Antibody-antigen complex affinity

Scheme 13 Electronic and optical transduction of the formation of antigen-antibody affinity complexes on transducers (A) ampero-metric transduction at an electrode (R+/Ris a redox label in the electrolyte solution), (B)...

Nielsen et al. used this method to separate antigen-antibody complexes of recombinant hGH with monoclonal antibody specific to hGH (12). The complexes were well resolved from the free antibody and antigen in solution. The strong binding affinity of the antibody for hGH results in strong, stable antibody-antigen complexes that form very rapidly in solution two complexes (IgG-hGH, IgG-(hGH)2) have been observed. 

The interaction between biological macromolecules can be of various degrees of complexity. These interactions may be of high specificity, as in antigen-antibody complexes, or of low specificity, as in protein-lipid associations. The complexes that are formed may be very stable, or highly unstable. These properties of a complex must be considered in selecting an affinity adsorbent to be used for the purification of a specific substance. 

Friguet, B., Chaffotte, A. F., Djavadi-Ohamance, L, and Goldberg, M. E (1985) Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent-assay. J Immunol Methods 77,305-319... 

The antibody capture affinity assay samples the free MAb binding sites by the addition of FITC-labeled antigen to the incubation mixture for 1 min, and the antigen-antibody complexes are immobilized on anti-IgG particles. The plates then are treated as above. 

Biotin has served this purpose well in both nucleic acid and antibody probe systems. As well as being easily detected with immunoglobulins specific for biotin, biotin may also be detected non-immunologically with avidin or streptavidin, two proteins which share a marked, highly specific affinity for biotin. The affinity constant for avidin-biotin interactions is approximately 10 - liters/mole, much higher than the range for antigen-antibody interactions which are commonly between 10 -10 liters/mole. Consequently, a vast number of detection complexes composed of avidin or streptavidin bound to a detection system are commercially available (e.g. streptavidin-alkaline phosphatase). 

If the antibody is unable to cross-link or aggregate the antigen of interest to the extent needed to induce precipitation of the complex (as is often the case with monoclonal antibodies), an alternative method may be employed. Protein A from Staphylococcus aureus is known to have a high affinity for immunoglobulins. If a soluble antigen-antibody complex is incubated with an insoluble matrix covalently conjugated with Protein A, the complex will be captured by the Protein A covalently attached to the matrix (Fig. IV-9). Because Protein A... 

Sepharose and elution with lactose (Fig. 23). The UV-absorbing fraction that was eluted with the lactose solution was collected and the protein precipitated by addition of an equal volume of saturated ammonium sulfate. On refrigeration of the sample overnight, a white precipitate formed. This precipitate was collected by centrifugation and redissolved in 0.2 mL of 0.02 M phosphate buffer of pH 7 in saline. The agar-diffusion test shown in the inset of Fig. 23 showed that high-titer antibodies were produced that formed a precipitin complex with the antigen. Several affinity runs were made. The antibody samples were combined and lyophilized. The yield of anti-lactose antibody was 50 mg from 20 mL of serum. 

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