Antibody Basics

Basic Antibody Structure

Immunoglobulins (Igs) are produced by B lymphocytes and secreted into plasma. The Ig molecule in monomeric form is a glycoprotein with a molecular weight of approximately 150 kDa that is shaped more or less like a Y. Basic structure of the Ig monomer (Figure 1) consists of two identical halves connected by two disulfide bonds. Each half is made up of a heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa, joined together by a disulfide bond near the carboxyl terminus of the light chain. The heavy chain is divided into an Fc portion, which is at the carboxyl terminal (the base of the Y), and a Fab portion, which is at the amino terminal (the arm of the Y). Carbohydrate chains are attached to the Fc portion of the molecule. The Fc portion of the Ig molecule is composed only of heavy chains. Fc regions of IgG and IgM can bind to receptors on the surface of immunomodulatory cells such as macrophages and stimulate the release of cytokines that regulate the immune response. The Fc region contains protein sequences common to all Igs as well as determinants unique to the individual classes. These regions are referred to as the constant regions because they do not vary significantly among different Ig molecules within the same class. The Fab portion of the Ig molecule contains both heavy and light chains joined together by a single disulfide bond. One heavy and one light chain pair combine to form the antigen binding site of the antibody. Each Ig monomer is capable of binding two antigen molecules.

Figure 1.

Antibody Classes and Subclasses

The main immunoglobulin (Ig) classes (Figure 2) are IgA, IgD, IgE, IgG, and IgM. Birds also produce IgY in egg yolks.

Figure 2.

Class identity is determined by class-specific sequences in the Fc region of the heavy chain which are designated by Greek letters corresponding to the Ig letter designation: alpha-IgA, delta-IgD, epsilon-IgE, gamma-IgG, mu-IgM. Light chains are universal among immunoglobulins and occur as two types — kappa or lambda. These are usually designated by the Greek letters kappa and lambda. Refer to Table A and Table B for Human and Mouse Immunoglobulin Properties, respectively.

Table A

Human Immunoglobulin Properties
Property	IgG				IgA		IgM	IgD	IgE
H Chain class (heavy chain)	γ				α		µ	δ	ε
H Chain Subclasses	γ1	γ2	γ3	γ4	α1	α2	None	None	None
H Chain MW	50 kDa	50 kDa	60 kDa	50 kDa	55 kDa	55 kDa	70 kDa	62 kDa	70 kDa
L Chain MW* (light chain k & λ )	23 kDa	23 kDa	23 kDa	23 kDa	23 kDa	23 kDa	23 kDa	23 kDa	23 kDa
Total MW	150 kDa	150 kDa	170 kDa	150 kDa	160 kDa (serum) 600 kDa (secretory)	160 kDa (serum) 600 kDa (secretory)	970 kDa	180 kDa	190 kDa
Ext. Coeff. 0.1% @280 nm	1.4	1.4	1.4	1.4	1.32	1.32	1.18	1.7	1.53
Complement fixation	weak	weak	strong	no	no	no	strong	no	no
Fc receptor binding	strong	weak	strong	weak	yes	yes	yes	no	yes
Mast cell/basophil degranulation	no	no	no	no	no	no	no	no	yes
Placental transfer	strong	weak	strong	strong	no	no	no	no	no

* Light chains are present on all Immunoglobulin classes. In humans, k. chains are found 67% of the time, and λ chains are found 33% of the time. For ratios in other species see Immunoglobulin Light Chain Ratios table.

Table B

Mouse Immunoglobulin Properties
Property	IgG				IgA	IgM	IgD	IgE
H Chain class	γ				α	µ	δ	ε
H Chain Subclasses	γ1	γ2a	γ2b	γ3	None	None	None	None
0% of total LPG	46	24	27	2
Light chain	Approximately 95% kappa (K LC) - 5% lambda (λ LC)
Molecular weight (kDa)	160				160 (monomer) 350-400	900	180	190
Normal Serum Concentration (mg/mL)	0.3-5.0	0.1-4.0	0.1-5	0.1-0.2	0.01-0.03	0.1-1.6	0.003-0.01	0.0001-0.001
Serum half-life (days)	8-11	3-12	2.6-3.5	4-8	0.5-1	0.5-1	<1	<1
Electropnoretic Mobility	fast	slow	slow	slow
Carbohydrate (%)	2-3				7-11	9-12	12-15	12
Complement fixation	-	+++	+++	+/-	-	+++	n.k	n.k
Ext. Coeff. 0.1% @280 nm	1.4				1.35	1.18
Sedimentaion Coeff.	6.6				6.7	19	6.8	8

nk. not known
The ratio of kappa to lambda found in the Ig population varies by species. Refer to Table C.

Table C

Immunoglobulin Light Chain Ratios
Species	% k / λ
Human	67/33
Mouse	99/1
Rat	99/1
Rabbit	90/10
Goat	1/99
Sheep	1/99
Pig	50/50
Bovine	1/99
Horse	1/99
Chicken	N/A

Antibody Forms

We offer antibodies as whole (intact) molecules and as F(ab)₂ fragments of antibodies. Table D highlights some common forms of antibodies.

Table D

Antibody Form	Description	Uses
Whole Antiserum	Whole antiserum contains specific antibody as well as all the other host serum proteins.
Fractionated Antiserum (Ig fraction)	Whole antiserum is fractionated to yield primarily the immunoglobulin fraction of antiserum. This fraction may contain small amounts of the other host serum proteins.	IgG fractions and fractionated antiserum may be considered useful in situations where very high affinity is required, most commonly when the antigen of interest is rare or present in low abundance. IgG fractions may have the benefit of containing very high affinity antibodies. Affinity isolation may remove some very high affinity antibodies because they bind so tightly to the affinity matrix that they are not eluted.
IgG Fraction of Antiserum	Whole antiserum is fractionated and then further purified by ion exchange chromatography to provide the IgG fraction of antiserum. This fraction is essentially free of other host serum proteins.	IgG fractions and fractionated antiserum may be considered useful in situations where very high affinity is required, most commonly when the antigen of interest is rare or present in low abundance. IgG fractions may have the benefit of containing very high affinity antibodies. Affinity isolation may remove some very high affinity antibodies because they bind so tightly to the affinity matrix that they are not eluted.
Affinity Isolated Antibody (AIA)	These products are the most purified, therefore, give the lowest amount of non-specific binding. Affinity isolated antigen specific antibody is obtained from antiserum by immunospecific purification using antigen-bound agarose, which removes essentially all the host serum proteins, including immunoglobulins, which do not specifically bind to the antigen.	Universal
Ascites Fluid	Ascites fluid is intraperitoneal fluid extracted from mice that have had hybridoma cells expressing specific monoclonal antibodies injected into their peritoneal cavity, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities secrete high-titered antibodies. Ascites fluid is clarified by centrifugation to remove the lipid layer and the cell pellet. It contains specific antibody as well as other host serum proteins including immunoglobulins. Specific antibody concentration range is 1-10 mg/mL. Total protein concentration is approximately 20 mg/mL	Universal
Tissue Culture Supernatant	Supernatant is the fluid resulting from centrifugation of hybridomas in tissue culture that are secreting specific monoclonal antibodies. Supernatants therefore typically contain culture medium and 5-10% fetal calf serum. Speicifc antibody concentration is in the range of 50 5g/mL.	Universal
Purified Immunoglobulin	Purified immunoglobulin typically refers to monoclonal antibodies that have been purified using protein A or protein G affinity chromatography.	Universal
F(ab)2 Fragment	F(ab)2 fragments are produced by digestion of IgG with pepsin yielding a divalent molecule (containing two antibody binding sites) but lacking the Fc portion.	F(ab) 2 antibody fragments are used in assay systems where the presence of the Fc region may cause problems. Samples such as lymph nodes, spleen, and peripheral blood preparations contain cells with Fc receptors (macrophages, B lymphocytes, and natural killer cells) which could bind the Fc region of intact antibodies, causing high background staining. Use of F(ab)2 fragments ensures that any antibody binding observed is not due to Fc receptors.1 These fragments may also be desirable for staining cell preparations in the presence of plasma, because they are not able to bind complement, which could lyse the cells. F(ab)2 fragments, because of their smaller size, are able to localize antigen more precisely than intact IgG, particularly in staining tissue for electron microscopy. The divalency of F(ab)2 fragments enables cross-linking of antigens, allowing use of these reagents in precipitation assays, rosetting assays2, or for cellular aggregation via surface antigens. 3

References

1. K S, T A, T T. 1998. Decreased Fcgamma receptor III (CD16) expression on peripheral blood mononuclear cells in patients with Sjögren's syndrome. 25 4 689-96. J Rheumatol..

2. DESAINTMARTIN J, SCHWARTZ J, MANNESSIER L, EYQUEM A. 1983. Idiotypic and anti-idiotypic determinants on lymphocytes during anti-Rh immunization. Revue Francaise de Transfusion et Immuno-hématologie. 26(6):573-583. https://doi.org/10.1016/s0338-4535(83)80072-5

3. De Reys S, Hoylaerts M, De Ley M, Vermylen J, Deckmyn H. 1994. Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation. 84(2):547-555. https://doi.org/10.1182/blood.v84.2.547.547