Monoclonal antibodies and their application in poultry

V. Thiagarajan
Tamil Nadu Veterinary and Animal Sciences University , Chennai - 600 051
Email: [email protected]

Monoclonal antibodies (Mabs) are antibodies, which have a single specificity and are continuously secreted by “immortalized” hybridoma cells. A hybridoma is a biologically constructed hybrid between an antibody-producing, mortal, lymphoid cell and a malignant, or “immortal”, myeloma cell. Following the discovery of hybridoma technology in 1975 by Kohler and Milstein, developments in Mab production and their application have had profound implications not only on medical research, diagnosis and therapy, but also on biology in general. Hybridoma technology represents a significant advance because, in principle, it provides a means for obtaining unlimited supplies of highly specific antibodies.

In the production of Mabs, animals (generally mice) first have to be immunized with the target antigen to obtain mortal antibody-producing cells. The biological construction of hybrids, and the selection of hybridomas, which produce antibodies with the desired specificities, is carried out in vitro. The hybridomas developed in vitro were then injected into the peritoneal cavity of an animal so that useful amounts of the desired Mab could be harvested from the ascitic fluid. This procedure was considered necessary at the time, since no efficient large- scale in vitro methods were available. By the middle of the 1980s, there were already serious doubts regarding the necessity of such painful animal procedure. Nevertheless, as a result of its early introduction as part of the hybridoma technology, ascites production of Mabs is now employed worldwide in spite of the ongoing developments of in vitro technologies and the growing public pressure to replace or reduce animal experiments.

Hybridoma Technology

There are essentially two stages in the production of Mabs

  1. the propagation of antibody-producing lymphoid cells in vivo and the selection of antibody producing hybridoma cells in vitro; and
  2. the in vitro / in vivo propagation of selected hybridoma clones.
  3. The first stage, the formation and selection of the hybridoma clone, involves the use of one or more animals and is carried out in the following way:

    1. The antigen is injected into mice. The antigen is often injected in combination with an adjuvant, to enhance the immune response.
    2. After an appropriate interval (5 – 21 days), the immunized animals are killed and lymphoid cells are isolated from the spleen.
    3. The lymphoid cells are fused with myeloma cells, which have been grown in vitro
    4. The two original cell types and the newly formed hybrids are cultured in a selective medium, such as HAT (Hypoxanthine Aminopterin Thymidine) medium, which only allows the hybridoma cells to grow.
    5. The supernatant media for the numerous in vitro micro cultures exhibiting a recognizable growth of hybridomas are screened for secretion of the desired antibody, by using various immunoassay procedures.
    6. Selected cells are subcultured in vitro, using special cloning procedures to ensure that each in vitro culture consists of hybridomas with single antibody specificity only.
    7. Hybridoma cells can be cryo preserved at this stage.

    The second stage, the propagation of cloned hybridoma cells, can be accomplished either by continuing to grow the cells in vitro or by propagating them in vivo, in the form of ascites tumors.

Current Demand for Mabs

The applications of Mabs are numerous and diverse, and they are extensively used in fundamental research, medicine and biotechnology. At present, four user groups can be identified, according to the amount of antibody required as summarized in Figure below.

User Group A: Less than 0.1 g

Approximately 60 % of the Mab users fall within this group, as do many of the current users of the in vivo (ascites) method. Small amounts of antibodies are produced for use in fundamental and applied research, the commercial production of special diagnostic kits for research and for analytical purposes.

User Group B: 0.1 – 0.5 g

This group accounts for approximately 30% of Mab users and encompasses a significant number of people still using the in vivo method. Antibodies in these amounts are required for the development and production of a wide range of in vitro diagnostic kits and reagents, as well as for evaluating the usefulness of novel therapeutic Mabs in animal experiments.

User Group C: 0.5 – 10 g

In this group, which accounts for approximately 10% of Mab users, adoption of the in vivo method is comparatively rare. The Mabs produced are used in routine diagnostic procedures and in pre clinical evaluation studies. Large biotechnology companies usually produce them but, during the last few years, the production of these Mabs has increasingly been contracted out to smaller facilities.

User Group D: > 10 g

Users in this group, who require Mabs for prophylactic and therapeutic purposes in vivo, make up less than 1% of all Mab users. The Mab production processes they use are first developed and validated by the pharmaceutical industry, and are then submitted to a regulatory body for approval.

Applications of Mabs

1. Immuno diagnostic reagents

  1. Antigen detection
    With the development of Mabs of various specificities, advances have been made in the detection of infectious agents (antigens) directly in tissue of body fluid specimens.
  2. Antibody detection
    The second use of Mabs in disease diagnosis is in the detection of antibody most commonly used as an antiglobulin reagent conjugated with a detection system (fluorochrome, enzyme or isotope).

2. Research purposes of Mabs

  1. Cells of immune system
    Mabs have been used extensively to characterize various cell population involved in immune responses of mice, man and other animals.
  2. Pregnancy and Sex determination
    Mabs have been used to detect pregnancy by the presence of progesterone in cow’s milk. Antibody to H-Y antigen, the male specific transplantation antigen is used to diagnose definitely the sex of an embryo.

3. Molecular structure of antigens

Mabs used in the study of tumor-causing viruses and the survival and function of tumor virus proteins in host cells.

4. Affinity chromatography

Because of their fine specificity, Mabs are suitable for use in purification of antigens.

5. Mabs as in vivo reagents for animal

The current use of monoclonals in domestic animals can be categorized as follows:

  1. Passive antibody administrated prophylactically or therapeutically for infectious diseases.
  2. Passive antibody to target on particular cell markers either alone or coupled to cytotoxic agents.
  3. Passive antibody to enhance the clearance of toxic compounds.
  4. Passive antibody to modulate the cellular or messenger components of the in vivo immune response.

Application of Mabs in Poultry Disease Diagnosis

A. Newcastle Disease

Mouse Mabs directed against strains of Newcastle Disease Virus (NDV) have been used in haemagglutination inhibition tests to allow rapid identification of NDV without the possible cross reactions with other paramyxo virus serotypes that may occur with polyclonal sera (Alexander et al., 1992).

Some workers have used Mabs to distinguish between specific viruses. For example, two groups have described Mabs, which distinguish between the common vaccine strains, Hitchner B1 and LaSota (Erdei, 1987; Meulemans et al., 1987) while other Mabs can separate vaccine viruses from epizootic virus in a given geographical area (Srinivasappa et al., 1986).

Panels of Mabs have been used to establish antigenic profiles of NDV isolates based on their ability to react or not with the viruses. This has proven to be a valuable method for grouping and differentiating isolates of NDV, which has been particularly valuable in understanding the epizootiology of outbreaks (Alexander et al., 1997).

B. Infectious Bronchitis

Excellent results were obtained when Mab-based procedures for diagnosis of infectious bronchitis (IB), such as different types of ELISA, immunoperoxidase test and immunofluorescence test were used, over the conventional tests such as virus neutralization (VN) and haemagglutination tests. An excellent correlation was found when the results of IBV serotyping by Mab based indirect ELISA were compared with those from the conventional VN tests and it will serve as valuable tools in epizootiological studies and serotype specific diagnosis of IBV infections (Karaca et al., 1992).

The Mab-based immunoperoxidase technique offers excellent potential to achieve the diagnosis of IBV infection. It is preferred over the Mab based immunofluorescence technique because it is simpler, allows evaluation of antigen bearing cells as well as general tissue morphology and the slides provide a permanent record (Naqi, 1990). The introduction of IBV serotype specific Mabs has allowed development of convenient assays such as indirect ELISA, antigen capture ELISA and antibody blocking ELISA for IBV diagnosis and serotype identification (Karaca et al., 1992; Karaca and Naqi, 1993).

C. Infectious Bursal Disease

Mabs have been used to classify the IBD viruses as very virulent (vv) pathotype based on their reactivity to two Mabs (Mabs 3 and 4) of eight developed against the IBD vaccine strain (Eterradossi et al., 1997). All the eight Mabs reacted with the F-52/70 classical virulent strain. However all the vvIBDV failed to react with Mabs 3 and 4. The weak or negligible reactivity of field isolates with Mabs 3 and 4 indicated their antigenic similarity with other vvIBDV strains.

D. Avian Influenza

A Mab-based dot ELISA was developed that detected epitopes specifically associated with avian influenza virus (AIV) (Lu, 2003). This dot-ELISA detected AIV antigen from allantoic fluid samples that contained a concentration of 0.4 HA units.

Mabs against poultry viruses have been prepared at the Department of Animal Biotechnology, Madras Veterinary College, Chennai. These Mabs were directed against the hexon protein of egg drop syndrome virus (EDS) (Dhinakar Raj et al., 2003), VP3 protein of IBD virus and S2 protein of IB virus. These Mabs have been employed in dot/ AC ELISA tests for antigen detection from field samples with high sensitivity and specificity. These Mabs could also be used in AC ELISA tests to quantify the antigenic content of inactivated vaccines and thus assess their potency.

In addition Mabs have also been prepared against chicken IgG and IgM, which were used in the study of class specific immune responses to IBD and EDS viruses. The Mabs against chicken IgG were used to assess total IgG content in egg yolks or chicken serum in an AC-ELISA format (Dhinakar Raj et al., 2004).

Application of Mabs in the detection of adulteration in meat

Poultry (Chicken and Turkey) tissue represents a major source of protein, generally less expensive than red meat, which is consumed and imported throughout the world. These factors together with the regulated increasing use of mechanically separated poultry meat produce a significant potential for the adulteration or substitution of red meat by poultry products.

Many of the ELISA methods currently available for meat speciation in kit form have used polyclonal antisera raised against various blood proteins present in meat such as albumin. Such antisera require special immunosorbent affinity purification to eliminate significant cross-reactions and it’s a problem for large-scale production. For meat speciation, Mabs of required specificity would then by suitable for various types of ELISA for example capture or competitive ELSIA.

Martin, (1989) produced and characterized Mabs against species-specific sarcoplasmic protein of chicken. Three Mabs, which are capable of distinguishing between muscle extracts of the most frequently, marketed avian (chicken and turkey) and mammalian (beef, pork, horse and lamb) species of meat animals. One of these antibodies has the added advantage of distinguishing between chicken and turkey extracts by ELISA. Since the Mabs were raised against native proteins they were not ideally suited for detection of heat denatured chicken meat materials.

It is hoped that Mab technology is bound to improve diagnosis and serotyping/pathotyping procedures in poultry disease management. As poultry becomes more and more of a large-scale industry the slightly increased cost of production of Mabs would be off set by their exquisite sensitivity and specificity in the long rum. The application of Mab-based assays in the lateral flow formats (immuno chromatography or strip tests) is bound to revolutionize the ‘on-farm’ testing in the near future, which the genome based methods, at its present state, can not offer.

References

Alexander, D. J. (1992). Use of monoclonal antibodies in the diagnosis of Newcastle disease and characterization of the virus. In Proc. Commission of the European Communities workshop on avian paramyxo viruses (E. F. Kaleta & U. Heffels-Redmann, Eds), 145 – 156.

Alexander, D.J., Manuvell, R., Kemp. P. A., Parsons, G., Collins, M. S., Brockman, S., Russel, P. H., Lister, S. A. (1997). Antigenic diversity and similarities detected in avian paramyxo virus type 1 (Newcastle disease virus) isolates using monoclonal antibodies. Avian Pathology 26: 399 – 418.

Dhinakar Raj, G., Sivakumar, S., Matheswaran, K., Chandrasekhar, M. S., Thiagarajan, V. and Nachimuthu, K. (2003) Detection of egg drop syndrome virus antigen or genome by enzyme linked immuno sorbent assay or polymerase chain reaction. Avian Pathology 32: 545-550

Dhinakar Raj, G., Bhavani Latha, Chandrasekhar, M. S. and V. Thiagarajan (2004) Production, characterization and application of monoclonal antibodies against chicken IgY. Veterinarskvi Archiv 74: 189 – 199

Erdei, J. (1987). Newcastle disease vaccine (LaSota) strain specific monoclonal antibody. Archives Virology 96: 265 – 269.

Eteradossi, N., G. Rivallan, D. Toquin and M. Guittet (1997) Limited antigenic variation among recent infectious bursal disease virus isolates from France. Archives of Virology 142: 2079-2087

Karaca, K., Naqi, S and Gelb Jr. J. (1992). Production and characterization of monoclonal antibodies to three infectious bronchitis virus antibodies. Veterinary Microbiology 34, 249 – 257.

Kohler, G. and Milstein, C. (1976). Derivation of specific antibody – producing tissue culture and tumor lines by cell fusion. European Journal of Immunology6: 511 – 519.

Lu, H. (2003) A longitudinal study of a novel dot-ELISA for detection of avian influenza virus. Avian Diseases 47: 361-369

Martin, M. (1989). Production and characterization of monoclonal antibodies to species-specific sarcoplasmic protein of chickens. Meat Science 25: 199 – 207

Meulemans, G., Gonze, M., Carlier, M., Petit, P., Burny, A. and Long, L. (1987). Evaluation of the use of monoclonal antibodies to heamagglutination and fusion glycoproteins of Newcastle disease virus for virus identification and strain differentiation purposes. Archives of Virology 92: 55-62.

Naqi, S. (1990). A monoclonal antibody – based immuno peroxidase procedure for rapid detection of infectious bronchitis virus in infected tissues. Avian Disease36: 903 – 915.

Naqi, S., Karaca, K. and Bauman, B. (1993). A monoclonal antibody – based antigen capture ELISA for identification of infectious bronchitis virus serotypes. Avian Pathology 22: 555 – 564.

Srinivasappa, G.B., Snyder, D.B., Marquardt, W.W. (1986). Isolation of a monoclonal antibody with specificity for commonly employed vaccine strains of Newcastle disease virus. Avian Disease30: 562 – 567.

Source : IPSACON-2005

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