Antibody Production 101
Why use antibodies?
Antibodies are extremely useful molecular tools for use in a wide range of biomedical research applications, due to their ability to bind tightly and selectively to a desired molecule. They can be used in a variety of assays where characterization, quantification, localization or verification of a protein is desired. Applications include diagnostic kits, such as pregnancy tests, protein microarrays, immunohistochemistry, and ELISA procedures.
What are polyclonal IgY antibodies?
Polyclonal IgY antibodies are the major circulating antibody type found in serum and egg yolks of chickens. In the serum, IgY antibodies are mixed with other antibody isotypes, including IgM’s. However in the egg yolks, IgY’s are the only antibody type present, and they are found at high concentrations (about twice the concentration of serum).
There is only one isoform of IgY (unlike IgG’s which arise from multiple gamma heavy chains isotypes, such as IgG1, IgG2a, etc.). Like mammalian IgG’s, though, chicken IgY’s consist of two heavy and two light chains, and contain two antigen binding domains.
Although there are some minor physicochemical differences between chicken IgY’s and rabbit IgG’s in terms of thermal- and acid-stability, chicken antibodies can be used in all of the same research applications. Specifically, chicken IgY’s can be used in Western Blots, Immunohistochemistry and Immunocytochemistry, Immunoprecipitation, ELISA’s, protein microarrays, and other common uses of mammalian IgG’s.
How long will it take to receive my antibodies?
The time frame for production of maximal-titre antibody production process is about the same in chickens as in rabbits – about 12 weeks. This is because the cell biology of antibody induction is the same in all vertebrates, including chickens. Consequently, any company that advertises faster production times is probably sacrificing high titres for speed. We don’t do that.
This 12 week production schedule including 4 injections spaced about 2-3 weeks apart, egg collection periods, and IgY purification schedules (for both immune and pre-immune antibodies). If you want us to synthesize peptides and conjugate them to carrier proteins, you would need to add on an additional 2 weeks (14 weeks total). If you also want us to perform affinity purification and/or ELISA analysis at the end, you would need to add on an additional 2 weeks (16 weeks total).
How come Aves’ IgY antibodies are purified from eggs? Why don’t we purify our antibodies from serum?
Although IgY antibodies are the major circulating antibody in chickens (like IgG antibodies in mammals), we purify the IgY antibodies from eggs exclusively because it is the animal-friendly way to go. We never bleed our hens, and we don’t have to perform cardiac puncture to obtain large quantities of antibodies – we simply collect eggs.
Hens naturally transfer their circulating IgY antibodies into their eggs as a means of conferring passive immunity to their offspring. Although the eggs used for antibody production at Aves Labs are not fertilized, they DO contain about 100 mg of IgY per yolk.
What are the down-sides of producing antibodies in chickens?
There is only one significant downside of producing antibodies in chickens – reliance on daily ovulation. As in mammals, the process of ovulation is sensitive to physiological and psychological stressors, including conditions that cause pain or distress. Consequently, we at Aves Labs are highly motivated to reduce procedures or conditions that cause the hens to be stressed. Basically, the hens can tell us when they are stressed by ceasing egg production.
In addition, chickens normally stop ovulating once a year for a period of about 5 weeks during their annual molt. Although we take great care to suppress this tendency by providing 16 hours of light per day, our hens do occasionally go into a molt, presumably due to other factors. Consequently, we cannot guarantee that any particular hen won’t go into a molt during a project. We do, of course, inform our clients of such delays and will substitute hens, if necessary. On the other hand, hens that come through a molt usually have higher titres of antibodies than hens that don’t go through the same process.
What are the advantages of chicken IgY antibodies as research tools?
There are 6 major advantages of chicken IgY antibodies over rabbit IgG antibodies as research tools.
(1) Since birds are so far removed evolutionarily from mammals, they tend to respond vigorously to most mammalian proteins. This is particularly important for production of antibodies against highly conserved mammalian gene products, where there are only a few differences in the amino acid sequence of the target protein and the hosts’ homolog.
(2) It is much simpler to perform double immunostaining studies with mouse or rabbit antibodies because secondary anti-chicken IgY antibodies don’t cross-react with mammalian IgG’s.
(3) The price of the antibodies is considerably cheaper if you compare equivalent degrees of purification. The price, for example, of about 1.5 grams of protein A-purified rabbit IgY is about TWICE that of 1.5 grams of purified chicken IgY.
(4) Since chicken IgY antibodies lack an “Fc domain,” they don’t bind “rheumatoid factors” found in the serum of animals (or humans) with chronic inflammatory diseases. In research applications using serum from such animals (or humans), the rate of “false positives” due to these rheumatoid factors is therefore reduced considerably.
(5) In applications that require binding of antibody to solid surfaces (e.g., ELISA’s, antibody microarrays), one can absorb about twice as much chicken IgY to a glass or plastic surface than rabbit IgG. This is presumably due to the higher carbohydrate content in chicken IgY’s (about 3% carbohydrate as compared to about 1% in rabbit IgG’s).
(6) The higher content of carbohydrate allows a higher degree of enzyme- or hapten-conjugation away from the antigen-binding regions of the antibody. For example, one can easily conjugate 15-20 biotins per chicken IgY using hydrazide carbohydrate chemistry, as compared to 7-10 per rabbit IgG.
What are the down-sides of chicken IgY antibodies as research tools?
There are two major downsides of chicken IgY antibodies, and a couple of minor inconveniences.
(1) The first major downside concerns labs that use birds as their primary experimental animal. This is, of course, the same reasons why mouse labs have difficultly using mouse antibodies – secondary antibodies cross-react with endogenous antibodies in the experimental animals. For this reason, we recommend that chicken labs use another host animal for their antibody production.
(2) The second major downside involves applications that require Fab fragments. It is much more expensive, for example, to produce Fab fragments from chicken IgY molecules than rabbit IgG’s (although it is still possible). This is because chicken IgY’s lack the sequence to which protein A binds, necessitating the use of anti-chicken IgY-stem antibodies to separate the IgY-stems from the Fab fragments.
(3) One inconvenience concerns labs that tend to use protein A- or protein G-immunoreagents for immunoprecipitation. Since chicken IgY’s don’t have sequences that bind proteins A or G, these reagents cannot be used. This inconvenience is easily overcome, however, by the use of PrecipHen® — an affinity purified goat anti-chicken IgY coupled to agarose.
(4) A second inconvenience concerns labs that use PVDF membranes for their western blots. Presumably due to their high carbohydrate content, PVDF and other nylon-based membranes bind chicken antibodies non-specifically. This inconvenient is easily overcome, though, by substituting pure nitrocellulose membranes for PVDF. For most purposes, nitrocellulose membranes substitute beautifully for other, nylon-based membranes.