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免疫不応答の抗原抗体反応を利用できるハプテン標識化試薬

 
K. Landsteinerらは1920~30年代に,本来抗原とはならない有機小分子を生体内タンパク質に結合させて動物に注射すると,有機小分子に対する抗体が得られることを明らかにしています1)。この抗体は,結合させたタンパク質に対する抗体とは独立して作られ,有機小分子と抗原抗体反応を示しますが免疫応答までは引き起こしません。この様な有機小分子はハプテン(不完全抗原)と呼ばれ,ジニトロフェノール(1)などの芳香族化合物や糖類が知られています。
6-(2,4-ジニトロアニリノ)ヘキサン酸 N-スクシンイミジル(2は2,4-ジニトロフェニル(DNP)基を有するハプテン標識化試薬で,スクシンイミジル基を介してペプチドやタンパク質などのアミノ基に結合させることができます。抗DNP抗体と共に利用することで,ラジオイムノアッセイ(放射免疫測定)やELISA,免疫組織化学的染色に応用されています2)。また,2を合成高分子に標識したドラッグデリバリーシステムの研究3)や,2を組み込んだ細胞表面の受容体モデルの研究4)なども行われています。

 

[1] (a) “Uber heterogenetisches antigen und hapten. XV. Mitteilung uber antigene”

K. Landsteiner, Biochem. Z. 1921, 119, 294.

(b) “Studies on the sensitization of animals with simple chemical compounds. II”

K. Landsteiner, J. Jacobs, J. Exp. Med. 1936, 64, 625.

[2] (a) “In vitro and in vivo targeting of radiolabeled monovalent and divalent haptens with dual specificity monoclonal antibody conjugates: enhanced divalent hapten affinity for cell-bound antibody conjugate”

J.-M. Le Doussal, M. Martin, E. Gautherot, M. Delaage, J. Barbet, J. Nucl. Med. 1989, 30, 1358.  PubMed

A method of pretargeted immunolocalization of a mouse cell subset, using dual specificity monoclonal antibody conjugates and labeled divalent haptens, is described. Conjugates were prepared by coupling F(ab’)2 fragments of an antibody specific for the allelic mouse B cell antigen Lyb8.2, to Fab’ fragments of an anti-2,4-dinitrophenyl antibody. Divalent and monovalent haptens were obtained by coupling dinitrophenyl to peptides or to diethylene-triamine-pentaacetic acid. In vitro, divalent haptens bind with higher affinity to mouse spleen lymphocyte-bound than to excess soluble conjugate (affinity enhancement). In vivo, localization of 125I- or 111In-labeled divalent haptens in mouse spleen is much higher than that of the monovalent analogs. Thus, using divalent haptens, a new kind of specificity to target cells was achieved, suggesting that affinity enhancement may improve target to background ratios in radioimmunoscintigraphy.

(b) “Determination of the binding of ligands containing the N-2,4-dinitrophenyl group to bivalent monoclonal rat anti-DNP antibody using affinity capillary electrophoresis”

M. Mammen, F. A. Gomez, G. M. Whitesides, Anal. Chem. 1995, 67, 3526. PubMed

Affinity capillary electrophoresis has been used to determine the two dissociation constants of the complex between anti-DNP rat monoclonal IgG2b antibody and charged ligands that contained a N-dinitrophenyl group. Singly and multiply charged ligands were used to establish the influence of the charge on the mobility of the complex between Ig and its ligand(s). Zwitterionic buffer additives lessened adsorption of protein to the walls of the capillary. A form of analysis of the binding data is derived that is more useful than Scatchard analysis for certain multivalent systems where cooperativity of binding is in question, but where it is also possible to make plausible assumptions about electrophoretic mobilities of protein and protein-ligand complexes. The uncertainties and assumptions of this analysis are contrasted with those of Scatchard analysis. For this antibody and these monovalent ligands, the dissociation of the ligands from the antibody occurred noncooperatively. The charge on IgG2b at pH 8.3 is estimated to be -8.0 +/- 0.2; this value is obtained by analysis of the electrophoretic mobilities of complexes IgG2bL2, where the ligands L are structurally similar but have different charges (the charges on the ligands were also determined by CE).

(c) “Development of an internally controlled antibody microarray”

E. W. Olle, A. Sreekumar, R. L. Warner, S. D. McClintock, A. M. Chinnaiyan, M. R. Bleavins, T. D. Anderson, K. J. Johnson, Mol. Cell. Proteomics 2005, 4, 1664. DOI:10.1074/mcp.M500052-MCP200

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1beta, IL-5, IL-6, and macrophage inflammatory proteins 1alpha and 1beta. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.

(d) “Application of fluorescence microscopy for investigation of cellular distribution of dinuclear platinum anticancer drugs”

G. V. Kalayda, G. Zhang, T. Abraham, H. J. Tanke, J. Reedijk, J. Med. Chem. 2005, 48, 5191. DOI: 10.1021/jm050216h

jm050216hn00001

The dinuclear platinum complexes with aliphatic diamines [{cis-Pt(NH3)2Cl}2(μ-H2N(CH2)6NH2)](NO3)2 (1,1/c,c) and [{trans-Pt(NH3)2Cl}2(μ-H2N(CH2)4NH2)](NO3)2 (1,1/t,t), which are known to be highly active in vitro against several cancer cell lines, have been modified with a fluorogenic reporter (carboxyfluorescein diacetate, CFDA) and a hapten (dinitrophenyl, DNP). These labeled complexes have been designed for fluorescence microscopy investigation of cellular pathways of promising dinuclear platinum anticancer drugs and present the first example of labeling biologically active dinuclear platinum complexes with a fluorescent reporter. The modified compounds interact with a guanine model base similarly to the label-free parent complexes. The uptake of the complexes with a fluorescent label and the respective unlabeled complexes in the U2-OS human osteosarcoma cell line and its cisplatin-resistant derivative, U2-OS/Pt cell line has been investigated. Cellular processing of the CFDA- and DNP-modified dinuclear platinum complexes in U2-OS and U2-OS/Pt cells has been studied.

[3] “Extracellular barriers to in vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver”

R. S. Burke, S. H. Pun, Bioconjugate Chem. 2008, 19, 693. DOI: 10.1021/bc700388u

Polyplex-mediated gene therapy is a promising alternative to viral gene therapy. One challenge to these synthetic carriers is reduced transfection efficiencies in vivo compared to those achieved in vitro. Many of the intracellular barriers to gene delivery have been elucidated, but similar quantification of extracellular barriers to gene delivery remains a need. In this study, the unpackaging of polyplexes by serum proteins, soluble glycosaminoglycans, and an extracellular matrix extract was demonstrated by a YOYO-1 fluorescence quenching assay. Additionally, exposing polyplexes to serum or proteoglycans before in vitro transfection caused decreased cellular uptake of DNA. Lastly, PEI polyplexes and PEGylated PEI polyplexes were injected into the portal vein of mice, and the intrahepatic distributions of labeled DNA and polymer were assessed by confocal microscopy. PEI polyplexes delivered DNA to the liver, but extensive vector unpackaging was observed, with PEI primarily colocalized with the extracellular matrix. PEGylated polyplexes mediated less DNA delivery to the liver, possibly due to premature vector unpackaging in the blood. Through this work, both the blood and the extracellular matrix have been determined to be significant extracellular barriers to polyplex-mediated in vivo gene delivery to the liver.

[4] “Synthetic mimics of small mammalian cell surface receptors”

S. Boonyarattanakalin, S. E. Martin, S. A. Dykstra, B. R. Peterson, J. Am. Chem. Soc. 2004, 126, 16379. DOI: 10.1021/ja046663o

ja046663on00001

Receptors on the surface of mammalian cells promote the uptake of cell-impermeable ligands by receptor-mediated endocytosis. To mimic this process, we synthesized small molecules designed to project anti-dinitrophenyl antibody-binding motifs from the surface of living Jurkat lymphocytes. These synthetic receptors comprise N-alkyl derivatives of 3β-cholesterylamine as the plasma membrane anchor linked to 2,4-dinitrophenyl (DNP) and structurally similar fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD) headgroups. Insertion of two β-alanine subunits between a DNP derivative and 3β-cholesterylamine yielded a receptor that avidly associates with cell surfaces (cellular t1/2  20 h). When added to Jurkat cells at 10 μΜ, this receptor enhanced uptake of an anti-DNP IgG ligand by 200-fold in magnitude and 400-fold in rate within 4 h (ligand internalization t1/2  95 min at 37 °C). This non-natural receptor mimics many natural receptors by dynamically cycling between plasma membranes and intracellular endosomes (recycling t1/2  3 min), targeting of protein ligands to proposed cholesterol and sphingolipid-enriched lipid raft membrane microdomains, and delivery of protein ligands to late endosomes/lysosomes. Quantitative dithionite quenching of fluorescent extracellular NBD headgroups demonstrated that other 3β-cholesterylamine derivatives bearing fewer β-alanines in the linker region or N-acyl derivatives of 3β-cholesterylamine were less effective receptors due to more extensive trafficking to internal membranes. Synthetic cell surface receptors have potential applications as cellular probes, tools for drug delivery, and methods to deplete therapeutically important extracellular ligands.

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