Antibody

‘Antibody’ the heart of defence system

Immunity—the state of protection from infectious disease has less specific component, innate immunity, provides the first line of defence against infection and are present before the onset of infection it involves skin, mucous membrane, physiologic barriers (Temperature, pH, Lysozyme, Interferon,) Phagocytic and endocytic barriers, Inflammatory barrier. In contrast to innate immunity other is more specific component, adaptive immunity (acquired immunity) does not come into play until there is an antigenic challenge to the organism it involves humoral immunity (antibody) and cellular immunity (T-cells and macrophage).

When an antigen invade into body it is digested by cells and presented by cells on its surface, this altered cells present antigen. To this altered cells MHC (major histocompatibility complex) molecules bound, MHC II bounded cells are responsible for the activation of T helper cell (TH). Activated TH cells release cytokines that contributes to the activation of B cells and cytotoxic T (Tc) cells. Activation of B cells differentiate into plasma cells and memory cells, plasma cells contributes to antibody production while memory cells are responsible for the memorizing of that antigen in future invading. MHC I molecules bounded cells activate Tc cells that bind to altered cells and kill them.

Antiboody

Antibody is a glycoprotein produces by plasma cells in response to an immunogen (Ig)/foreign materials. 

Human have five Igs class and all have ‘Y’ shaped structure with two identical heavy chain and two identical light chain held together by bisulfide linkages and non-covalent interaction as shown in figure 1.

There are five types of heavy chain and antibody has the named on the basis of heavy chain which they contains while light chains are only two types κ and λ and an antibody contains a single type of light chain never a mixture of these. Light chain and hinge regions are responsible for the subclass of antibodies like IgG have four subclass on the basis of hinge region eg. IgG1, IgG2, IgG3, IgG4 etc. Constant region of antibody is glycosylated and this region only responsible for immune response eg. If antibody produces in human ingested to mouse than mouse produces mouse-antihuman-antibody due to constant region of that antibody while the paratope (antigen binding region) of variable region is responsible for binding to the epitope (antibody binding region) of antigen.

These five classes of antibodies are:     

IgG  have heavy chain γ, percentage 75-80% and monomer in structure having ability to cross placenta and transfer from mother to foetus,

IgA have heavy chain α, percentage 10-15%, dimer in structure and predominant in clostrum mainly found in secretion,

IgM have heavy chain μ, percentage 5-10%, pentamer and first antibody produces during an infection and the first antibody produces by neonant,

IgE have heavy chain ε, percentage 0.002%, monomer in structure and tightly bound to mast cells and in response to allergen release histamine and causes allergy,

IgD have heavy chain δ, percentage 0.2%, and monomer in structure, found on B cell surface, and serve as B cell receptor,

Antibody IgM and IgE do not have hinge region but they contain one extra constant region thus these two antibodies have four constant region in heavy chain while IgA, IgD, IgG has hinge region and have three constant region. 

Polyclonal Vs Monoclonal antibody

Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body whereas monoclonal antibodies come from a single B cell lineage. 

Most antigens encountered naturally (e.g. proteins, viruses, bacteria, etc.) contain hundreds, if not thousands, of different epitopes. A typical epitope region on a protein surface would comprise five to seven amino acid residues. A specific B-lymphocyte produces each specific antibody, which recognizes a specific epitope. If one single antibody-producing cell could be isolated and cultured in vitro, then it would be a source of monoclonal (monospecific) antibody. However, B-lymphocytes die after a short time when cultured in vitro and, hence, are an impractical source of long-term antibody production so it fused with myeloma cell (cancerous immune cell) and produce hybrid cell be immortal by hybridoma techniques.

HYBRIDOMA techniques:

Hybridoma technology was discovered in 1975 by two scientists, Georges Kohler of West Germany and Cesar Milstein of Argentina. In this, an antigen against which we are interested in isolating an antibody first infects experimental animals (eg. mice). Once splenocytes are isolated from the mammal, the B cells are fused with immortalized myeloma cells - which lack the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) gene - using polyethylene glycol or the Sendai virus. Fused cells are incubated in the HAT (Hypoxanthine Aminopetrin Thymidine) medium. Aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine (a purine derivative) and thymidine (a deoxynucleoside) which are intermediates in DNA synthesis.  In HAT medium unfused myeloma cells die, as they cannot produce nucleotides by the de novo or salvage pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die. Unfused B cells die as they have a short life span. Only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells).  This medium is diluted and pick up a single clone, which produces a single type antibody called monoclonal antibody.

Polyclonal Antibody

Advantages:

·         Recognize multiple epitopes on any one antigen.

·         Peptide immunogens are often used to generate polyclonal antibodies.

·         Most polyclonals are either goat or rabbit polyclonals. However, other species can be used (e.g. mouse, rat, chicken).

·         Polyclonals are more tolerant of minor changes in the antigen

·         polyclonal antibodies can be used when the nature of the antigen in an untested species is not known

·         Polyclonal antibodies are often the preferred choice for detection of denatured proteins.

·         Multiple epitopes generally provide detection that is more robust.

·         Disadvantages:

·         Batch-to-batch variability.

·         Produce large amounts of non-specific antibodies.

·         Chances of cross-reactivity.

·         Cannot use for specific domains of antigen, because antiserum will usually recognize many domains.

Monoclonal Antibody

Advantages:

·         High specificity. Detect only one epitope on the antigen.

·         They will consist of only one antibody subtype (e.g. IgG1, IgG2, IgG3). Where a secondary antibody is required for detection.

·         Monoclonals are typically rat or mouse monoclonals. However, monoclonals can be generated from various species such as rabbit and goat. 

·         Monoconal antibody can be produced by recombinant techniques.

·         No batch to batch variation

·         Can use as primary as well as secondary antibody

·         Very high homogeneity  

Disadvantages:

·         Due to high specificity  (e.g. less likely to detect in across a range of species).

·         More vulnerable to the loss of epitope through chemical treatment of the antigen than polyclonal antibodies are.

Therapeutic classification of Monoclonal antibody (mAb)

Therapeutically mAb classified into four classes as shown below Figure 2.

Fig.2 Different classes of mAb

Murine

Initial therapeutic antibodies were murine analogues (suffix -omab). These antibodies have a short half-life in vivo (due to immune complex formation), limited penetration into tumour sites and inadequately recruit host effector functions.

Chimeric

Chimeric antibodies (suffix -ximab) are composed of murine variable regions fused onto human constant regions (Figure 2). Taking human gene sequences from the kappa light chain and the IgG1 heavy chain results in antibodies that are approximately 65% human. This reduces immunogenicity, and thus increases serum half-life.

Humanized

Humanised antibodies (suffix -zumab) are produced by grafting murine hypervariable regions (CDR-complementry determining region) on amino acid domains into human antibodies. This results in a molecule of approximately 95% human origin.

Human

Human monoclonal antibodies (suffix -umab) are produced using transgenic mice or phage display libraries by transferring human immunoglobulin genes into the murine genome and vaccinating the transgenic mouse against the desired antigen, leading to the production of appropriate monoclonal antibodies.Murine antibodies in vitro are thereby transformed into fully human antibodies.