    Systemic Lupus
Anti-dsDNA, anti-Smith               Specific for Systemic Lupus
Anti-histone                                 Drug-induced Lupus
Anti-IgG                                       Rheumatoid arthritis
Anti-neutrophil                             Vasculitis
Anti-centromere                           Scleroderma (CREST)
Anti-Scl-70                                   Sclerderma (diffuse)
Anti-mitochondria                         1^oary biliary cirrhosis
Anti-gliadin                                   Celiac disease
Anti-basement membrane            Goodpasture’s syndrome
Anti-epithelial cell                          Pemphigus vulgaris
Anti-microsomal                            Hashimoto’s thryoiditis

Enzymes:

Serum lysozyme:

Provides innate & nonspecific immunity
Lysozyme is a hydrolytic enzyme capable of digesting bacterial cell walls containing peptidoglycan 
•    In the process of cell death, lysosomal NZs fxn mainly to aulolyse necrotic cells (NOT “mediate cell degradation”)
•    Attacks bacterial cells by breaking the bond between NAG and NAM.
•    Peptidoglycan – the rigid component of cell walls in most bacteria – not found in archaebacteria or eukaryotic cells
•    Lysozyme is found in serum, tears, saliva, egg whites & phagocytic cells protecting the host nonspecifically from microorganisms

Superoxide dismutase: catalyzes the destruction of O2 free radicals protecting O2-metabolizing cells against harmful effects 

Catalase:

- catalyzes the decomposition of H2O2 into H2O & O2
- Aerobic bacteria and facultative anaerobic w/ catalase are able to resist the effects of H2O2
- Anaerobic bacteria w/o catalase are sensitive to H2O2  (Peroxide), like Strep
- Anaerobic bacteria (obligate anaerobes) lack superoxide dismutase or catalase
- Staph makes catalase, where Strep does not have enough staff to make it

Coagulase

- Converts Fibronogen to fibrin
•    Coagulase test is the prime criterion for classifying a bug as Staph aureus – from other Staph species
•    Coagulase is important to the pathogenicity of S. aureus because it helps to establish the typical abscess lesion 
•    Coagulase also coats the surface w/ fibrin upon contact w/ blood, making it harder to phagocytize

THE PLASMIDS

The extrachromosomal genetic elements, called as plasmids are autonomously replicating , cyclic ,double stranded DNA molecules which are distinct from the cellular chromosome 

Classification

Plasmids can be broadly classified as conjugative and nonconjugative. 

Conjugative plasmids are large and self-transmissible i.e. they have an apparatus through which they can mediate their own transfer to another cell after coming in contact with the same. Example:  RF and certain bacteriocinogen plasmids.

Nonconjugative plasmids are small in size and can be mobilised for transfer into another cell only through the help of a conjugative plasmid. To this group belong some ‘r’ determinants and few bacteriocinogenic plasmids. Plasmids can also be transferred without cell contact by the process of transfection.

Properties of plasmids

Double stranded DNA , Autonomously replicate in host cell, Plasmd specific, Free DNA is transferred b transfection

Significance of Plasmids :The spread of resistance to antibiotics is one such well known example. These also play an important  role in the geochemical  cycle by spreading genes for the degradation of complex organic compounds.
 

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.
 

PHYSICAL AGENTS

Heat occupies the most important place as a physical agent.

Moist Heat : This is heating in the presence of water and can be employed in the following ways:

Temperature below 100°C: This includes holder method of Pasteurization where 60°C for 30 minutes is employed for sterilization and in its flash modification where in objects are subjected to a temperature of 71.1°C for 15 seconds. This method does not destroy spores.

Temperatures Around 100°C : Tyndallization is an example of this methodology in which steaming of the object is done for 30 minutes on each of three consecutive days. Spores which survive the heating process would germinate before the next thermal exposure and would then be killed.

Temperatures Above 100°C : Dry saturated steam acts as an excellent agent for sterilization. Autoclaves have been designed on the principles of moist heat.

Time-temperature relationship in heat sterilization
Moist heat   (autoclaving)

121°C       15 minutes
126°C         10 minutes
134 C          3 minutes

Dry heat

>160°C    >120 minutes
>170°C    >60minutes
>180°C    >30 minutes

Mechanism of microbial inactivation 

The autoclaving is in use for the sterilization of many ophthalmic and parentral products. surgical dressings, rubber gloves, bacteriological media as well a of lab and hospital reusable goods.

Dry Heat: Less efficient,  bacterial spores are most resistant. Spores may require a temperature of 140° C for three hours to get killed.
Dry heat sterilization is usually carried out by flaming as is done in microbiology laboratory to sterilize the inoculating loop and in hot air ovens in which a number of time-temperature combinations can be used. It is essential that hot air should circulate between the objects to be sterilized. Microbial inactivation by dry heat is primarily an oxidation process.

Dry heat is employed for sterilization of glassware glass syringes, oils and oily injections as well as metal instruments.    -

Indicators of Sterilization:  
These determine the efficacy of heat sterilization and can be in the form of spores of Bacillus stearothermophilus (killed at 121C in 12 minutes) or in the form of chemical indicators, autoclave tapes and thermocouples.

Ionizing Radiations

Ionizing radiations include X-rays, gamma rays and beta rays, and these induce defects in the microbial DNA synthesis is inhibited resulting in cell death. Spores are more resistant to ionizing radiations than nonsporulating bacteria.

The ionizing radiations are used for the sterilization of single use disposable medical items.

Mechanism of microbial inactivation by moist heat

Bacterial spores

•    Denaturation of  spore_epzymes
•    Impairment of germination
•    Damage to cell membrane
•    Increased sensitivity to inhibitory agents
•    Structural damage
•    Damage to chromosome

Nonsporulating bacteria

•    Damage to cytoplasmic membrane
•    Breakdown of RNA
•    Coagulation  of proteins
•    Damage to bacterial chromosome

Ultraviolet Radiations : 
wave length 240-280 nm have been found to be most efficient in sterilizing. Bacterial spores are more resistant to U.V. rays than the vegetative forms. Even viruses are sometimes more resistant than vegetative bacteria.

Mechanism of Action :

Exposure to UV rays results in the formation of purine and pyrimidine diamers between adjacent molecules in the same strand of DNA. This results into noncoding lesions in DNA and bacterial death.
Used to disinfect drinking water, obtaining pyrogen free water, air disinfection (especially in safety laboratories, hospitals, operation theatres) and in places where dangerous microorganisms are being handled.

Filteration

Type of Filters

Various types of filters that are available are    /
Unglazed ceramic filter (Chamberland and Doulton filters)
Asbestos filters (Seitz, Carlson and Sterimat filters)
Sintered glass filters

Membrane filters

Membrane filters are widely used now a days. Made up of cellulose ester and are most suitable for preparing_sterile solutions. The range of pore size in which these are available is 0.05-12 µm whereas the required pore size for sterlization is in range of 0.2-0.22 p.m.

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.