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A Manual of Online Molecular Biology Techniques

The "Lunchbox" Immunoabsorbent Technique

Ed Rybicki

copyright 1990, 2000

I have explored the possibilities of using nitrocellulose and other adsorptive membranes as immunoadsorbents for simple, reliable "low tech" procedures for the purification of a specific antibody. The technique does not require the prior fractionation of an antigen mixture by electrophoresis, and requires only very basic laboratory equipment, including nitrocellulose membrane, simple benchtop centrifuges, a plastic freezer container or "lunch box," and some form of simple immunoassay system. The suitability of the method for the small-scale preparation of antibodies for immunoassays such as ELISA, immunodot blot, and western blotting, is discussed.  

Another techniques page will cover the specific absorption of Ab to, and elution from, electrophoretically-purified proteins ( Monospecific Ab Production ).

Contents

Absorption of antigens to membrane ("blotting")

Nitrocellulose paper (0.45um pore; Schleicher and Schuell) is cut into 10 x 10 cm squares, which fit into 12.5 x 17 x 8 cm plastic freezer boxes ("lunchboxes") with room for movement during shaking. Other membranes such as polyvinyllidene difluoride (PVDF, Millipore) or other nylon based material can also be used (E.P. Rybicki, unpublished). I have used crude Brome mosaic virus (BMV) preparations to make monospecific antibody preparations: semipurified virus was made by resuspension of centrifugally pelleted clarified plant extract in O.1M phosphate pH 7.0 (1/10 original volume) and used to coat nitrocellulose. This sort of extract contains a large amount of plant proteins, including cell wall and membrane components. I and others in the lab have used a variety of other crude preparations, including E coli preparations with foreign proteins, with great success. In the E coli case, a sonicated soup or French-pressed mush works fine.

Water-wetted nitrocellulose is added to ± 25 ml volume of extract, and the boxes agitated at room temperature (22oC) for at least 2 hours. Alternatively, the boxes are left on the bench and agitated by hand every 10 min. It does not appear necessary to "fix" antigen to the membrane by heat or other treatment.

Membranes are washed in 3x changes of saline containing 0.05% (v/v) Tween-20 or Triton X-100 for 5 min, then "blocked" overnight using 20 ml phosphate-buffered saline (PBS) containing 0.05% Tween or Triton and 2% (w/v) bovine serum albumin or 1-5% (w/v) skimmed milk powder (blocking buffer).

Absorption of anti-host plant antibodies from serum

Sera to be absorbed are diluted - depending on titre, pre-tested using a simple precipitin assay - in saline/detergent, and 20 ml added to a drained blot of healthy plant extract (made exactly as for the virus blot, above) in a freezer box. The box is agitated at room temperature for 2 hours, and the serum then poured off. This is also a useful way to absorb serum dilutions immediately prior to their use in western blottests.

Attachment and elution of specific antibodies

Appropriately diluted absorbed serum is added to a drained virus extract blot in a freezer box. This is incubated at 37oC (or at room temperature) for 1-2 hr with shaking. The blot is then washed as above, with a final rinse in water.

Bound antibodies are eluted using about 20 ml of a 0.1 M glycine/HCI, buffer, pH 2.9. The box was agitated for 10 min, the liquid drained off and immediately neutralized by addition of a predetermined amount of 0. 1 M NaOH. Essentially all specifically bound antibody may be removed from a blot by a single elution (Rybicki, 1986 and unpublished), with yields of up to 1 mg for BMV extracts. The antibody attachment and elution steps may be repeated up to three times with the same BMV-infected sap blot with only slight drop in yield, as long as a fresh dilution of serum is used each time (E.P. Rybicki, unpublished).

Antibody preparations are concentrated by dialysis against water and lyophilisation. Preparations are resuspended in small volumes of saline.

Immunoassay techniques

Antibody preparations may be tested for specific activity by indirect ELISA (eg: Rybicki and von Wechmar 1981) and western blotting (eg: Rybicki and von Wechmar 1982).  An example of a blot - taken from the source reference below - is shown here:

omvblot.jpg (29474 bytes)

Discussion

As mentioned above, 1 mg or more antibody can be prepared from a single elution of a single BMV-infected sap extract-coated membrane; this is sufficient to prepare enzyme conjugates for DAS-ELISA, and would provide materials for many assays. In any case, re-use of the antigen-coated membrane enables accumulation of enough antibody to allow many serological tests. If antisera are absorbed with host plant antigens prior to application to blots, eluted antibodies react almost exclusively with virus coat protein, with very little "background" reaction with other polypeptides in Western blot tests (see Figure). Eluted anti-BMV antibody could be diluted by a factor of 1/50 for use in Western blots, and up to 1/500 for indirect ELISA. This is a far better yield than can be obtained with individual excised polypeptides.

An important consideration is that cheap materials may be used. Nitrocellulose or other membrane is the only expensive component, and that is far cheaper, far easier to use, and far better as an adsorbent than CNBr- Sepharose, or other comparable column chromatography materials commonly used as immunoadsorbents. Up to 100 ug protein/cm2 may be adsorbed onto nitrocellulose; polyvinyllidene fluoride's capacity is even higher.

The use of crude extracts to purify antibodies is important when no facilities exist for purification of low-yielding viruses: clarified sap extracts could be concentrated by PEG treatment; non-infected extracts could be used to absorb antisera; and infected extracts to purify monospecific antibodies. Even if antibody preparations are not exactly monospecific, their preparation in this way represents an important purification and concentration step, as relatively specific antibodies are purified in one step from raw serum, with a consequent increase in activity of the preparation once most of the extraneous serum proteins and immunoglobulins are removed.

This has worked very sucessfully with detection of plant virus coat protein bands against a background of whole plant, using antisera that reacted with EVERYTHING before absorption; also with antisera raised against purified proteins either from, or cloned into, E coli: in latter case, as anyone who has done it knows, when you do a Western, ALL the bands light up, as rabbits are immune to E coli and related gut microflora - and no-one thought to tell you...!

You can use the same technique to mass-absorb / elute Ab to a particular purified protein, without having to go to the trouble of making up an expensive column immunoabsorbent: soak NC or other memb in protein of interest, wash, block, soak in AS. Wash thoroughly, then elute Ab with preferred elution mix (I use 0.1M Glycine/HCl/0.15M NaCl pH 2.9). You can repeat the absorption/elution several times, and yield is quite high - certainly enough for labelling specific Ab for immunofluorescence, ELISA, etc. We have used it in our labs to make monospecific Ab to plant viruses, and to E coli proteins or proteins cloned in E coli, as long as one has a background free of the protein of interest.

You can also combine two techniques: pre-absorb antisera with membrane with complex mixture NOT containing protein of interest, then pour off antisera onto memb with complex mixture CONTAINING protein of interest, preferably at highish concentration. First absorption takes out Ab reacting with "host protein", in second, what is left is hopefully relatively monospecific, and can be eluted as above, for labelling, etc.

Very simple, very easy, VERY CHEAP!!! - and originally published here:

Source Reference:

EP Rybicki, MB von Wechmar and JT Burger (1990)

Monospecific antibody preparation for use in the detection of viruses. pp. 149-153 in "World Perspectives on Barley Yellow Dwarf" (PA Burnett, ed.); CIMMYT, Mexico DF, Mexico.

References

Rybicki, E.P. 1986. Affinity purification of specific antibodies from plant virus capsid protein immobilised on nitrocellulose. Journal of Phytopathology 116:30-38.

Rybicki, E.P. and M.B. von Wechmar. 1981. The serology of the bromoviruses. 1. Serological interrelationships of the bromoviruses. Virology 109:391-402.

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.