Production and Characterization of a Monoclonal Antibody to Capture Proteins Tagged with Lithocholic Acid

Ikegawa, Shigeo; Yamamoto, Tetsushi; Miyashita, Takahiro; Okihara, Rika; Ishiwata, Shunji; Sakai, Toshihiro; Chong, Rung-Hwa; Maeda, Masako; Hofmann, Alan F.; Mitamura, Kuniko

doi:10.2116/analsci.24.1475

Cited by 8 publications

(5 citation statements)

References 27 publications

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“…This contrasts with the moderate sensitivity to free antigen (micromolar range) observed for the cyclic aliphatic and nonpolar COP. Improvement in binding sensitivities have been reported in other studies following the chemical addition of polar OH or HO 3 SO groups at the carbon position 3 of COP (lithocholic acid or glycolithocholic acid sulfate) [ 47 , 48 ]. It would appear therefore, that the presence of polar groups within the POR structure was more important in the generation of higher sensitivity binders than structural rigidity alone.…”

Section: Discussionmentioning

confidence: 63%

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction

Qaraghuli

Palliyil

Broadbent³

et al. 2015

BMC Biotechnol

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BackgroundLow molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes.MethodsDifferent hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential.ResultsThe simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL.ConclusionsThe best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.

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Section: Discussionmentioning

confidence: 63%

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction

Qaraghuli

Palliyil

Broadbent³

et al. 2015

BMC Biotechnol

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“…Bile acids are often attached as pendent groups to polysaccharides (chitosan [50][51][52] , glycol chitosan [53][54][55][56] , heparin [57] , dextran [58][59][60] or cellulose [61] ) or proteins (bovine serum albumin) [62,63] via ester or amide bonds (Figure 9) [64] . Polymers can be modified to introduce -CHO groups, followed by derivation with bile acids (Figure 10) [59][60] .…”

Section: Bile Acids As Pendent Groups Of Polymersmentioning

confidence: 99%

Biomaterials made of bile acids

Zhang

Zhu

2009

Sci. China Ser. B-Chem.

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The use of natural compounds in the preparation of new materials can improve the biocompatibility of the materials and avoid any potential toxicity of the degradation products when used for biomedical applications. Bile acids are amphiphilic molecules biosynthesized in the liver. They are used to prepare various polymers and oligomers. These polymers made of bile acids are promising materials in both biomedical and pharmaceutical fields.

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“…有趣的是, 如在胆酸的 24 位接上热敏性的高分子, 然后再开环聚合, 则可以得到热敏性的药物载体 [46] . [61] 等多糖上或牛血清白蛋白 [62,63] 等蛋白质上. 通常是利用高分子活泼的-OH、 -NH 2 与胆酸分子的 3 位或 24 位形成酯键或酰胺键 (图 9) [64] ; 也可以将高分子氧化, 通过所得的-CHO 与胆酸相连(图 10) [59,60] .…”

Section: 以胆酸为核的星型高分子unclassified