Polyclonal antibody catalytic variability

Abstract

We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

Development of New Antibodies and an ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine

“…To obtain the serum, all of these blood samples were left at room temperature (22 °C) for 30 min and then centrifuged at 1191× g for 10 min, after which the supernatant was collected. In accordance with a previous report [ 17 ], antibody titers in these sera were confirmed by direct ELISA. For the present study, we ensured the collection of sufficient antibody titers, as the results of ELISA performed with 40,000-fold diluted serum after 5 times immunization had previously shown absorbance values of 1.5–2.0 at 450 nm/630 nm wavelengths.…”

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the ‘flexibility’ hypothesis

“…Both anxieties have been shown to be without foundation. Thus, as far as the first is concerned, catalysis by all of the polyclonal IgG preparations investigated to date in different laboratories has not deviated from single-site (Michaelis-Menten) saturation kinetics (e.g [2][3][4][10][11][12][13][14][15][16][17][18]). Given the heterogeneous nature of the polyclonal antisera, this observation may be regarded as counter-intuitive.…”

An active immunization approach to generate protective catalytic antibodies

“…This approach avoids the problematic immune responses associated with monoclonal catalytic antibodies. Several studies have demonstrated that PCAbs active in itro can be generated relatively simply, rapidly and cheaply [10][11][12][13][14]. Here, we provide the first evidence that enzymically active PCAbs can be useful as therapeutic reagents in i o.…”

“…Furthermore, this strategy can be used for treatment of enzyme deficiencies and cardiovascular diseases. Although the catalytic activity of PCAbs generated in past studies was modest [10,13], improvements in hapten design methods and immunization strategies may lead to an increase in the catalytic activity of antibodies and broaden the application of catalytic antibodies for therapeutic strategies in i o.…”