In this article we will discuss about:- 1. Background and Objective of Monoclonal Antibody 2. Procedure of Monoclonal Antibody 3. Use in Taxonomy 4. Properties.
Antibodies are tools widely applicable to studies of structure, nomenclature and function of the molecule they specifically recognize—the antigen. Classically, an antigen is defined as an organism, a molecule or part of a molecule that is recognised by the immune system. The organism is identified by using monoclonal antibody technique which is of great importance for taxonomic use.
Monoclonal antibody is a homogenous preparation of antibody molecules, produced by a single clone of B-lineage cells, often a hybridoma, all of which have the same antigenic specificity. Serological taxonomy is a current and advanced biochemical branch. It is used in the field of biotechnology and parasitology on the basis of immune response to distinguish the strain. Monoclonal antibody is one of them.
Background and Objective of Monoclonal Antibody:
In 1975, C. Milstein and G. Kohler discovered that large amounts of homogenous antibody of nearly any desired specificity can be obtained by fusing an antibody producing cell with a myeloma cell (cancerous plasma cell).
The main objective of this technique is the production of a specific antibody (Monoclonal antibody, McAb) against a predefined immunogen. For the purpose the fusion of 2 cells from a suitable immunized animal gives a permanent cell lines of hybrid cells which produce monoclonal antibodies of desired properties.
For the production of a specific antibody Kohler and Milstein fused the spleen cells containing the B-lymphocytes and myeloma cells of the same strain and after fusion was raised in the HAT (Hypoxanthine aminopterine and thymine) medium. This technique is known as Hybridoma technique (Hybrid of myeloma).
Significance of HAT Medium:
The role of aminopterine in this medium used to block do novo synthesis of nucleotides; hypoxanthine can’t be used by the unfused myeloma cell because they lack HGPRT (Hypoxanthine- Guanosine-Phosphoribosyl-transferase) for the synthesis of inosinate in the salvage pathway of the DNA synthesis.
So the unfused cell line readily die in tissue culture because they are not able to proliferate in vitro. In contrast, hybrid cells survive because they have neoplastic character of the myeloma parent cell and contain HGPRT genes of their spleen parent cell. They are called hybridoma cells.
Procedure of Monoclonal Antibody:
1. Myeloma cells and spleen cells are taken from BALB(c) mice and LOU rat which are suitably immunized.
2. The spleen cells die quickly within ordinary tissue culture medium whereas myeloma cells are adapted to grow permanently in the culture.
3. So for the production of a specific antibody against a predetermined antigen, the myeloma ceils and the spleen cells are fused and are raised in the culture medium which is known as HAT medium.
4. In the HAT medium, mutant forms lacking HGPRT are eliminated from culture.
5. In HAT medium, aminopterine usually blocks the de novo DNA synthesis.
6. In the culture salvage pathway could be used to proper cells having HGPRT cell components. Mutants lacking HGPRT are unable to utilize salvage pathway.
7. The fused cells are cultured in pots and these hybrids are actively multiplied in the HAT medium.
8. From growing hybrids, +ve clone is identified by characterisation process like radio-immuno assay (RIA) or enzyme linked immunosorbent assay (ELISA) to find out this immunological characters.
9. The cultures are diluted serially and grown further against a particular cell.
10. The other steps in between are kept in liquid N2 for further use.
11. The selected +ve clones secrete the desired antibody. Such antibodies are monoclonal in origin as they originated from a single type of hybrid cell.
12. Monoclonal probes are prepared from the selected +ve clones and are maintained in the lab both in vivo and in vitro conditions.
Use in Taxonomy (Applications in Biology) of Monoclonal Antibody:
In the New World, two types of Leishmania sp. are found; L. braziliensis causes mucocutaneous leishmaniasis which eats into the cartilage core and L. mexicana which causes subcutanieous leishmaniasis (plane skin disease).
These 2 spp. can be separated from each other by:
1. Buoyant density of nuclear and kinetoplast DNA.
2. Electrophoretic mobility of different isozymes.
3. Different characteristic growth patterns in the Sand-fly and Hamster and in vitro.
4. Size in EM.
But unfortunately there is no available method for rapid and accurate diagnosis of fresh isolates of New World Leishmania sp.
Furthermore, immunological cross-reaction of Leishmania with Trypanosoma cruzi, (causing American sleeping sickness) which is co-endemic with the former, has posed a serious problem in the use of serodiagnostic test for Leishmaniasis.
To solve this problem and develop a specific diagnostic test, Pratt and David (1981) have produced McAb specific for either L. mexicana or L. braziliensis.
One of these McAb is specific for a subgroup of L. braziliensis, none is cross reactive with T. cruzi epimastigotes.
These McAbs should be used in taxonomic identifications of different spp. of New World Leishmania as well as for the direct diagnosis of Leishmaniasis.
Pratt and McBridge (1983) used an indirect radio-immune binding assay for monoclonal study.
(i) They inject a ♀ BALB/C mice with membrane rich preparation isolated from either L. braziliensis or L. mexicana promastigotes. The membrane preparation were obtained by disrupting the promastigotes.
(ii) Antibody production was asserted between day 14 and 17 after fusion by an indirect radioactive binding assay using Leishmanial membrane hydrophobical fixed to PVC microtitre plates. Cultures secreting antibodies was then studied.
(iii) In the RIA study it was found that the CPM count for L. braziliensis is 510 and in L. mexicana it is 340.
Thus the 2 different spp. of Leishmania can easily be differentiated with McAb technique.
Properties of Monoclonal Antibody and Analysis of their Significance:
1. The Abs secreted by myeloma hybrids can contain light chains and heavy chains from both parental cells and these can associate at random. For this reason, only some of the Abs are functional (binds antigens) and thus parental cells were derived which make no Ig chains of their own. Thus it is now possible to derive hybridomes which secrete McAbs, which are 100% pure.
2. Hybridomes do not represent random samplings from all the lymphoid cells of the immune donor but a random representation from the Ab producing cells or precursor. It is believed, therefore, that by driving hybridomes, one is able to dissect the Ab response of the animal.
3. Each McAb is a unique protein with its own biochemical and immunochemical characteristics unlike conventional antisera which are composed of Abs of different class affinity and specificities. The biochemical uniformity of Mc reagent results in this requisite specificity and high titres (strength of the solution) obtained after growth in clonal hybridomes.
4. Meal immunosorbents—If a McAb binds well to protein, it can easily be used as solid phase immuno-absorbent for antigen purification and analysis.