[Antibody engineering for advanced molecular recognition].
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Animals↗
Biomedical subjects
Norihiro Kobayashi
Publications and source records attributed to Norihiro Kobayashi.
[Antibody engineering-based approach for hapten immunometric assays with high sensitivity].
The trace characterization of physiologically active substances with low molecular weight (e.g., steroids, catecholamines, prostaglandins, and oligopeptides), which are classified as "haptens", is an important subject in clinical analysis, and competitive immunoassays have conventionally been used for this purpose. However, the subfemtomole-range determination of haptens is very difficult, as the sensitivity of competitive immunoassays is essentially limited by the affinity of the anti-hapten antibodies that barely reaches the range of 10(11) (l/mol) as the affinity constant (K(a)). Although a noncompetitive "immunometric assay" format, the two-site immunometric assay (sandwich immunoassay), enables even subattomole-range measurements of macromolecules such as proteins, this principle can not be directly applied to haptens, as their low molecular mass prohibits simultaneous binding by two antibody molecules. To overcome such limitations, we are required either to create artificial antibodies showing ultrahigh affinity to haptens by protein engineering of antibody molecules ("antibody engineering") or establishment of novel immunometric assay formats applicable to haptens. This review surveys the background and recent approach for subfemtomole-range determination of haptens using novel immunometric assay methods. Our studies for the development of hapten immunometric assays are also described.
Antibodies↗
Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies.
To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable.
Animals↗
Antiallergic activity of aqueous extracts and constituents of Taxus yunnanensis.
The H2O, H2O/MeOH (1:1) extracts from the wood of Taxus yunnanensis showed a remarkable inhibitory effect on induced histamine release from the human basophilic cell line, KU812. The eleven constituents purified from the wood extracts of Taxus yunnanensis were tested by an in vitro histamine release inhibition assay. Among them, secoisolarciresinol and taxiresinol were found to show inhibitory activities. A new neolignan, 2-[2-hydroxy-5-(3-hydroxypropyl)-3-methoxyphenyl]-1-(4-hydroxy-3-methoxyphenyl)propane-1,3-diol, was isolated from the wood of Taxus yunnanensis.
Anti-Allergic Agents↗
Correlation between reduction potentials and inhibitory effects on Epstein-Barr virus activation by emodin derivatives.
As a continuation of studies using natural and synthetic products as cancer chemopreventive agents, we examined the reduction-oxidation potentials of hydroxylated emodin derivatives prepared from emodin in phosphate buffer at pH 7.2 using cyclic voltammetry. A significant correlation was found between the reduction potentials and number of the hydroxyl groups and the inhibitory effects of the hydroxylated emodin derivatives on Epstein-Barr virus early antigen activation. The electronic properties, i.e. LUMO energy and atomic charges of carbon at the 9-position (C(9)) and oxygen at the 11-position (O(11)), may also be useful for estimating the inhibitory effect on EBV-EA activation.
Antigens, Viral↗
Dissociation-independent selection of high-affinity anti-hapten phage antibodies using cleavable biotin-conjugated haptens.
The engineering of hapten-specific antibodies with affinity constant higher (K(a) values >10(10)M(-1)) than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity, based on the conventional competitive assay principle. Here we report a simple method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), selected as a model target hapten, was covalently conjugated to biotin via a spacer that included a reductively cleavable disulfide bond. Phage particles displaying high-affinity, single-chain Fv fragments (scFvs) specific for 11-DC (K(a)1.3 x 10(10)M(-1)) were incubated with the "cleavable biotin"-conjugated 11-DC, and the resulting complexes was captured on immobilized NeutrAvidin. Mild reductive conditions that did not decrease phage infectivity easily cleaved the disulfide bond, allowing the recovery of target phage particles; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC phage (specific phage ratio >90%) from among a 100,000-fold excess of nonspecific phage particles. This method will be applicable for selection of extra-high-affinity phage antibodies against a wide variety of haptens.
Animals↗
Structure-activity relations of azafluorenone and azaanthraquinone as antimicrobial compounds.
Antimicrobial activities of two azafluorenones, four 1-azaanthraquinones, five 2-azaanthraquinones, and one 2-azaquinone were tested. Several azaanthraquinones possessed broad, potent activity, while the azafluorenones demonstrated weak activity. The following structure-activity relationship was postulated: (1) activity decreased in the order 2-azaanthraquinones>1-azaanthraquinones>azafluorenones; and (2) a hydroxyl group at the peri-carbonyl group enhanced activity. In addition, correlations among reduction potential, hydrophobic parameter, and antimicrobial activity were discussed.
Anthraquinones↗
Generation of a single-chain Fv fragment for the monitoring of deoxycholic acid residues anchored on endogenous proteins.
A subset of lipophillic bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA), are thought to be biologically transformed into reactive intermediates forming covalently modified, "tissue-bound" bile acids that can exert several toxic effects. We have generated a single-chain Fv fragment (scFv) as a probe to monitor DCA residues anchored on proteins. DNA fragments encoding the variable heavy (V(H)) and light (V(L)) domains of a mouse antibody raised against a DCA hapten (Ab #88) were cloned by rapid amplification of cDNA 5'-ends. These sequences were combined via a common linker sequence coding (Gly(4)Ser)(3) to construct a single scFv gene with the gene segments in the following order: 5'-V(H)-linker-V(L)-3'. This construct was subcloned into an antibody-expression vector, pEXmide 5; soluble scFv protein was then expressed in the bacterial periplasm of the XLOLR Escherichia coli strain. In a competitive enzyme-linked immunosorbent assay using DCA-coated microtiter plates, the scFv provided a dose-response curve for free DCA ranging between 2 and 5000 pg/assay. The scFv reacts similarly with the l-lysine adduct of DCA (cross-reactivity, 72%), while bile acids having a modified DCA steroid skeleton were well-discriminated (cross-reactivity, <1%). This scFv could also monitor trace amounts of DCA residues anchored on a protein through DCA acyl adenylate reactions, the likely reactive intermediate. The present scFv may be a useful tool for trace characterization of tissue-bound bile acids; this usefulness may be significantly enhanced by fusion with signal-generating proteins, such as alkaline phosphatase or green fluorescent protein.
Amino Acid Sequence↗
Simultaneous determination of anthraquinones, their 8-beta-D-glucosides, and sennosides of Rhei Rhizoma by capillary electrophoresis.
The simultaneous separation and determination of major anthraquinones (emodin, chrysophanol, rhein and their glucosides, aloe-emodin, sennoside A, and sennoside B) of Rhei Rhizoma were achieved by cyclodextrin modified capillary zone electrophoresis. The running electrolyte used in this method was 0.005 M alpha-cyclodextrin in 0.03 M borate buffer (pH 10.0) containing 20% acetonitrile, with an applied voltage of 20 kV.
Anthraquinones↗
Correlation between reduction potentials and inhibitory effects on Epstein-Barr virus activation of poly-substituted anthraquinones.
As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the reduction potentials of some poly-substituted anthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the reduction potentials and the inhibitory effects of the poly-substituted anthraquinones on Epstein-Barr virus early antigen activation. It has been further shown that the correlation can be enhanced by introducing log P as an additional parameter.
Anthraquinones↗
A nonenzymatic modification of the amino-terminal domain of histone H3 by bile acid acyl adenylate.
Although it has been proposed that the secondary bile acids, deoxycholic acid and lithocholic acid, increase the number of aberrant crypt foci in the colon and may act as colon tumor promoters, there is little evidence detailing their mechanism of action. Histones play an important role in controlling gene expression, and the posttranslational modification of histones plays a role in regulation of intracellular signal transduction. In particular, the amino-terminal tail domain of histone H3 is sensitive to several posttranslational modifications, and acetylation of this domain changes its electrostatic environment and results in the loss of native folding. Therefore, we studied the modification of epsilon-amino groups on human histone H3 by deoxycholyl adenylate, which is an active intermediate in deoxycholyl thioester biosynthesis. After incubation of recombinant human histone H3 with a smaller amount of acyl adenylate, followed by enzymatic digestion, the peptide fragment mixtures were analyzed by matrix-assisted laser desorption ionization mass spectrometry. These data showed the formation of only one adduct fragment, which corresponded to amino acids 3-8 with a deoxycholate adduct, suggesting that the epsilon-amino group of Lys(4) had the highest reactivity. This novel modification, formation of a bile acid adduct on the histone H3 amino-terminal tail domain through an active acyl adenylate, may relate to the carcinogenesis-promoting effects of secondary bile acids.
Amino Acid Sequence↗
Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of 2-azaanthraquinones.
As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some 2-azaanthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the 2-azaanthraquinones on Epstein-Barr virus early antigen (EBV-EA) activation. It has been further shown that the correlation can be enhanced by introducing an electronic properties, i.e. the atomic charges at the C5 and O12 atoms in the quinone skeleton ring and the HOMO energy as additional parameters.
Anthraquinones↗
Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.
To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.
Animals↗
Analysis of the antigen binding site of anti-deoxycholate monoclonal antibody using a novel affinity labeling reagent, acyl adenylate.
Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site.
Adenosine Monophosphate↗
Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain.
Using liquid chromatography/electrospray ionization mass spectrometry, we have found three unconjugated bile acids [cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA)] in the rat brain cytoplasmic fraction. CDCA was detected only upon extraction with high concentrations of guanidine, indicating that it is bound noncovalently to protein in the brain. The most abundant of the three, it was present at a concentration of 1.6 nmol/g wet weight (approximately 15 mg of protein) of brain, corresponding to almost 30 times its serum concentration. CA and DCA were present at 1/30th the concentration of CDCA. Bile acids conjugated with amino acids, sulfuric acid, and glucuronic acid were not detected. These data clearly demonstrate that unconjugated CDCA and, to a lesser extent, CA and DCA, exists in the rat brain.
Animals↗
Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of naphthoquinones.
As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some naphthoquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the naphthoquinones on Epstein-Barr virus early antigen activation. It has been further shown that the correlation can be enhanced by introducing an electronic property, i.e. the atomic charges at the C(4) and O(10) atoms in the quinone skeleton ring as additional parameters.
Animals↗
Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay.
Anti-idiotype antibodies recognizing the variable regions of a particular anti-hapten antibody are valuable tools, which can be used in sensitive hapten immunoassays based on a noncompetitive format. Here, we describe the production and characterization of monoclonal anti-idiotype antibodies against idiotopes on the variable regions of an antibody showing high affinity and specificity to 11-deoxycortisol (11-DC). 11-DC is the biosynthetic precursor of cortisol and a diagnostic index for the assessment of pituitary-adrenal function. BALB/c or A/J mice were repeatedly immunized with the anti-11-DC antibody conjugated with keyhole limpet hemocyanin and their spleen cells were then fused with P3/NS1/1-Ag4-1 myeloma cells. Seven kinds of anti-idiotype antibodies were generated, one of which was a beta-type antibody recognizing the paratope and others which were alpha-type antibodies recognizing the framework region. A noncompetitive ELISA based on idiotype-anti-idiotype reactions was established using one of these alpha-type antibodies in combination with the beta-type antibody and with the anti-11-DC antibody. This noncompetitive assay system provided improved sensitivity (detection limit: 1.0 pg=2.9 fmol), which is approximately 10 times higher than the corresponding competitive enzyme immunoassay, and offered a practical specificity for clinical use. Appropriate serum 11-DC levels were obtained for normal subjects [0.16+/-0.09 (S.D.) microg/l (n=6), ranging from 0.086 to 0.316 microg/l] using the present assay system.
Animals↗
Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity.
We have established a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the group-specific determination of 7-N-acetylglucosaminide of ursodeoxycholic acid (UDCA 7-NAG) and its glycine- and taurine-amidated metabolites (UDCA 7-NAGs) in human urine. These metabolites are expected to be a diagnostic marker for patients with primary biliary cirrhosis (PBC). This assay is based on the idiotype-antiidiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a beta-type antiidiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled alpha-type antiidiotype antibody. The amount of bound biotin, increasing proportionally to the increase in the analyte, was colorimetrically determined using a peroxidase-labeled streptavidin. This assay provided subfemtomole range sensitivity (detection limit 118 amol) and allowed group-specific measurement of the UDCA 7-NAGs in urine without any pretreatment. The present ELISA revealed that significant amounts of UDCA 7-NAGs are excreted even in healthy subjects. Daily excretion rates for healthy males were determined to be 246+/-184 (S.D.) microg (n=5) as the glycine-amidated UDCA 7-NAG equivalent. Randomly collected urine specimens from patients with PBC (n=7) were also measured, and the assay values (standardized to creatinine excretion) ranged from 1.82 to 13.4 microg/mg Ucre with the average of 5.41+/-4.53 (S.D.) microg/mg Ucre.
Acetylglucosamine↗