Immunoglobulin (Ig)

Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field

FUNCTION
1. Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.

2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

STRUCTURE OF IMMUNOGLOBULINS

The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.

A. Heavy and Light Chains

All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)

B. Disulfide bonds

1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.

2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions

When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:

1. Light Chain - VL (110 amino acids) and CL (110 amino acids)

2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.

E. Domains

Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.

1. Light Chain Domains - VL and CL

2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

F. Oligosaccharides

Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations. 

IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS

Immunoglobulin fragments produced by proteolytic digestion –

A.  Fab 
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

Antigen binding – These fragments are  called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. 

B. Fc 
Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.

Effector functions – The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment . 

Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.
 

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.

Immunofluorescence

This is precipitation or complement fixation tests. The technique can detect proteins at concentrations of around 1 µg protein per ml body fluid. Major disadvantage with this technique is frequent occurrence of nonspecific fluorescence in the tissues and other material.
The fluorescent dyes commonly used are fluorescein isothocyanate (FITC). These dyes exhibit fluorescence by absorbing UV light between 290 and 495 nm and emitting longer wavelength coloured light of 525 nm which gives shining appearance (fluorescence) to protein labelled with dye. Blue green (apple green) fluorescence is seen with FITC and orange red with rhodamine.

Enzyme Immunoassays

These are commonly called as enzyme linked immunosorbent assays or EL1SA. It is a simple and versatile technique which is as sensitive as radioimmunoassays. It is now the
technique for the detection of antigens, antibodies, hormones, toxins and viruses.

Identification of organisms by immunofluorescence

Type of agent         Examples

Bacterial            Neisseria gonorrhoeae, H. influenzae ,Strept pyogenes, Treponema pallidum
Viral                  Herpesvirus, Rabiesvirus, Epstein-Barr virus
Mycotic             Candida albicans

Enzymatic activity results in a colour change which can be assessed visibly or quantified in a simple spectrophotometer.

Bacteria

A bacterial cell has a nuclear apparatus which is a loose arrangement of DNA This is surrounded cytoplasm which contains ribosomes, mesosomes and inclusion granules. The cytoplasm is enclosed within a cytoplasmic membrane. Bacterium has a rigid cell wall  Fimbriae and flagella are the surface adherents. Some bacteria may have a capsule (or loose slime) around the cell wall.

Shape and Size of Bacteria

The bacteria can be spheroidal (coccus), rod or cylindrical (bacillus) and spirillar (spirochaete). Very short bacilli are called as coccobacilli  Some of the bacilli may be curved or comma shaped (Vibrio cholerae).

Arrangement of Bacterial Cells

Streptococci are present in chains; staphylococci in grape-like clusters Cocci in pairs (diplococci) are suggestive of pneumococci, gonococci or menigococci.
Bacilli do not exhibit typical arrangement pattern except the Chinese letter arrangement shown by Corynebacterium diphtheriae

Surface Adherents and Appendages

CAPSULE The gels formed by the capsule adhere to the cell Capsule can be detected by negative staining ,with specific antiserum and observing the capsular swelling phenomenon called as Quellung reaction
Usually weakly antigenic Capsule production is better in vivo as compared to in vitro environment.
Eg. Capsules seen in Pneumococci,  Klebsiella, Escherichia coli, Haemophilus influenzae

Flagella : provide motility to the bacterium. 
Motile organisms: vibrios, pseudomonas, Esch.coli, salmonellae, spirochaetes and spirilla. 
Pathogenic cocci are nomotile.
Flagella measure in length from 3 to 20 µm and in diameter from 0.01 to 0.0 13 µm.
 
Arrangement

Bacteria with one polar flagellum are known as monotrichous; 
Tuft of several polar flagellae is known as lophotrichous
Presence of  Flagellae at both the ends of organism is amphitrichous 
Flagellae distributed all over the surface of the bacterium, it is called peritrichous.
•    Filament is composed of a protein-flagellin. The flagellar antigen is called as H (Hauch) antigen in contrast to somatic antigen which is called as O (Ohne haunch)

PILI (fimbriae) : hair like structures help in attachment also called sex pilli, transfers genetic material through conjugation , Present in Certain Gram negative bacteria. Only Composed of protein pilin  
Gram positive bacterium that has pili is Cornebacterium renale

The Cell Wall

The cell wall of  bacteria is multilayered structure. The external surface of cell wall is smooth in Gram positive bacteria  Gram negative bacteria have convoluted cell surfaces. The average thickness of cell wall is 0.15 to 0.50 .µm. Chemically composed of mucopeptide scaffolding formed by N acetyl glucosamine and N acetyl muramic acid
The cell wall is a three layered structure in Gram negative bacteria: outer membrane middle layer and plasma membrane. The outer membrane consists of lipoprotein and 1ipoppolysaccaride component

Functions of bacterial cell wall

 Provides shape , Gives rigidity , Protection, Surface has receptor sites for phages, Site of  antibody action,  Provides attachment to complement, Contains components toxic to host
 
Cytoplasmic Structures

The Plasma Membrane: This delicate membrane separates rigid cell wall from cytoplasm. It accounts for 30% of total cell weight. Chemically, it is 60% protein, 20-30% lipids and remaining carbohydrates.

 Mesosomes: 
 
 Principal sites of respiratory enzyme , Seen well in Gram positive bacteria as compared to Gram negative batcteria. Attachement of mesosomes to both DNA chromatin and membrane have been noticed thus help in cell division
 
Ribosomes: 

sites of protein synthesis. These are composed of RNA and proteins and constitute upto 4 of total cell protein and 90% of total cellular RNA.
Cytoplasmic Granules: Glycogen  :  Enteric bacteria
Poly-beta & hydroxy Butyrate : Bacillus & Pseudomonas
Babes-Ernst  :Corynebacterium & Yersinia pestis

Nuclear Apparatus

Bacterial DNA represents 2-3% of the cell weight and 10% of the volume of bacterium. Nucleous can be demonstrated by staining it with DNA specific Fuelgen stain .Consists of a single molecule of  double stranded DNA arranged in a circular form. Bacterial chromosome is haploid and replicates by binary fission, the bacteria may have  plasmid an extrachromosomal genetic material.
 

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

ANTIGEN-ANTIBODY REACTIONS

Affinity of the antigen-antibody reaction refers to the intensity of the attraction between antigen and antibody molecule.
Antigen-antibody reactions

Reaction test            Modified test

Precipitation  -> Immunoelectrophoresis, Immunoprecipitation
Agglutination -> Latex agglutination, Indirect, Haemagglutination , Coagglutination ,Coombs test

Neutralization  -> Measurement of LD, Plaque assays

Complement fixation  -> Conglutination

Immunofluorescence ->  Indirect immunofiuorescence, Immunoofluoremetric Assay

Enzyme immunoassay -> Enzyme linked, Immunosorbent assay

Radioimmunoassay -> Immunoradiometric assay

Avidity is the strength of the bond after the formation of antigen-antibody complex.

Sensitivity refers to the ability of the test to detect even very minute quantities of antigen or antibody. A test shall be called as highly sensitive if false negative results are absent or minimal.

Specificity refers to the ability of the test to detect reactions between homologous antigens and antibodies only, and with no other. In a highly specific test, false positive reactions will be minimal or absent.