Takeyuki Kohno

Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the sensitivity.

Development of ultrasensitive enzyme immunoassays for antigens, haptens and antibodies is reviewed with emphasis on factors which limit the sensitivity. One of the most important conditions for ultrasensitive immunoassays is the use of non-competitive solid phase assay systems rather than competitive ones. Although non-competitive immunoassays are available for antigens and antibodies, there are only competitive immunoassays for hapten molecules which can not be bound simultaneously by two different antibodies. In order to overcome this difficulty, methods have been developed to derivatize haptens with amino groups so that the derivatized haptens may be measured by two-site assays. The other condition for ultrasensitive immunoassays is to minimize the non-specific binding of labelled reactants. This has been achieved by developing methods to transfer the complex of analytes and labelled reactants from solid phase to solid phase without dissociation. Thus, the sensitivity for antigens, haptens and antibodies has been markedly improved. However, the sensitivity for the detection of label enzymes remains to be improved.

Methods for Enzyme-Labeling of Antigens Antibodies and their Fragments

A number of reagents have been used for enzyme-labeling of antigens and antibodies during the past two decades. However, most of them suffer from serious disadvantages, and only a few reagents are in current use, including glutaraldehyde, periodate, maleimide compounds and pyridyl disulfide compounds (Ishikawa et al., 1983a).

Development and applications of ultrasensitive enzyme immunoassays for antigens and antibodies

Some enzymes can be measured at lower molar concentrations than the radioisotopes used as labels in radioimmunoassay. Therefore, enzyme immunoassay should be capable of higher sensitivity than radioimmunoassay, if appropriate techniques are used for enzyme-labelling of antibodies and formulation of assay protocols. Techniques required for enzyme immunoassay with attomole sensitivity have been developed, and successfully applied to the measurement of clinically important ligands at levels below those possible using radioimmunoassay. In addition, a novel enzyme immunoassay technique (immune complex transfer enzyme immunoassay technique) is being developed to provide higher sensitivity.

Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich Transfer Enzyme Immunoassay) for Antigens

Abstract A novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens is described. Antigens were reacted with dinitrophenyl monoclonal mouse antibody IgG1 and rabbit antibody Fab′-s-D-galactosidase conjugates. The complex formed of antigens with dinitrophenyl monoclonal mouse antibody IgG1 and rabbit antibody Fab′-s-D-galactosidase conjugates was trapped onto affinity-purified rabbit (antidinitrophenyl bovine serum a1bumin) IgG-coated polystyrene balls. After eliminating excess of the conjugates, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. s-D-Galactosidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed by fluorimetry. Nonspecifically bound s-D-galactosidase activity considerably decreased with less decrease in specifically bound s-D-galactosidase activity. As a result, the detection limits of ...

Antibodies to food antigens in Japanese patients with type 1 diabetes mellitus.

To examine humoral and mucosal immune responses to food antigens and their relation to the pathophysiology of type 1 diabetes mellitus, IgA and IgG antibodies to cows milk antigens (bovine serum albumin (BSA) and beta-lactoglobulin (BLG)) and another food antigen (ovalbumin, (OVA)) in human serum were assessed by enzyme-linked immunosorbent assay (ELISA). If anti-idiotype antibodies to the antibodies were present in serum, they might interfere with the ELISA assay, so suitable microtiter plates were employed to minimize such interference. The levels of IgA and IgG antibodies to the above antigens (P<0.001-P<0.01) and the prevalence of positive sera (P<0.001-P<0.05) in the patient group (n=52, aged 14.5+/-4.1 (S.D.) years) were significantly higher than those in the control group (n=41, aged 13.3+/-6.8 (S.D.) years). Interestingly, the levels of IgA antibodies to all the food antigens examined were elevated in 26 (50%) patients, while the elevation was seen in 3 (7%) healthy controls. The elevation of IgA antibodies in the patients was well correlated with increased concentrations of IgA and transforming growth factor (TGF)-beta, which induces IgA-producing B-cells, in serum. Although the cytokine TGF-beta is secreted from regulatory T-cells (Th3), and is related to oral tolerance, the interleukin-2 (IL-2, Th1)/IL-4 (Th2) ratio in the patient group was significantly elevated (P<0.001), which might indicate that the oral tolerance is impaired in patients. Thus, we demonstrated that both IgA and IgG antibodies to several food antigens are elevated in patients. We suggest that impairment of oral tolerance might be related to the pathogenesis of type 1 diabetes mellitus.

Therapeutic approach for type 1 diabetes mellitus using the novel immunomodulator FTY720 (fingolimod) in combination with once‐daily injection of insulin glargine in non‐obese diabetic mice

Aims/Introduction:  The therapeutic effectiveness against type 1 diabetes mellitus (DM) of the novel immunomodulator FTY720 (fingolimod), alone and in combination with insulin glargine, was examined in the non‐obese diabetic (NOD) mouse model.

Development of a highly sensitive and specific two-site enzyme immunoassay for parathyroid hormone (1-34): Application to pharmacokinetic study on intranasal parathyroid hormone (1-34) in human

A highly sensitive and specific two‐site enzyme immunoassay for parathyroid hormone (1‐34) (PTH(1‐34)) and its usability for the pharmacokinetic study are described. Plasma samples were incubated simultaneously with 2,4‐dinitrophenylated anti‐PTH(1‐34) IgG and anti‐PTH(1‐34) Fab′‐ β‐D‐galactosidase conjugate. The immune complex formed of the three components was trapped onto (anti‐2,4‐dinitrophenyl group) IgG‐coated polystyrene balls. β‐D‐ Galactosidase activity bound to the polystyrene balls was assayed by fluorometry. The practical detection limit of PTH(1‐34) was 50 fg (12 amol)/0.05 ml of sample and 1 pg/ml as the concentration and practically no interference occurred by PTH(1‐84) and PTH‐related protein (1‐34) up to 300 pg/ml and 10 ng/ml, respectively. The application of this method has enabled us to directly estimate the bioavailability of PTH(1‐34) dosed intranasaly at the prescribed level (0.090 mg). The pharmacokinetic parameters of the intranasal PTH(1‐34) (n = 4) thus estimated were as follows: the area under the plasma concentration‐time curve (AUC) = 20,500 ± 15,900(SD) pg·min/ml; the mean residence time (MRT) = 194 ± 16.3(SD) min; and the maximal concentration (Cmax) = 98 ± 51(SD) pg/ml with the maximal time (Tmax) = 35.0 ± 12.2(SD) min. J. Clin. Lab. Anal. 12:268–275, 1998.

Oral therapy for type 1 diabetes mellitus using a novel immunomodulator, FTY720 (fingolimod), in combination with sitagliptin, a dipeptidyl peptidase‐4 inhibitor, examined in non‐obese diabetic mice

Aims/Introduction:  The therapeutic effectiveness against type 1 diabetes mellitus of a novel immunomodulator, FTY720 (fingolimod), in combination with sitagliptin, a dipeptidyl peptidase‐4 inhibitor, was examined in the non‐obese diabetic (NOD) mouse model.

Purification of Bacillus subtilis Spore Coat Protein by Electrophoretic Elution Procedure and Determination of NH2-Terminal Amino Acid Sequences

Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS‐PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS‐PAGE, both retaining antigenic activity against anti‐spore coat protein serum on immunoblot analysis. The N‐terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.

A sandwich transfer enzyme immunoassay for salmon calcitonin: determination of the bioavailability of intranasal salmon calcitonin in human.

A sandwich transfer enzyme immunoassay for salmon calcitonin (SCT) and its usability for the pharmacokinetic study are described. The assay procedure consisted of the reaction of SCT with 2,4‐dinitrophenyl biotinyl anti‐SCT IgG and anti‐SCT Fab′‐β‐D‐galactosidase conjugate, trapping onto (anti‐2,4‐dinitrophenyl bovine serum albumin) IgG‐coated polystyrene balls, eluting with ϵN‐2,4‐dinitrophenyl‐L‐lysine and transferring to streptavidin‐coated polystyrene balls and fluorometric detection of β‐D‐galactosidase activity. The practical detection limit of SCT was 0.05 pg (15 amol)/50 μl of sample and 1 pg/ml as the concentration. The application of this method has enabled us to directly estimate the bioavailability of SCT dosed intranasally at the therapeutic level (160 IU, 31 μg) for its anti‐osteoporotic effect as compared to an intramuscular dose (10 IU, 1.9 μg). The pharmacokinetic parameters of the intranasal SCT (n = 6) thus estimated were as follows: the area under the blood concentration‐time curve (AUC) = 9400 ± 5400 (SD) pg·h/ml, and the mean residence time (MRT) = 42 ± 14 (SD) min, when the AUC for the intramuscular SCT (n = 3) = 5600 ± 2000 (SD) pg·h/ml and the MRT = 39 ± 19 (SD) min. J. Clin. Lab. Anal. 11:380–387, 1997.