Kanji Tomioka

Homogeneous immunoassay of antibody by use of liposomes made of a model lipid of archaebacteria

Liposomes made of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPhyPC), which was synthesized as one of the model lipids existing in archaebacterial halophiles, showed excellent stability. Because of this high stability, DPhyPC liposomes could be constituted high ratios (50%) of N-[4-(p-maleimidophenyl) butyryl] dipalmitoyl phosphatidylethanolamine (MPB-DPPE), and consequently could bind large amounts of antigen (alpha-chymotrypsinogen A) on the liposome surface in comparison with those made of ordinary lipids, such as dipalmitoylphosphatidylcholine (DPPC). Though the characteristics of the DPhyPC liposomal membranes in lysis by the classical complement pathway were similar to those of DPPC liposomes, a high sensitivity and a low detection limit in the liposome immune lysis assay (LILA) of antibodies were attained by binding large amounts of the antigen. Further, by coupling sufficient amounts of antigen, almost all the DPhyPC liposome surface was covered with the antigen, and such liposomes showed higher resistance against non-specific lysis caused by complement activity in serum samples, which may be effective in reducing positive-false errors in LILA.

Use of peptide immunization for porcine insulin of a very high homology with a host protein

An anti-peptide antibody was obtained against a peptide corresponding to the 12 amino acids of the C-terminal region of porcine insulin B chain. The adsorption characteristics of this antibody were compared with those of anti-porcine insulin and anti-sheep insulin antibodies. Immunization of rabbits using the peptide corresponding to the C-terminal region of porcine insulin B chain produced an anti-peptide antibody which reacted with native porcine insulin with a much higher association constant than that of an anti-porcine insulin antibody. In addition, this immunization technique did not cause any observable physiologically harmful effects, such as hypoglycemia, in the immunized rabbits. Thus, peptide immunization may be a useful strategy when target proteins have biological activity and/or toxicity, and also have a very high degree of homology with the corresponding proteins of the immunized animal, which may inhibit the production of antibodies with a high association constant.

Insulin delivery system based on an affinity chemical valve

Abstract A very simple insulin delivery system is proposed which is based on the concept of competitive binding between glucose and dextran to concanavalin-A ligands bound to the membrane surface of hollow fibers. The delivery rate of the hormone from the donor phase to the acceptor phase increased in the presence of glucose. In this system, the dextran behaved as a chemical valve which was directly opened by glucose.

Comparison of Liposome Immune Lysis Assay (LILA) and Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Antibodies

Abstract Anti-α-chymotrypsinogen A antibody was assayed by both enzyme-linked immunosorbent assay (ELISA) and liposome immune lysis assay (LILA). The detection limit was slightly affected by the measurement conditions in ELISA; however, it was possible to control the detection limit and to achieve a lower level by adapting the measurement conditions in LILA. LILA is believed to offer a simple and highly sensitive method for measuring the concentration of antibody in serum.

Immune lysis assay of antibodies by use of antigen-coupled liposomes

The concentrations of antibodies were measured by the use of immunoliposomes coupled with the corresponding antigens. The addition of complement under conditions of the existence of the antigen-antibody complex on the surface of the liposomes caused lysis of the liposomes. The degree of marker release from the liposomes was increased with the concentration of the antibodies, as well as the amount of antigen coupled to the liposomes, and was proportional to the amount of antigen-antibody complex formed. The liposomes made of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPhyPC) showed excellent stability and consequently could bind large amounts of antigen on the surface of the liposomes in comparison with liposomes made of ordinary lipids such as dipalmitoylphosphatidylcholine (DPPC). Although the characteristics of liposomal membranes consisting of DPhyPC in lysis by the classical pathway of complement were similar to those of DPPC liposomes, the DPhyPC liposomes showed higher resistance against non-specific lysis caused by the complement activity in serum samples, which may be effective to reduce false-positive errors in liposome immune lysis assay.

Characterization of antigen-coupled liposomes for homogenous immunoassay of polyclonal antibodies

Abstract The effects of the preparation conditions of antigen-coupled liposomes on their characteristics in liposome immune lysis assay (LILA) of specific antibodies were studied. The amount of antigen coupled to liposomes depended on the molar ratio of N-[4-( p -maleimidophenyl)butyryl] dipalmitoyl phosphatidylethanolamine (MPB-DPPE) constituting the liposomes as well as on the molar ratio of modified antigen to MPB-DPPE in the reaction mixture. In depended also on the molecular weight of the antigen. With increases in the amount of antigen coupled to liposomes higher sensitivity and a wider dynamic range were obtained. Though an increase in the average association constant between antigen and antibody lowered the detection limit, the wide dynamic range, which prevents false positive results in LILA, can be controlled mainly by the amount of coupled antigen.

Use of a substrate as an encapsulated marker for liposome immunoassay

Abstract For spectrophotometric liposome immunoassay of a specific antibody, antigen (cytochrome c)-coupled liposomes containing the substrate glucose-6-phosphate (G6P) as a marker were prepared from 1,2-di(3 RS ,7 R ,11 R -phytanyl)- sn -glycero-3-phosphocholine (DPhyPC). DPhyPC liposomes showed good stability against G6P leakage, which was below 8% after incubation for 70 d at 4°C. When cytochrome c-coupled liposomes were incubated with anti-cytochrome c antibody and complement, after which glucose-6-phosphate dehydrogenase and NAD + were added, the absorbance at 340 nm increased with the amount of anti-cytochrome c antibody added.

Peptide-coupled liposomes for homogeneous immunoassay of polyclonal antibodies against proteins

Abstract The concentration of anti-native insulin antibodies was measured by liposome immune lysis assay (LILA) using liposomes coupled with a peptide which corresponds to residues 19–30 of the B-chain of porcine insulin. Although native insulin was slightly soluble in the reaction solution used in binding the antigens to the liposomes, this peptide was readily soluble in the solution. Thus liposomes coupled with a sufficient amount of the antigen could be easily obtained. Antibodies against native insulin from several species bound to the peptide on the surface of the liposomes, and the antigen-antibody complexes activated the complement system. By using the peptide-coupled liposomes, the concentrations of antibodies against insulin from several species were measured by the LILA method without the limitation of the solubility of the antigens.

Production and purification of soluble human CD2 secreted from recombinant Pichia pastoris

Abstract The solubilized form of human CD2 (sCD2), which consists of D1 and D2 domains and can bind specifically to LFA-3, was produced by secretion from Pichia pastoris by use of the AOX1 promoter and the α-factor signal sequence. An anti-peptide antibody against the C-terminal region of sCD2 (anti-PC-CD2-12 antibody) was used for ELISA, purification and characterization of sCD2. Secreted sCD2 was purified from fermentation broth with an affinity column coupled with anti-PC-CD2-12 antibody. The concentrations of sCD2 in fermentation broth and purified fractions were measured with the anti-peptide antibody and a monoclonal antibody recognizing the D1 domain. These results suggest that a large portion of secreted sCD2 lacked several amino acids in the C-terminal region probably because of digestion by proteases. Therefore, purification by the anti-peptide antibody recognizing the C-terminal region is a promising method for the purification of sCD2 having the complete C-terminal region.

Immunogenicity differences between intact insulin and a peptide representing the C-terminal region

Abstract Immunization of rabbits using a peptide corresponding to the 12 amino acids of the C-terminal region of porcine insulin B-chain produced an anti-peptide antibody reactive with the native insulin, as well as with the peptide. It was also found that the adsorption characteristics of the anti-peptide antibody against insulin and peptides, including its reactivity against the insulin A-chain, were different from those of antinative insulin antibodies. The reactivity against the insulin A-chain was raised by maturation of the anti-peptide antibody. These results show that peptide immunization may cause different immuno-responses, and have some advantages for obtaining antibodies possessing desirable characteristics.