Authors: Kishor U Tribhuvan, Antara Das, Sandhya Sanand, Anshul Watts, Alim Junaid
ICAR-National Research Centre on Plant Biotechnology, New Delhi-110012
Summary
Antibodies are group of glycoprotein produced by B-lymphocyte of vertebrate and it is the primary protection from pathogen infections. Ability of antibody to bind specific substrate increase its use as probe in research and diagnostics. Nowadays, production of antibodies is largely depend upon experimental animal which limit the quantity and also create ethical concern. Alternatively, transgenic plants having antibody producing genes can effectively synthesize heavy and light chain polypeptides and assembled into mature antibody called plantibody.
Introduction
Immunoglobulin proteins also called antibodies, are group of glycoprotein produced by B-lymphocyte and present in the blood serum and tissue fluids of vertebrate in the form of plasma cell (white blood cell). They act as a part of humeral immune response by specifically recognizing and binding to foreign particles (antigen), such as bacteria and viruses, enabling it to clear from circulation. As vertebrate body sense the attack of pathogen, specific type of antibodies bind to combat and eliminate the pathogen. Simultaneously, the antibodies which are engaged in elimination of pathogen proliferate in B-lymphocyte and secreted in the tissue fluids of the vertebrate. The characteristics capacity antibodies to bind its specific substrate allows to use it in diverse application of the medical field such as diagnosis, prevention and treatment of disease. Antibodies are extensively use in medical and cell biology research. It also utilized to manufacture probes for detection of molecules of interest in research and diagnostics. No other current technology allows researcher to design and synthesize such highly specific molecular recognition tool.
Structure and classification of antibody
Antibody is Y shaped molecule in which two identical heavy chain and light chain are joined together through a disulfide linkage. The amino terminal ends of both heavy and light chains shows considerable variation in amino acid composition called variable region and which is responsible for specific binding to antigen. Each light chain consists of one variable domain (VL) and one constant domain (CL). The heavy chain consist of one variable domain (VH) and three constant domain CH1, CH2, CH3. A single antibody contain two antigen binding site and is said to be bivalent.
Antibodies are classified into five classes viz. IgG, IgM, IgA, IgD and IgE based on heavy chain found in molecule. Difference in heavy chain allow these antibodies to function in different type and stage specific immune responses. Although there are five different type of heavy chains, there are only two main types of light chains: kappa (κ) and lambda (λ). Both kappa (κ) and lambda (λ) light chain functionally similar and either type of light chain may be found in antibodies of any of the five major classes.
Antibody production using animals
Animal such as chicken, goats, guinea pigs, hamsters, horse, mice, rats, sheep and rabbit are most frequently used for production of antibodies. However, the rabbit is the most commonly used laboratory animal for this purpose.
An antigen/adjuvant is injected in the body of animal to initiate and proliferate immune response. After series of injection over a specific period of time, the animal is expected to have create an antibody against the conjugate and accumulate in blood serum. Blood is then extracted from animal then purified to obtain antibody of interest. As rabbit is most preferable animal for antibody production, young adult animal (2.5-3.0 Kg) should be used for primary immunization for vigorous antibody response. At puberty Immune production is on peaks and primary responses to new antigens decline with age. Female rabbits are generally preferred for an antibody production because they are more docile and are reported to mount a more vigorous immune response than males.
Purification of antibody from blood serum
Purification of antibodies form animal blood serum involved followings methods:
- Physiochemical fractionation: antibodies are purified based upon their characteristic differential precipitation, size exclusion or solid phase binding of immunoglobulines based on size, charge, or other chemical characteristics.
- Class specific affinity: Use of immobilized biological ligand (protein, lectins, etc) that have specific affinity to immunoglobulins.
- Antigen specific affinity: purification of antibody is based upon their affinity to bind particular antigen molecule through their specific antigen binding domain.
- Rearing of animal at large scale for antibody production is difficult task.
- Limited quantity of blood serum produced from each animal, harvesting blood beyond the limit cause death of animal.
- Ethical concern related to experimental animal.
- Production of antibody is limited by certain factors viz, health, Age, Sex of animal and surrounding environment.
Huge demand of antibodies in therapeutic, diagnostic veterinary, pharmaceutical industry put the extra burden on the experimental animal for its production. Researcher assessed out potential of bacteria and other prokaryotes for production of antibodies by transforming genes coded for heavy and light chains, but lack of posttranslational protein modification system in these organism unable to assembled mature antibody. The transgenic plants having transgenes for heavy and light chains of antibody can efficiently produce mature antibodies. Posttranslational modification system facilitate N-glycosylation of polypeptides and assembled of heavy and light chain together by forming weak hydrogen bond and disulfide bond between the chains. In such way functional antibodies produced in the plant is termed as plantibody.
Advantages of Plantibody
- Load of large scale production of antibodies on experimental animals are reduced
- Plants are cost effective medium for antibody production than animal
- Multiplication of transgenic plant is easy, storage and further use of seeds are possible.
- Purification of plantibodies are easy from plant parts as compared to antibodies from animal blood.
- No ethical issues related to production of plantibody using model plants.
- Plantibodies used as edible vaccine will reduces cost of vaccination and also help in irradiation of certain dangerous disease from human as well as animal population.
CaroRx a secretory immunoglobulin A is the first plantibody created from tobacco having anti-Streptococcus mutans for protecting human from dental caries and now it is in human trial. Plantibody glycoprotein B against herpes simplex virus was expressed in soybean. The experiment on mice showed that engineered plantibodies extracted from tobacco showed effective in fighting the B. anthracis strain and bodes well for the future if ever there is an anthrax epidemic, as there will be a cheap and effective prevention of the disease. Recently, anti-Ebola virus antibodies has been explored in plants for mass scale production. The plantibodies like DoxoRx and RhinoRx were develop for post-cancer therapy and anti-rhinoviruses respectively and are in various stages of completion. Transgenic plants that express antigens in their edible tissue might be used as an inexpensive oral vaccine production and delivery system. Thus, along with edible tissue of plants ‘edible vaccine’ consumption will provide effective way immunization against various pathogens.
References:
- Oluwayelu DO, Adebiyi AI (2016) Plantibodies in human and animal health: a review. Afr Health Sci. 16(2): 640-645
- Stoger E, Sack M, Nicholson L, Fischer R, Christou P (2005) Recent progress in plantibody technology. Current pharmaceutical Design, 11: 2439-3457
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