The Elisa guidebook
.pdfThe purpose of immunization is to obtain high-titer antisera that bind strongly to antigen (high avidity). The properties of antisera are determined by the genetic composition of the animal injected (particularly the Ir genes). This means that there can be great variation in the quantitative and qualitative aspects of antisera from among species and even among individuals of the same species. This should be borne in mind when considering the use for which the serum is being made. In preparing sera one should (1) always obtain a preimmunization serum, and (2) never automatically pool sera. Point (2) is particularly important if a defined property of an antiserum is required (e.g., in discrimination of antigens).
Up to a certain degree, an increase in the dose (weight) of antigen will increase antibody titer; however, this may also increase crossreactivity. Adjuvants also increase the immunogenicity of proteins. Haptens should be labeled with carrier proteins to elicit an immune response. The carrier protein should be foreign to the host to be recognized by the T-cells. For most immunogens, the interaction of T- and B- cells is essential for antibody production.
The animal species chosen can be important. The animal species most often used in laboratories are rabbits, goats, guinea pigs, pigs, sheep, and rats. Commercial companies may favor horses and donkeys for large-scale preparations. Many animals contain crossreactive antibodies in their serum before immunization; this could complicate their use in ELISA, and some simple absorption technique may be required (or may have been performed in commercial preparations) to block such reactions. Another point to remember is that many smaller animals can be immunized as compared to only a few larger animals,
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owing mainly to cost considerations. Because of the variations in sera from the previously mentioned animals, smaller animals offer advantages in which relatively small volumes of serum are required.
For most immunization regimes, the immunogen (at about 2 mg/mL) in an isotonic salt solution is mixed vigorously with an equal volume of Freund's complete or incomplete adjuvant, to obtain an emulsion that is stable in water. It is essential that the antigen be added in small aliquots in a stepwise fashion to the adjuvant, with vigorous mixing between each addition (e.g., using a vortex mixer). On complete addition of the antigen, the emulsion must be tested for stability. This is easily done by placing a drop of the emulsion on to the surface of some distilled water in a beaker. This should spread out over the surface. However, a second drop added (or sometimes a third) should not spread and remain as a distinct drop, and the edges of the drop should show no signs of dissolution.
4.2¡ª Immunogen Dose
The amount of immunogen needed to induce an immune response depends on the exact nature of the antigen and the host species used. A typical dose/ response is sygmoidal in nature, whereby very large and very small doses of material elicit a weak or no response. Generally, the lowest effective dose of an antigen is preferred when raising antisera for immunoassays since this tends to elicit antibodies of highest affinity and produce polyclonal sera of high avidity. General values can be given; however, a range of concentrations of antigen is often needed to allow an estimation of its potency. Rabbits and guinea pigs usually require approx 100 µg of protein with an adjuvant, whereas doses of between 500 and 1000 µg are required for larger animals. Generally, subsequent booster doses are lower and given without adjuvant.
4.3¡ª
Improving Antigenicity of Antigens
Adjuvants are substances that enhance the immune response. A range of adjuvant methodologies is available, including the following:
1.Water-in-oil emulsions such as Freund's complete adjuvant, Freund's incomplete adjuvant. The complete adjuvant contains heat-killed Mycobacterium tuberculosis bacteria whereas the incomplete does not. Both have the basis of an immune modulator, e.g., branched glucose polymers or methylated bovine serum albumin (Pierce's AdjuPrimeTM).
2.Minerals in which the antigen can be absorbed on aluminum hydroxide, bentonite, or quaternary ammonium salts.
3.Bacterial species such as Bacille Calmette-Gu¨¦rin, Bordetella pertussis, or Corynebacterium parvum.
4.Bacterial products such as endotoxins, lipopolysaccharides, and liposomes.
5.Polynucleotides such as poly I-poly C, and poly (A-U).
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The adjuvant actions generally stimulate immune responses nonspecifically by increasing antigen presentation and the number of collaborating cells involved. This effectively reduces antigen doses required and enhances immunogenicity of proteins. Adjuvant may alter the spectrum of antibodies produced in terms of both isotypes and specificities. Adjuvants also act to produce depots of antigen that are released slowly, thereby promoting continuous stimulation of the immune system. In addition, adjuvants may protect the immunogen from rapid removal and cleavage from host enzymes (see ref. 9 for review of adjuvants).
In cases in which there is no response in animals after multiple injections, alternative animal species should be tried and the dose of antigen(s) increased. If this does not succeed, then attempts to enhance the antigenicity by direct modification methods can be tried. An excellent practical description of these techniques is found in ref. 7. Common methods include the following:
1.The addition of small modifying groups such as dinitrophenol or arsenate.
2.Denaturation of antigen by heat treatment and/or sodium dodecyl sulfate treatment.
3.Coupling of antigen to small synthetic peptides that are sites for T-cell receptor class II protein binding.
4.Coupling of antigens to large particles such as sheep red blood cells or agarose beads.
5.Purification of antigens with other antibodies and injection of immune complexes.
References
1.Harlow, E. and Lane, D., eds. (1988) Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
2.Avrameas, S. (1972) Enzyme markers: their linkage with proteins and use in immuno-histochemistry.
Histochem. J. 4, 321¨C330.
3.Farr, A. G. and Nakane, P. K. (1981) Immunochemistry and enzyme labeled antibodies: a brief review. J. Immunol. Methods 47, 129¨C144.
4.Nakane, P. K. and Kawaoi, A. (1974) Peroxidase-labelled antibody: a new method of conjugation. J. Histochem. Cytochem. 22, 1084¨C1091.
5.Tijssen, P. and Kurstak, E. (1984) Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays. Anal. Biochem. 136, 451¨C457.
6.Avrameas, S. and Ternynck, T. (1969) The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunoadsorbents. Immunochemistry 6, 53¨C66.
7.Avrameas, S. and Uriel, J. (1966) Methode de marquage d'antigen et anticorps avec des enzymes et son application en immunodiffusion. Cr. Acad. Sci. D 262, 2543¨C2545.
8.Immunogens, Ag/Ab Purification, Antibodies, Avidin-Biotin, Protein Modification: PIERCE Immunotechnology Catalog and Handbook, Volume 1. Pierce and Warriner, UK (published yearly).
9.Spier, R. and Griffiths, J. B. (1985) Adjuvants in Animal Cell Biotechnology, vols. 1 and 2, Academic, New York.
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11¡ª
Test Questions
This chapter consists of a test concerning ELISA and the sciences needed to perform assays. The test can be used to gage knowledge on ELISA. Suggested points are given for the benefit of educators. Of course, the test can be adapted. All the answers can be obtained by reading this book. It may be useful to test oneself before and after referring to the book. Answers follow the test as a guide to marking.
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