Enzyme Linked Immuno Sorbant Assay Biology Essay

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Recombinant protein estimation is generally done by any one of the protein quantification methods such as Lowry's or Bradford's or BCA method or even by UV spectroscopy. These quantification methods are rather non-specific, not accurate and laborious. Hence detection and quantification can be done by the method of ELISA, where the results are specific to the target protein. Rather, more number of samples (atleast 96) can easily be processed in a single hand. Our proposed fusion protein consists of a SUMO protein and a 6xHis tag. The presence of these fusions can be exploited for detection and quantification of the target protein as given in figure 1. During or after fermentation process, the culture medium is harvested and centrifuged to obtain supernatant. This supernatant can directly be used for the detection and protein quantification by the ELISA method.

Generation of antibodies

Monoclonal and polyclonal antibodies are of great value in biological research and medicine, where they serve as essential components in a variety of diagnostic systems used for the qualitative and quantitative determination of a wide range of substances.

Antibodies presently available for research, diagnostic and therapies are mostly mammalian monoclonal or polyclonal antibodies. Antibody production normally requires the use of laboratory animals (mostly rabbits, mice, rats and guinea pigs) or larger mammals, such as horses, sheep and goats. The procedure involves two steps, each of which causes distress to the animals involved: a) The immunization and b) bleeding, which is a perquisite for antibody preparation (Schande et al., 1996).

The major problem of monoclonal antibody production is that some antigens are weakly or not at all immunogenic for mice. In polyclonal antibody production, purification of antibodies from mammalian blood has been found low yielding and laborious in many cases. Both technologies also involve some steps, each of which causes distress to the animals involved (Narat, 2003).

Figure 1: ELISA method for detection and quantification of recombinant protein produced in E.coli

Chicken Antibodies - an alternative source

Chickens, as a source of desired antibodies, represent an alternate animal system that offers some advantages with respect to animal care, high productivity and special suitability of antibodies for certain applications. Chickens are known to produce higher levels of antibodies than mammals, thus minimizing the number of animal needed.

Further the yolk of the egg receiving the same antibodies from the maternal chicken serum can be a suitable alternative source; it can avoid bleeding of the animal. Further the egg provides a continuous daily source of mono specific polyclonal antibody and can be stored at 4°C up to 1 year. Despite being an excellent counterpart to mammalian IgG, chicken IgY antibodies still represent an underused resource.

Egg yolk - the IgY reservoir

The yolks of eggs laid by immunized chickens have been recognized as an excellent source of polyclonal antibodies for over a decade (Jensenics et al., 1981). The serum IgG antibodies of immunized chicken were efficiently transported and accumulated in the egg yolk (Patterson et al., 1962; Rose et al., 1974; Larsson et al., 1993). The main type of immunoglobulin isolated from egg yolk is generally referred to as IgY. Other Ig classes are present, but only in negligible amount. The term IgY is originally coined to refer to the larger molecular weight IgY found in egg yolk, but it is now accepted that IgY is the major antibody in both the blood and yolk in concentration of around 10-20 mg/ml.

The studies carried out by Leslie and Clem (1972) has shown that chicken, like rabbits, can produce high levels of precipitating antibodies to a streptococcal carbohydrate. Immunization of chickens for polyclonal antibody production is comparable to that of rabbits with respect to the route of injection, the amount of antigen used and the kinetics of specific antibody generation (Song et al., 1985). Specific antibodies produced in chickens offer several important advantages over antibodies produced in mammals, because a single egg contains as much antibody as an average bleed from a rabbit, this simple, noninvasive approach presents an appealing alternative to conventional polyclonal antibody production methods.

Purification of chicken egg yolk immunoglobulin Y (IgY) does not require animal bleeding. In addition, the eggs from immunized chickens provide a continuous, daily source of polyclonal antibody and this convenient approach offers greater compatibility with animal protection regulations (Gottstein and Hemmeler, 1985). High levels of antibody activity in egg yolk were maintained for several months by periodic immunization (Polson et al., 1980). Furthermore, vaccination of small animals such as chickens can be performed easily.

Chicken antibodies recognize more epitopes on a mammalian protein than the corresponding rabbit antibody does, making it advantageous to use IgY in immunological assays of mammalian proteins. This is especially true when he antigen is a highly conserver protein, such as a hormore (Gassmann et al., 1986 and Gourele et al., 1990). Another advantage of chicken IgY over mammalian antibodies is that it does not activate the complement system; the latter has been shown to reduce antigen binding and cause false negative results (Koch and Jongenelen, 1988). Chicken IgY does not react with anti-mammalian antibodies in human serum, such as rheumatoid factors and human anti-mammalina anti-IgG. In immunological assays, the interference caused by these antibodies can be problematic, particularly as the sensitivity of the assay increases.

Table 1 - Comparison of the characteristics of mammalian IgG and avian IgY (Schade et al., 1991)

Properties

Mammalian IgG

Avian IgY

Antibody sampling

Invasive

Non-invasive

Antibody amount

200mg IgG per bleeding

(40 mL blood)

50-100mg IgY per egg

(5-7 eggs per week)

Amount of antibody per month

200mg

1500mg

Amount of Specific antibody

5%

2-10%

Protein-A/G binding

Yes

No

Interference with mammalian IgG

Yes

No

Activation of mammalian complement

Yes

No

Thus, if chicken IgY is used, interference by anti-mammalian IgG antibodies is eliminated (Lindmark et al., 1983). Due to the phylogenetic distance between birds and mammals when using chickens there is a greater potential of producing a higher percentage of specific antibody against mammalian antigens, (Akita and Nakai, 1992). Highly conserved mammalian proteins sometimes fail to elicit a humoral immune response in animals such as rabbits that are traditionally used for generating polyclonal antibodies. Since Chicken IgY does not cross-react with mammalian IgG and does not bind bacterial or mammalian Fc receptors, non-specific binding is reduced, and the need for cross-species immunoabsorptions is also limited. Polson et al. (1980) also corroborated the production of chicken antibodies at lower cost and convenience.

Stability

Experience with egg yolk antibodies is that, they are stable over time, which contradicts some rumors that chicken antibodies are labile. Egg yolk antibodies are stable and can be stored for over ten years at 4°C without any significant loss in antibody activity. Chicken antibodies have also retained their activity after 6 months, at room temperature or 1 month at 37°C. Chicken antibodies are also useful in immunoprecipitation assays in agar. Chicken antibodies offer many advantages over mammalian antibodies, and a change from mammalian to chicken antibodies improves an immunological assay.

Isolation and purification methods

Several methods can be used for the extraction of IgY from egg yolk, and commercial extraction kits are available (Van regnmortel, 1993). The most frequently used procedures involve protein precipitation with ammonium sulphate, dextran sulphate or polyethylene glycol (PEG) and separation by ion exchange chromatography. According to Chang et al., (1996) isolation of immunoglobulin from egg yolk by anionic polysaccharides was followed for the best recovery of IgG. In practice, the choice of a specific extraction procedure is usually influenced by the intended application of the antibody, as well as the experience of the laboratory concerned (Lvsch et al., 1986).

A practically efficient method consists of two successive precipitations in PEG, using 3.5% PEG to remove fatty substances, and then 12% PEG to precipitate the IgY. An improvement of this method incorporates an emulsification step, adding one volume of chloroform rather than using 3.5% PEG (Polson et al., 1980 and Polson, 1990). According to Schwarzkopf and Thiele (1996), the ammonium sulfate precipitation proved to be the most environmentally compatible and economical one. The advantage of this method becomes particularly obvious when the method is followed by affinity chromatography.

Applications of chicken antibodies

Chicken antibodies can be used in both diagnostic and therapeutic purposes. The yolk antibodies also used in a double antibody sandwich ELISA for detecting African horse sickness virus (Du Plessis et al., 1999). Chicken antibodies used in the development of snake antivenom antibodies (Almeida et al., 1998).

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