Labeling antibodies with enzymes, fluorochromes, or biotin provides a signal for visualization or quantitation of the target molecule. Antibody bound to agarose is useful for separating a target antigen from a complex mixture. To avoid excessive background staining and to improve sensitivity, only purified antibodies should be used for staining. At the minimum, the IgG fraction, which contains naturally occurring immunoglobulin as well as specific antibody, should be isolated from the antiserum. This can be done by various methods, usually involving a combination of fractionation and chromatography. Preferably, the antibody should be affinity isolated on a column containing the antigen bound to a solid support. This will eliminate all serum proteins, including immunoglobulins that do not specifically bind to the antigen. Protocols for antibody purification can be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) Chapters 8 and 13 (Product Code A 2926).
Labeling with Enzymes
Antibodies may be labeled with various enzymes to provide highly specific probes that both visualize the target and amplify the signal by acting on a substrate to produce a colored or chemiluminescent product. Horseradish peroxidase and alkaline phosphatase are the most commonly used enzymes for this purpose. Antibody-enzyme conjugates may be used for detecting proteins in immunohistology and immunocytology1,2, immunoblotting3,4, and ELISA5,6.
Protocols for conjugating enzymes to antibodies may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 342-352 (Product Code A 2926).
- Hanas, J.S., et al., expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and p53 tumor suppressor in dysplastic progression and adenocarcinoma in Barrett esophagus. Cancer, 86, 756-763 (1999).
- Heider, H., and Schroeder, C., Focus luminescence assay: macroscopically visualized foci of human cytomegalovirus and varicella zoster virus infection. J. Virol. Methods, 66, 311-316 (1997).
- Krajewski, S., et al., Detection of multiple antigens on western blots. Anal. Biochem., 236, 221-228 (1996).
- Roe, I.H., et al., Changes in the evolution of the antigenic profiles and morphology during coccoid conversion of Helicobacter pylori. Korean J. Intern. Med., 14, 9-14 (1999).
- Sanz, C. et al., An enzyme-linked immunosorbent assay applicable to screen blood donors for IgA deficiency. Haematologica, 84, 887-890 (1999).
- Lundberg, B.B., et al., Conjugation of an anti-B-cell lymphoma monoclonal antibody, LL2, to long-circulating drug-carrier lipid emulsions. J. Pharm. Pharmacol., 51, 1099-1105 (1999).
Labeling with Fluorochromes
Fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and R-phycoerythrin (PE), represent the fluorescent probes most commonly cited. Labeling proteins with fluorochromes provides a colored reagent that can be observed directly. The intensity and narrow wavelength of the fluorochromes makes them useful in immunohistochemistry and immunocytochemisty, using both visible and fluorescence microscopy,1 and in flow cytometry.2
Protocols for labeling antibodies with FITC and TRITC may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 353-355 (Product Code A 2926). A procedure for labeling antibodies with PE has been published by Kronick, et al.3
Note: Also see FITC-1, FITC Conjugation Kit
- Sender, S., et al., Localization of carbonic anhydrase IV in rat and human heart muscle. J. Histochem. Cytochem., 46, 855-861 (1998).
- Lloyd-Evans, P., et al., Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry. Transfus. Med., 9, 155-160 (1999).
- Kronick, M.N., The use of phycobiliproteins as fluorescent labels in immunoassay. J. Immunol. Meth., 92, 1-13 (1986).
Labeling with Biotin
Biotinylated antibodies are widely used in systems wher signal amplification is desired. Biotin binds avidin with a high degree of affinity and specificity. Avidin, ExtrAvidin®, or streptavidin labeled with enzymes or fluorochromes can bind biotinylated antibodies, amplifying the signal and allowing detection of antigens present in small amounts. This system may be used in immunohistochemistry and immunocytochemistry,1 immunoblotting,2 ELISA,3 and flow cytometry.4
A protocol for the biotinylation of antibodies is available in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 340-341 (Product Code A 2926).
Note: Also see BK-101 and B-TAG, Biotin Conjugation Kits
- Erdamar S., et al., Levels of expression of p27KIP1 protein in human prostate and prostate cancer: an immunohistochemical analysis. Mod. Pathol., 12, 751-755 (1999).
- Soderberg, A., et al., Monoclonal antibodies to human thioredoxin reductase. Biochem. Biophys. Res. Commun., 249, 86-89 (1998).
- Payette, P.J., et al., Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C. Clin. Diagn. Lab. Immunol., 4, 671-675 (1997).
- Dale, G.L., Rapid production of quasi-stable antibody-phycoerythrin conjugates for use in flow cytometry. Cytometry, 33, 482-486 (1998).
Labeling with Gold
Antibodies conjugated to colloidal gold are used primarily in electron microscopy (EM)1 because of the electron-dense nature of the gold particles. Gold-antibody conjugates may also be used for immunohistochemistry and immunocytochemistry,2 and immunoblotting.3 In some cases silver enhancement may be used to amplify the signal.4 Sigma offers colloidal gold in three particle sizes, 5, 10, and 20 nm, for use in these applications. In general, 5 nm particles are recommended for intracellular staining because they are able to penetrate the cell membranes more easily, and for high resolution EM because the small size allows more exact localization of antigen. 10 nm particles are recommended for cell surface staining and for light microscopy because the larger size makes the stain more visible. 20 nm particles are recommended for blotting and for some histochemical applications. These are suggestions only — the end user will have to determine the best particle size for each application.
A protocol for preparation of gold-antibody conjugates can be found in Immunochemistry, A.C. Cuello, ed., John Wiley & Sons (Chichester, England, 1983), pp. 353-358.
- Wildenburg, G., et al., onchocerca volvulus: ultrastructural localization of two glutathione S-transferases. Exp. Parasitol., 88, 34-42 (1998).
- Wan, L.H., and Xing, M., Immunolocalization of actin in intact and DNA- and histone-depleted nuclei and chromosomes of allium cepa. Cell. Res., 8, 51-62 (1998).
- Boyle, D.L., and Takemoto, L.J., Localization of MIP 26 in nuclear fiber cells from aged normal and age-related nuclear cataractous human lenses. Exp. Eye Res., 68, 41-49 (1999).
- Brandt, B., et al., Isolation of blood-borne epithelium-derived c-erbB-2 oncoprotein-positive clustered cells from the peripheral blood of breast cancer patients. Int. J. Cancer, 76, 824-828 (1998).
Attaching Antibodies to Agarose
Antibodies attached to agarose have a variety of applications. They can be used to isolate proteins and other compounds from sera or from cell and tissue homogenates by affinity chromatography for quantitation or further analysis.1,2 They have also been used for immunoprecipitation of proteins from cell lysates,3 and for reduction of serum immunoglobulin in autoimmune diseases4 and transplantation experiments.5
A protocol for attachment of antibodies to cyanogen bromide-activated agarose is available in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 536-537 (Product Code A 2926).
- Miller, C.H., et al., Diagnosis of von Willebrand disease type 2N: a simplified method for measurement of factor VIII binding to von Willebrand factor. Am. J. Hematol., 58, 311-318 (1998).
- Boudjennah, L., et al., Immunopurification and characterization of a collagenase/gelatinase domain issued from basement membrane fibronectin. FEBS Lett., 391, 52-56 (1996).
- De Corte, V. et al., Identification of Tyr438 as the major in vitro c-Src phosphorylation site in human gelsolin: a mass spectrometric approach. Protein Sci., 8, 234-241 (1999).
- Schoen, H., et al., Immunoapheresis in paraneoplastic pemphigus. Arch. Dermatol., 134, 706-710 (1998).
- Pascher, A., et al., Impact of immunoadsorption on complement activation, immunopathology, and hepatic perfusion during xenogeneic pig liver perfusion. Transplantation, 65, 737-740. (1998).
In some assays it is preferable to use only the antigen binding (Fab‘) portion of the antibody. For these applications, antibodies may be enzymatically digested to produce either an Fab or an F (ab) 2 fragment of the antibody. To produce an F(ab’) 2 fragment IgG is digested with pepsin, which cleaves the heavy chains near the hinge region. One or more of the disulfide bonds that join the heavy chains in the hinge region are preserved, so the two Fab regions of the antibody remain joined together, yielding a divalent molecule (containing two antibody binding sites), hence the designation F (ab‘) 2. The light chains remain intact and attached to the heavy chain. The Fc fragment is digested into small peptides. Fab fragments are generated by cleavage of IgG with papain instead of pepsin. Papain cleaves IgG above the hinge region containing the disulfide bonds that join the heavy chains, but below the site of the disulfide bond between the light chain and heavy chain. This generates two separate monovalent (containing a single antibody binding site) Fab fragments and an intact Fc fragment. The fragments can be purified by gel filtration, ion exchange, or affinity chromatography. The properties of these fragments are summarized in the Table as follow:
Protocols for antibody digestion and purification of antibody fragments can be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988 (Product Code A 2926).
Fab and F (ab’) 2 antibody fragments are used in assay systems wher the presence of the Fc region may cause problems. In tissues such as lymph nodes or spleen, or in peripheral blood preparations, cells with Fc receptors (macrophages, monocytes, B lymphocytes, and natural killer cells) are present which can bind the Fc region of intact antibodies, causing background staining in areas that do not contain the target antigen. Use of F (ab‘) 2 or Fab fragments ensures that the antibodies are binding to the antigen and not 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, and to a greater extent Fab, fragments allow more exact localization of the target antigen, i.e. in staining tissue for electron microscopy. The divalency of the F(ab ’) 2 fragment enables it to cross-link antigens, allowing use for precipitation assays, cellular aggregation via surface antigens,2 or rosetting assays. 3
- Saito, K. et al., Decreased Fc gamma receptor III (CD16) expression on peripheral blood mononuclear cells in patients with Sjogren's syndrome. J. Rheumatol., 25, 689-696 (1998).
- De Reys, S., et al., Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation. Blood, 84, 547-555 (1994).
- de Saint Martin, J., Idiotypic and anti-idiotypic determinants on lymphocytes during anti-Rh immunization. Rev. Fr. Transfus. Immunohematol., 26, 573-583 (1983).