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

Sigma offers 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 assays,2 or for cellular aggregation via surface antigens.3

 

References

K Saito, T Abe, T Takeuchi
Journal of Rheumatology 1998-04-01
To examine the expression of surface structures important in natural killer (NK) cell function and the roles of serum factors affecting the expression of surface antigens on these cells in patients with Sjögren's syndrome (SS). Peripheral blood mononuclear cells (PBMC) of 18 patients with SS were analyzed by immunofluorescence o...Read More
J de Saint Martin, J Schwartz, L Mannessier, A Eyquem
Revue Française de Transfusion et Immuno-hématologie 1983-12-01
Evolution of idiotypic determinants on lymphocytes membrane and presence of other lymphocytes carrying anti-idiotypic determinants, were studied in Rh negative human volunteer blood donors during immunization towards Rh factor. For this purpose E-Rh Rosettes, direct immunofluorescence, inhibition of E-Rh Rosettes by anti-idiotyp...Read More
S De Reys, M F Hoylaerts, M De Ley, J Vermylen, H Deckmyn
Blood 1994-07-15
A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose-dependently induces platelet aggregation and serotonin release in an Fc-independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that...Read More