Originally Published IVD Technology April 2002
Assay Development
Duck
antibodies for IVD applications
A naturally existing analog to mammalian antibodies offers an alternative with
broad applications.
Victor Chiou
In manufacturing
immunological reagents for IVD applications, antibodies are the key components.
For the most part, IVD companies employ mammalian antibodies to manufacture
immunological reagents. Such antibodies include polyclonal antibodies, which
are easy to produce, and monoclonal antibodies, which provide the analytical
advantage of recognizing only one epitope on the target antigen.
However, in addition
to the high cost of monoclonal antibodies, all mammalian antibodies have limitations.
For example, the Fc domain of such antibodies is responsible for cross-reactions
with interfering factors or Fc receptors, which constitute a family of cell-surface
molecules that are expressed on almost every cell of the immune system.
In immunoassays, such mammalian antibodies can be nonspecifically bound by either human antianimal antibodies that arise as a result of exposure to a monoclonal antibody therapeutic or imaging agent (e.g., human antimouse antibodies), or rheumatoid factors that are present in the specimens of rheumatoid arthritis patients and some healthy individuals.1,2 Such endogenous interfering antibodies usually exhibit broad reactivity and can result in false-positive or false-negative results in immunoassays. The consequences are that a patient may be required to undergo additional diagnostic testing or even unnecessary treatments. An antibody that can avoid such cross-reactions mediated by the Fc domain clearly has an advantage.
Avian Alternatives
) |
|
Figure
1. Molecular structures of mammalian IgG, chicken IgY, and duck IgY
(DFc). |
With the purpose
of finding a "better" antibody, research on avian antibodies has been
going on since the 1960s. Avian antibodies refer to the antibodies that are
produced in the serum or eggs of birds. Among the avian antibodies, immunoglobulin
Y (IgY) has been the most extensively studied. IgY is the functional equivalent
and evolutionary ancestor of mammalian immunoglobulin G (IgG) and is found in
some birds, reptiles, and amphibians.3 IgY contains molecules that possess two
heavy and two light chains, with the heavy chains having one variable and four
constant region domains (see Figure 1). It has a molecular weight of ~180 KDa
and a sedimentation coefficient of 7.8 Svedberg units (S).
Chicken egg yolk
IgY specifically has been advocated as an alternative to mammalian sources of
antibodies because it offers a cheap, bloodless, and productive source.4 IgY
derived from chicken egg yolk also offers the advantage of not cross-reacting
with mammalian antibodies, hence eliminating interferences in immunoassays.
) |
|
Figure
2. SDS-PAGE of duck egg yolk protein showing whole egg yolk protein (lane
1), egg yolk antibody (lane 2), and the IgY (DFc) antibody (lane
3). |
A truncated version
of IgY— IgY(DFc) or IgY(–Fc)—is found in ducks, some turtles,
and lungfish. This truncated form of IgY, which may have evolved as a result
of alternative mRNA splicing, lacks the two C-terminal domains in the heavy
chains and is the structural equivalent of an F(ab′)2 fragment.5
Due to the absence of the Fc domain, IgY(DFc) has a molecular weight of
~120 KDa and a sedimentation coefficient of 5.7 S. This unique DFc or
–Fc structure makes IgY(DFc) a potentially useful tool in producing
reagents for use in immunoassays and other immunological applications.
Despite their potential,
IgY antibodies are not extensively used in industry because of the difficulties
involved in isolating them from chicken egg yolk. No chicken IgY-based IVD reagents
are currently available on the market.6 Similarly, the high lipid content in
duck eggs makes duck egg yolk antibodies difficult to isolate, thereby hampering
research into possible applications for IgY(DFc).
Overcoming Isolation
Problems
) |
|
Figure
3. Stability of antibodies to pH. |
Recently, however,
a procedure for isolating duck egg yolk antibodies has been developed. Through
this procedure, which involves a short protocol of delipidization, salting out,
desalting, and concentration, bulk-quantity duck IgY (DFc) can be
isolated. The duck IgY(DFc) thus isolated has a purity of approximately
95% as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see
Figure 2). Duck eggs serve as a cost-effective source of antibodies since a
mature duck lays about 250 eggs each year, and 75 to 120 mg of antibody can
be isolated from each egg yolk.
) |
|
Figure
4. Stability of antibodies to temperature. |
The isolated IgY(DFc) is a natural F(ab')2 analog that has the ability to recognize specifically an immunizing antigen. In thiocyanate elution assays, which determine the binding avidity between antibodies and antigens, mammalian albumin-specific IgY(DFc) exhibits higher avidity to mammalian albumin than corresponding rabbit antibodies. This strong recognition and binding specificity of duck IgY(DFc) with mammalian antigens is perhaps due to phylogenetic differences between mammals and birds. For example, the differences between human and avian proteins are greater than those between human and chimpanzee proteins. This characteristic makes avian antibodies especially suitable for use in immunological assays involving mammalian specimens.
Stability Issues
) |
|
Figure
5. Nonspecific binding of antibodies to human rheumatoid factor. |
Since the application
of antibodies in biotechnology and medicine places a high premium on stability,
experiments were conducted to compare the stabilities of rabbit, goat, and duck
antibodies. In each case, antibodies to C-reactive protein (CRP) were selected
for testing. These antibodies were stored at 40°C in an incubation chamber
for up to 80 days, and subjected to low- and high-pH conditions for 4 hours,
followed by a determination of remaining titers using single radial immunodiffusion.
) |
|
Figure
6. Nonspecific binding of antibodies to human complement C3. |
In the pH experiments,
all of the rabbit IgG, goat IgG, and duck IgY(DFc) antibodies remained
stable during the 4-hour incubation period at pH 3.5 to 9. The antibody solution
was adjusted from 3.5 to 9 by adding an HLL/NaOH glycine buffer, and then neutralized
to pH 7. The capacity of the antibodies to form antigen-antibody complexes was
then determined by single radial immunodiffusion. At pH levels of less than
3.5 or greater than 10 all of the antibodies gradually lost the ability to form
an antigen-antibody complex (see Figure 3). In extremely acidic or basic conditions,
rabbit IgG was the most stable, while goat IgG was the least stable. At pH 2.5,
duck IgY(DFc) showed comparable stability to rabbit IgG.
As for thermal stability, the three assayed antibodies demonstrated similar results after storage for 80 days at 40°C (see Figure 4).
Cross-Reactivity
) |
|
Figure
7. Nonspecific binding of antibodies to staphylococcal protein A. |
The most distinctive
characteristic of IgY(DFc) is the absence of the Fc domain. The immunoglobulin
Fc domain is known to react nonspecifically with some molecules of mammalian
or bacterial origin.7 Some substances, such as animal heterophile
antibodies and bacterial proteins, are known to react with the immunological
Fc domain. Because IgY(DFc) antibodies lack the equivalent Fc domain,
an IgY(DFc) coating on microplates does not bind with molecules of
rheumatoid factor, human complement C3, human complement C4, or staphylococcal
protein A (see Figures 5–7).
By contrast, mammalian-antibody-coated
plates exhibit high nonspecific binding to these molecules. In these experiments,
normal human serum (for the complement C3 or C4 assay) or purified protein (for
the rheumatoid factor or protein A assay) was used to nonspecifically react
with the coated antibodies, followed by a determination of nonspecific binding
with antiinterfering molecule antibodies.
) |
|
Figure
8. Binding of a rabbit antimouse antibody to different species’ antibodies. |
To evaluate possible
cross-reactions caused by mammalian heterophilic antibodies, a sandwich ELISA
test was performed as a surrogate model. In this assay, rabbit IgG, mouse IgG,
chicken IgY, or duck IgY(DFc) was first coated onto microplates. A
rabbit antimouse antibody was then added to bind the coated antibodies. Finally,
peroxidase-conjugated mouse IgG and a substrate were added sequentially.
The strong signal on the mouse IgG-coated microplates indicated specific capture of the peroxidase-conjugated mouse IgG by rabbit antimouse IgG. The signal on the rabbit-IgG-coated plates indicated nonspecific cross reactions with these antibodies. No signal was observed to indicate the presence of cross-reactions between duck IgY(DFc) and mammalian antibodies (see Figure 8).
Applying Duck
Antibodies to IVDs
) |
|
Figure
9. Correlation of IgY(DFc)-based CRP assay reagent and an
FDA-approved commercial reagent. |
In order to evaluate
the applicability of duck IgY(DFc) in IVD reagents, an IgY(DFc)
antibody was coupled with latex microparticles to make latex-enhanced immunoturbidimetric
(LIT) reagents. As IgY(DFc)-sensitized latex particles encounter a
target antigen, the agglutination reaction, or the clumping together of antibody-bearing
particles in the presence of specific antigens, increases the turbidity of the
reaction mixture, which can then be measured by automated clinical chemistry
analyzers. Based on this principle, the concentration of an analyte in sample
specimens can be determined automatically through an interpolation against calibrators.
The prepared high sensitivity C-reactive protein assay reagents have assay ranges of 0.01 to 2 mg/dl with less than a 5% coefficient of variation. Compared with an FDA-approved latex immunoturbidimetric assay, the IgY(DFc)-based LIT assay reagent showed good correlation, suggesting that IgY(DFc) is suitable for IVD applications (see Figure 9).
Conclusion
Searching for a suitable antibody is top priority for immunological IVD reagent manufacturers. An ideal antibody should possess the following characteristics:
- High specificity or affinity.
- No interference from molecules present in sample specimens.
- Stable assay or storage conditions.
- Easily obtained with continuing supply.
- Cost-effective.
- Minimal animal suffering.
Mammalian polyclonal
antibodies, monoclonal antibodies, and avian antibodies each have their own
unique advantages as well as limitations. Choosing the most suitable antibody
depends on a thorough consideration of application fields, assay methods, and
budget.
The overlooked IgY(DFc) could provide an option in both research and commercial fields, as well as in state-of-the-art biotechnology devices (e.g., antibody chips or biosensors that utilize antibodies as the main biological detection unit). The key biological features of the duck antibodies described above—namely the lack of Fc and the corresponding low interference and cross-reactivity with mammalian systems—make these antibodies alternative candidates for diagnostic applications in which such factors play a significant role.
References
1. LJ Kricka, "Human
Anti-Animal Antibody Interferences in Immunological Assays," Clinical Chemistry
45, no. 7 (1999): 942–956.
2. N Despres
and AM Grant, "Antibody Interference in Thyroid Assays: A Potential for Clinical
Misinformation," Clinical Chemistry 44, no. 3 (1998): 440–454.
3. GW Warr,
KE Magor, and DA Higgins, "IgY: Clues to the Origins of Modern Antibodies,"
Immunology Today 16, no. 8 (1995): 392–398.
4. A Cipolla et al., "Campylobacter fetus Diagnosis: Direct Immunofluorescence
Comparing Chicken IgY and Rabbit IgG Conjugates," Altex 18, no. 3 (2001):
165–170.
5. DA Higgins
and GW Warr, "Duck Immunoglobulins: Structure, Functions and Molecular Genetics,"
Avian Pathology 22, no. 2 (1993): 211–236.
6. L Davalos-Pantoja
et al., "Colloidal Stability of IgG- and IgY-Coated Latex Microspheres," Colloids
and Surfaces B: Biointerfaces 20, no. 2 (2001): 165–175.
7. E Medina et al., "Fc-Mediated Nonspecific Binding Between Fibronectin-Binding
Protein I of Streptococcus Pyrogenes and Human Immunoglobulins," Journal
of Immunology 163, no. 6 (1999): 3396–3402.
Victor Chiou is the founder and CEO of Good Biotech Corp. (Taichung, Taiwan). He can be reached via gbc@good-biotech.com.
Copyright ©2002 IVD Technology
