Relative merits of solid phases

The simple manipulations required to separate free antibody or antigen from immune complexes immobilized non-covalently on plastic solid phase is probably the most important reason for the rapid increase in popularity of EIA. Desired traits of the solid phase are (i) high capacity for binding immunoreactants (high surface/ volume ratio) (ii) possibility of immobilization of many different immunoreactants (iii) minimal dissociation (iv) negligible denatura-tion of immobilized molecule and (v) orientation of immobilized antibody with binding sites towards the solution and the Fc to the solid phase (e.g.. Section 13.2.2). 

Plastic is by far the most popular solid phase, since it makes the procedures extremely simple. However, plastics may also have some important limitatons (i) they are immunoreactant-consumptive, i.e. often require 10 times more reactants than particulate solid phases or membranes (ii) the avidity of immobilized antibodies for large antigens decreases by 1-2 orders of magnitude (Zwolinski, G. Jo-sephson, L. cited by Parsons, 1981), probably due to the wide spacing of epitopes or paratopes (iii) the rate of antibody-antigen interactions is slower than in solution or with particulate solid phases (hours instead of minutes), due to the necessity of the free immuno-reactant to diffuse to the solid phase (association kinetics is largely dictated by diffusion rate Section 8.4) and, (iv) few suitable antibod- 

Nitrocellulose membranes are not yet widely used but should replace plastic in many investigations in which (i) only the presence of an immunoreactant is to be established and not its quantity (though the latter is also possible) (ii) only very small samples (e.g., less than 1 pi) are available and, (iii) ionic detergent-solubilized antigens are to be tested. Nitrocellulose binds close to 100% of most antigens or antibodies. 

Particulate solid phases (agarose, cellulose, polyacrylamide, dex-tran) are very efficient since they may be dispersed throughout the reaction mixture and have a much higher ratio of surface area/ volume. Moreover, the immunoreactant is covalently bound. 

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