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1.
Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies. 总被引:5,自引:0,他引:5
Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies. 相似文献
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Regina M. Santella Dezider Grunberger Alfred Nordheim Alexander Rich 《Biochemical and biophysical research communications》1982,106(4):1226-1232
Poly(dG-m5dC)·poly(dG-m5dC) was modified by treatment with N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) and its conformation examined by circular dichroism (CD) and susceptibility to S1 nuclease digestion. A sample with a modification level of 10% shows a CD spectrum characteristic of the Z form and is resistant to digestion by S1 nuclease. The relative reactivity of several polymers with N-Aco-AAF was shown to follow the order of ease of formation of Z DNA: poly(dG-m5dC)·poly(dG-m5dC) > poly(dG-dC)·poly(dG-dC) > poly(dG)·poly(dC). This suggests that AAF reacts more readily with Z DNA than B DNA. 相似文献
4.
The kinetics of the electrostatically induced phase transition of dimyristoyl phosphatidic acid bilayers was followed using the stopped-flow technique. The phase transition was triggered by a fast change in the pH or the magnesium ion concentration and followed by recording the time dependence of the absorbance. When the phase transition was induced by a pH jump the time course of the absorbance could be described by two exponentials, their time constants displaying the for cooperative processes characteristic maximum at the transition midpoint. The time constants are in the 10 and 100 ms range for the H+ triggered transition from the fluid to the ordered state. A third slower process shows no appreciable temperature dependence and is probably caused by vesicle aggregation. For the OH--induced transition fron the ordered to the fluid state the time constants are in the 100 and 1000 ms range. The fluid-ordered transition could also be triggered by addition of magnesium ions. Of the several observed processes only the fastest in the 10–100 ms time range could definitely be assigned to the fluid-ordered transition while the others are due to aggregation phenomena. The experimental data were compared with results obtained from pressure jump experiments and could be interpreted on the basis of theories for non-equilibrium relaxation. 相似文献
5.
Ronald J.A. Wanders Carlo Van Roermund Cor Lof Alfred J. Meijer 《Analytical biochemistry》1983,129(1):80-87
A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP+, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present. 相似文献
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Lower Miocene siliciclastic sediments of the Vöckla Schichten and Atzbacher Sande, Austria, contain a shallow-water trace-fossil assemblage of the Cruziana ichnofacies includingSkolithos isp.,Planolites ? beverleyensis, Ophiomorpha annulata, O.nodosa, Rosselia socialis, Cylindrichnus concentricus, Scolicia isp., andBichordites monastiriensis. The ichnogenusRosselia and the ichnospeciesRosselia socialis are revised. Analysis ofScolicia isp. andB. monastiriensis provides new information about their morphology and ichnotaxonomy. 相似文献
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B Vernet-der-Garabédian E Morel J F Bach 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1984,298(16):449-456
Inhibitory effect of myasthenic patients antibodies on alpha-bungarotoxin binding to the human acetylcholine receptor has been demonstrated by radioimmunoassay. By using decamethonium, an acetylcholine agonist, we have shown the existence of two antibody sub-groups reacting with the toxin-binding site: one sub-group is represented by antibodies which block the binding directly, the other by antibodies that inhibit the binding, only in the presence of decamethonium. 相似文献
10.
Michael F. Seldin Lawrence A. D'Hoostelaere Alfred D. Steinberg Yumiko Saga Herbert C. Morse III 《Immunogenetics》1987,26(1-2):74-78
Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus. 相似文献