| Autor: |
J F, Schildbach, D J, Panka, D R, Parks, G C, Jager, J, Novotny, L A, Herzenberg, M, Mudgett-Hunter, R E, Bruccoleri, E, Haber, M N, Margolies |
| Rok vydání: |
1991 |
| Předmět: |
|
| Zdroj: |
The Journal of biological chemistry. 266(7) |
| ISSN: |
0021-9258 |
| Popis: |
Two spontaneous variants of the murine anti-digoxin antibody-producing hybridoma cell line 26-10 were isolated by two-color fluorescence-activated cell sorting on the basis of altered hapten binding. The variable region sequences of the antibodies produced by the mutant lines revealed that each contains a single amino acid change in the heavy chain second complementarity determining region. A Tyr to His change at position 50 leads to a 40-fold reduction in affinity for digoxin. A Ser to Phe mutation at position 52 results in a 300-fold reduction in affinity for digoxin. A competition assay involving 33 digoxin analogues was used to examine the specificity of hapten binding of 26-10 and the two mutant antibodies. The position 50 mutant has a distinct specificity change; it exhibits a preference for digoxin congeners containing a hydroxyl group at the steroid 12 position, whereas the 26-10 parent does not. The affinities of all three antibodies for hapten are progressively lowered by substitutions of increasing size at the digoxin steroid D ring 16 position. Although 26-10 binds digoxin and its genin form equally, 12 and 16 steroid position substitutions which lower affinity also confer a preference for a sugar at the steroid 3 position. These results suggest that position 50 contributes to specificity of the antibody and that alterations of the hapten can lead to differences in recognition, possibly through a shift in hapten orientation within the binding site. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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