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
 

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

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.
 

NON-SPECIFIC KILLER CELLS

Several different cells including NK and LAK cells, K cells, activated macrophages and eosinophils are capable of killing foreign and altered self target cells in a non-specific manner. These cells play an important role in the innate immune system.

A. NK and LAK cells

Natural killer (NK) cells are also known as large granular lymphocytes (LGL) because they resemble lymphocytes in their morphology, except that they are slightly larger and have numerous granules.

NK cells can be identified by the presence of CD56 and CD16 and a lack of CD3 cell surface markers.

NK cells are capable of killing virus-infected and malignant target cells but they are relatively inefficient in doing so.

However, upon exposure to IL-2 and IFN-gamma, NK cells become lymphokine-activated killer (LAK) cells, which are capable of killing malignant cells.

Continued exposure to IL-2 and IFN-gamma enables the LAK cells to kill transformed as well as malignant cells. LAK cell therapy is one approach for the treatment of malignancies.

NK and LAK cells have two kinds of receptors on their surface – a killer activating receptor (KAR) and a killer inhibiting receptor (KIR). 

When the KAR encounters its ligand, a killer activating ligand (KAL) on the target cell the NK or LAK cells are capable of killing the target. However, if the KIR also binds to its ligand then killing is inhibited even if KAR binds to KAL. 

The ligands for KIR are MHC-class I molecules. Thus, if a target cell expresses class I MHC molecules it will not be killed by NK or LAK cells even if the target also has a KAL which could bind to KAR. 

Normal cells constitutively express MHC class I molecules on their surface, however, virus infected and malignant cells down regulate expression of class I MHC. Thus, NK and LAK cells selectively kill virus-infected and malignant cells while sparing normal cells.

B. K cells 

Killer (K) cells are not a morphologically distinct type of cell. Rather a K cell is any cell that mediates antibody-dependent cellular cytotoxicity (ADCC). 

In ADCC antibody acts as a link to bring the K cell and the target cell together to allow killing to occur. K cells have on their surface an Fc receptor for antibody and thus they can recognize, bind and kill target cells coated with antibody. 

Killer cells which have Fc receptors include NK, LAK, and macrophages which have an Fc receptor for IgG antibodies and eosinophils which have an Fc receptor for IgE antibodies.

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.

COMPLEMENT

The complement system primarily serves to fight bacterial infections. 

The complement system can be activated by at least three separate pathways. 
1) alternative pathway -
- The alternative pathway of complement activation starts with the spontaneous hydroysis of an internal thioester bond in the plasma complement component C3 to result in C3(H2O).

- The smaller cleavage products C3a, C4a, C5a, sometimes called "anaphylatoxins", act as phagocytes, they cause mast cell degranulation and enhance vessel permeability, thereby facilitating access of plasma proteins and leukocytes to the site of infection

- alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defense against a number of infectious agents.

2) Lecithin Pathway 

The lectin pathway of complement activation exploits the fact that many bacterial surfaces contain mannose sugar molecules in a characteristic spacing. The oligomeric plasma protein mannan-binding lectin (MBL; lectins are proteins binding sugars) binds to such a pattern of mannose moieties, activating proteases MASP-1 and MASP-2 (MASP=MBL activated serine protease, similar in structure to C1r and C1s). These, by cleaving C4 and C2, generate a second type of C3 convertase consisting of C4b and C2b, with ensuing events identical to those of the alternative pathway.

3) classical pathway

The classical pathway usually starts with antigen-bound antibodies recruiting the C1q component, followed by binding and sequential activation of C1r and C1s serine proteases. C1s cleaves C4 and C2, with C4b and C2b forming the C3 convertase of the classical pathway. Yet, this pathway can also be activated in the absence of antibodies by the plasma protein CRP (C-reactive protein), which binds to bacterial surfaces and is able to activate C1q.

Pharmacology cross reference: humanized monoclonal antibody Eculizumab binds to complement component C5, inhibiting its cleavage and preventing activation of the lytic pathway. This is desirable when unwanted complement activation causes hemolysis, as in paroxysmal nocturnal hemoglobinuria or in some forms of hemolytic uremic syndrome. For the lytic pathway's importance in fighting meningococcal infections, Eculizumab treatment increases the risk of these infections, which may be prevented by previous vaccination.

 BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION

Activation of complement results in the production of several biologically active molecules which contribute to resistance, anaphylaxis and inflammation.

Kinin production
C2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs from the C1qrs complex (Figure 10). A genetic deficiency of C1-INH results in an overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases plasmin activity.

Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA).

Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells.

Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.
Other Biologically active products of C activation
Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their functions.