Affinity Purified Monospecific Antibodies to Gel-Purified Proteins
copyright 1986, 2000
- Materials and Methods
- The routine use of the technique
- Preparation of immunoadsorbent
- Adsorption of antibody
Since the introduction of sensitive immune assays, it has become increasingly apparent that previous criteria of protein purity are inadequate for the production of truly specific antisera. For example, I found that rabbit antisera raised against highly-purified Brome mosaic virus (BMV) reacted sufficiently well with plant components as to obscure the virus protein reaction in western blotting tests with infected plant samples (RYBICKI and VON WECHMAR 1982), even though only traces of plant proteins were visible after silver staining of SDS-PAGE fractionated purified virus used for rabbit immunisation (RYBICKI, unpublished). Because this problem necessitated the thorough and relatively laborious absorption of all sera to be used in such tests, I decided to investigate the feasibility of using preparative electroblotting to purify virus capsid protein to a sufficiently rigorous extent as to ensure the absence of any of the common host protein contaminants, and then to apply the method of OLMSTED (1981) for the affinity purification of virus-specific antibodies from antisera that also reacted with plant components. The latter technique entails using the virus protein immobilised on a porous support as a specific immunoadsorbent, from which antibodies may be eluted after specific binding. This investigation was designed to test the applicability of the outlined procedures to the routine small-scale production of antibodies suitable for use in enzyme-linked immunosorbent assays (ELISA), for viruses that occur only at very low concentration in plant sap, or which are extremely hard to purify because of their lability. There are also obviously many other applications!
Nitrocellulose paper eg: BA 85, 0.45 um (Schleicher and Schuell, Keene, NH, USA).
Goat- anti-rabbit IgG horse radish peroxidase conjugate (GAR-HRP) eg: from BioRad Laboratories, as for the 4-chloro-l-naphthol-containing colour reagent. I have also used alkaline phosphatase conjugate and appropriate colour reagents ( see here for methods ).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and sample preparation techniques are described elsewhere (RYBICKI and VON WECHMAR 1982).
For the Olmsted technique, the sample containing the protein of interest is electrophoresed as identical aliquots in separate gel lanes, as a continuous "smear" - detectable by immunoblotting or silver staining - results from running samples as one wide lane (RYBICKI, unpublished). Electrophoresis is best performed at 4 'C in an apparatus with a heat-exchanger (such as the Hoefer SE-600) so as to avoid the 'smile" effect that results from uneven heating of the gel during electrophoresis.
The routine use of the technique:
Dilute samples 1 : 3 in SDS-PAGE sample buffer, heat to 95 'C for 5 min, and load20 ul (20 ug) into each of the 10 slots of a 1.5 mm-thick 4.5 %/12 % discontinuous acrylamide gel cast in a Hoefer SE-600 apparatus. Samples are run at 9 mA/gel at 4 'C for 16 h with coolant circulating. The gels are notched with a scalpel at the top of each track, the stacking gel is removed and the resolving portion laid onto a sheet of pre-wetted nitrocellulose, Electrophoretic transfer by the method of RYBICKI and VON WECHMAR (1982) - using large carbon electrodes in a home-built apparatus - is performed, eg: for 3 h with a current of 1A, at 22 'C.
Vertical strips corresponding to lanes 1 and 10 (alignment strips) are excised from the stacked blots. All the nitrocellulose pieces are soaked in 10 mM Tris/150 mM NaCl/2 % BSA/0.05 % Nonidet P-40 (NP- 40 blocking buffer; also use Triton X-100 or Tween 20, and 1-5 % non-fat dry milk suspension) for 12 h to block protein-adsorption sites. The two alignment strips from each blot are then shaken for 90 min with antiserum diluted (eg) 1/1000 in blocking buffer, washed on a shaker with 4 x changes of 150 mM NaCl/0.05 % NP-40 (wash buffer) for 5 min, and probed with a 1/ 1000 dilution of GAR-HRP or other conjugate for 90 min. The immunoblots are developed in a substrate solution made by mixing eg: 1 volume of 3 mg/ml BioRad colour reagent in methanol, 5 volumes of 50 mM Tris/ 200 mM NaCl pH 7.4, and 1/2000 volumes of 30 % H202 (0.015 % final concentration). The reaction is stopped by rinsing in water.
In a test experiment with BMV the two side strips each had faint protein bands at 40 kd, strong bands at 20 kd, and minor bands smaller than 20 kd (see Fig 1 .). The 20 kd band is the virus coat protein monomer; the 40 kd band has been presumed to be a coat protein dimer, and the minor bands to be derived by proteolysis from the coat protein (RYBICKIand VON WECHMAR 1982).
The strips were aligned with the parent blot, and three horizontal strips of 5 mm width - corresponding to the 20 kd capsid protein monomer (strip BM), the 40 kd (presumed) capsid protein dimer (strip BD), and a region of the blot containing no polypeptides (strip BC) (see Fig. 1) - were excised.
Adsorption of Antibody:
The strips were shaken in 1/40- diluted BMV antiserum for 90 min, then washed in 4X changes of wash buffer, and 2X changes of saline. The preparative strips were placed coiled in 50 ml beakers, and 7.5 ml of 0.1 M glycine-HCI buffer pH 2.9 added to each. The beakers were agitated for 10 min, the contents decanted and quickly neutralised by the addition of 1.4 ml of 0.1 M NaOH. The entire process was repeated twice - to give preparations 1, 2 and 3 - using the same serum dilution as antibody source. Essentially all IgG is removed after two elution steps with the glycine-HCI buffer (result not shown); blots may be re-used up to six times (EP Rybicki, unpublished) for successive serum absorption / elution.
Fig. 2. Purification of BMV-specific antibody from a mixed antiserum
All immunoblot strips had 10 ug of BMV (left lane) and 20 ul of barley homogenate (right lane). Strip 1 was probed with a 1/5-diluted 1:1 mixture of anti-BMV and anti-"Fraction 1" (Rubisco) antisera; strip 2 with antibodies eluted from an electroblotted strip of BMV monomer protein (strip BM, see Fig 1); and 3 with the eluate of control strip BC. Monospecific antibody preparations were used unconcentrated.
Fig. 3. Illustration of the R
eactivity of Antibodies Eluted from Low Molecular-Weight Barley Protein.
Immunoblot 1 contains 5 ug of BMV (left lane) and 10 ul of barley homogenate (right lane), 2 contains only barley homogenate and is the right-hand "alignment strip' from a blot of identical samples used to prepare specific antibodies for low-MW proteins. The dotted lines indicate the region of the whole blot that was excised. 2 was probed with I/50-diluted anti-'Fraction 1' serum, and 1 with unconcentrated eluate from the excised strip. Molecular weights are indicated at the side of the Figure
This result illustrates the most obvious potential use of the technique: using a protein band from an electrophoretically fractionated crude extract to purify antibodies from an antiserum made against the crude extract.
The technique described here and by OLMSTED (1981) is an obvious develop- ment from "Western blotting" techniques in general, and a simple extension of well-established affinity adsorption techniques that are widely used for either the removal of unwanted antibodies from a serum, or the purification of mono- specific antibodies using a highly purified immobilized antigen. The technique has already found application in this laboratory in the production of specific antibodies for the proteins of low-yielding plant viruses, for specific baterial enzymes, and for the different capsid subunits of an insect picornavirus (E. P. RYBICKI, R. T. MEW, C. WILLIAMSON, unpublished results).
Although I have used nitrocellulose and electroblotting to immobilize SDS- PAGE-resolved polypeptides, there are no obvious objections to the use of other kinds of electrophoretic fractionation, or to non-electrophoretic transfer techniques, or to the use of other blotting papers (See OLMSTED 1981). My choice of a glycine-HCI buffer for antibody elution was based on familiarity only; other elution media could be equally or more efficient. I have found that inclusion of any of the detergents Tween-20, Triton X-100, and NP-40 helps both to reduce background staining of electroblots, and to increase the specificity of antibodies eluted from nitrocellulose, Incubation of blot strips in too high a concentration of antiserum leads to greatly increased non-specific adsorption of antibodies: a useful rule of thumb practised here is to determine the highest concentration of serum that produces minimal background staining on blots, and to use no more than double that concentration for adsorption in preparative experiments. It is evident that the technique cannot be used for large-scale production of antibody. Accumulation of electroblots from many gels would enable preparation of at most a few milligrams of specific antibody, if the blots were used repeatedly as immunoadsorbents. Rather, it is a relatively simple small-scale procedure best suited to the preparation of the small amounts of specific antibody required for use in Western blotting or iIndirect ELISA, or for labelling with isotopes, enzymes, fluorescent compounds or other ligands, for the sensitive detection of specific proteins in complex mixtures. I feel that this type of technique, which utilises relatively simple and even home-made equipment, provides a viable alternative to the far more expensive and complicated monoclonal antibody technology, especially for the less well-endowed laboratory.
Rybicki, E.P. 1986. Affinity purification of specific antibodies from plant virus capsid protein immobilised on nitrocellulose. Journal of Phytopathology 116:30-38.
Olmsted, J.B. 1981. Affinity purification of antibodies from diazotised paper blots of heterogeneous protein samples. Journal of Biological Chemistry 256:11955-11957.
Rybicki, E.P. and M.B. von Wechmar. 1982. Enzyme-assisted immune detection of plant virus proteins electroblotted onto nitrocellulose paper. Journal of Virological Methods 5:267-278.