Precipitation 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
 

NITRIC OXIDE-DEPENDENT KILLING

Binding of bacteria to macrophages, particularly binding via Toll-like receptors, results in the production of TNF-alpha, which acts in an autocrine manner to induce the expression of the inducible nitric oxide synthetase gene (i-nos ) resulting in the production of nitric oxide (NO) . If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric oxide will be produced (figure 12). Nitric oxide released by the cell is toxic and can kill microorganism in the vicinity of the macrophage.

Neutralization Test

These are basically of two types:

•    Toxin neutralization
•    Virus neutralization

In toxin neutralization homologous anti-bodies prevent the biological effect of toxin as observed in vivo in experimental animals (e.g. detection of toxin of Clostridia and Corynebacterium diphthenae) or by in vitro method (e.g. Nagler’s method).

In virus neutralization test various methods are available by which identity of virus can be established as well as antibody against a virus can be estimated.

Variant Forms of Bacteria

Prortoplast ; surface is completely devoid of cell wall component,

Spheroplast : Some residual cell wall component is present 

Autoplast: protoplasts which are produced by the action of organisms’ own autolytic enzymes.

L Form: replicate as pleomorphic filtrable elements with defective or no cell wall These are designated as L forms after the Lister Institute where these were discovered by Klineberger-Nobel.

Bacterial Spores: Gram positive bacilli and actinomycetes form highly resistant and dehydrated forms which are called as endospores. The surrounding mother.cell which give rise to them is known as Sporangium. These endospores are capable of survival under adverse conditions
Structure :smooth walled and ovoid or spherical. 

In bacilli, spores usually fit into the normal cell diameter except in Clostridium where these may cause a terminal bulge. (drum stick ) or central. , these look like areas of high refractilitv under light microscope.

Germination : This is the process of converting a spore into the vegetative cell. It occurs in less than 2 hours and has three stages:Activation, Germination, Outgrowth
 

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