Monoclonal antibodies to bovine serum albumin: Affinity and specificity determinations

Morel, Guillemette; Yarmush, David M.; Colton, Clark K.; Benjamin, David C.; Yarmush, Martin L.

doi:10.1016/0161-5890(88)90085-5

Cited by 21 publications

(12 citation statements)

References 17 publications

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“…Competitive ELISAs were performed to evaluate the effect of labeling on the antigenicity of lysozyme and BSA. The antibodies MAb 5.1 and MAb 9.1 are known to bind to domains I‐C and III‐C of BSA, respectively (25). As can be seen in Figure 1, the presence of the IAEDANS label did not affect the binding of BSA to antibody MAb 5.1 and only slightly affected the binding to MAb 9.1.…”

Section: Resultsmentioning

confidence: 99%

“…Anti‐BSA monoclonals used in this study (6.1 and 9.1) were selected from a panel of MAbs which had been previously characterized in this laboratory according to their physicochemical and binding properties (25, 26) as well as their pressure behavior upon immobilization. The cell lines were donated by NEN Research (Boston, MA) and grown in mouse ascites, as previously described (25). The anti‐HEWL monoclonals (HyHEL‐5, HyHEL‐10) used in this study were a gift from Dr. Sandra Smith‐Gill (National Institutes of Health, Bethesda, MD) and were also grown in mouse ascites.…”

Section: Methodsmentioning

confidence: 99%

“…The anti‐HEWL monoclonals (HyHEL‐5, HyHEL‐10) used in this study were a gift from Dr. Sandra Smith‐Gill (National Institutes of Health, Bethesda, MD) and were also grown in mouse ascites. All anti‐BSA and anti‐HEWL MAbs were murine IgG1, κ. Anti‐BSA MAbs were isolated by affinity chromatography as previously described (25, 27).…”

Section: Methodsmentioning

confidence: 99%

“…The BSA antibodies used here have been extensively characterized and found to exhibit noncooperative binding such that there are ca. 2 binding sites for each antibody molecule (25). For each protein, we assume that M T is equal to twice the antibody concentration.…”

Section: Methodsmentioning

confidence: 99%

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Pressure-Induced Dissociation of Antigen-Antibody Complexes

1998

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Pressures on the order of 1000-4000 bar have been reported to reversibly dissociate a number of oligomeric protein complexes without gross changes in protein structure. Here, we report that hydrostatic pressure can also dissociate some antigen-antibody complexes in solution. The association of fluorescent-labeled antigens with monoclonal antibodies was monitored via increases in the fluorescence anisotropy upon binding. Previously, we had found that pressures of 2000 atm were able to dissociate bovine serum albumin (BSA) from immunoadsorbents formed from certain antibodies but not others. In this study, we have found that the sensitivity to pressure in solution is present for the interaction of BSA with MAb 9.1 and absent for the interaction of BSA with MAb 6.1; this behavior is consistent with the immunoadsorbent study. The interaction of hen egg white lysozyme with two monoclonal antibodies was also measured. Interestingly, the complex with the greater electrostatic character (HyHEL-5) did not exhibit pressure sensitivity, as would be expected due to electrostriction effects, whereas the more hydrophobic complex (HyHEL-10) exhibited a strong pressure sensitivity. In each of the systems displaying pressure sensitivity, the free energy of association was found to increase linearly with pressure, indicating a constant change in volume between the free and bound states. Overall, these results indicate that some antigen-antibody complexes exhibit significant sensitivity to pressure, whereas others do not; the mechanisms that discriminate between these cases remain unresolved. Understanding and manipulation of this phenomenon may prove useful in a variety of processes involving the recovery from antigens of antibodies.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pressure-Induced Dissociation of Antigen-Antibody Complexes

1998

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“…First, the preparation of anti-BSA mAbs has been difficult. 16 Previously, it was difficult to raise hybridomas that secrete high affinity specific mAbs against BSA using routine techniques because such hybridomas were found hard to raise in normal bovine serum medium. However, in some subsequent studies employing serum-free Hymedium 606, some hybridomas were also difficult to culture.…”

Section: Discussionmentioning

confidence: 99%