健康科学研究所::研究室紹介::Seiichi Hashida

Seiichi Hashida

2017/03/22
Division of Lifestyle-Related Disease

Research Summary
We have been studying for development of ultrasensitive enzyme immunoassay and for its application throughout. Ultrasensitive enzyme immunoassay methods can be developed by noncompetitive solid phase assay system rather than competitive ones for antigens and antibodies. For ultrasensitive noncompetitive solid phase immunoassay, the nonspecific binding of labeled reactants (background noise) should be minimized. This has been achieved by developing methods to transfer the complex of analyte and labeled reactants from solid phase to solid phase with minimal dissociation of the complex (the immune complex transfer method). Thus, the sensitivity for antigens, haptens and antibodies has been markedly improved and some applications have been made.

1. Urtrasensitive Enzyme Immunoassay for antigens.
We developed a new method for enzyme labeling of antibodies and a new system for measurement methods. As a result, attomole amounts of clinically important antigens that cannot be detected by competitive radioimmunoassay were measured by sandwich enzyme immunoassay. Actually, growth hormone in urine of healthy children and patients of dwarfism, thyroid-stimulating hormone in plasma of patients with Graves' disease and luteinizing hormone in serum of children under the age of 9-10 years could be first measured directly by enzyme immunoassay without extraction and concentration. Furthermore, we developed the new assay system that was an ultrasensitive. This new assay, enzymatic immune complex transfer assay (ICT-EIA) are the determination systems which decreased nonspecific bond drastically of enzyme labeled antibody, and these methods are the determination systems which were quite different from a traditional approach. The detection limit by this ICT-EIA has been reached zeptomole (10-21 mole, approximately 600 molecules). By this ICT-EIA, in HIV-1 seroconversion serum panels, p24 antigen was detected as early as the detection of HIV-1 RNA by RT-PCR and contributed to early diagnosis of HIV-1 infection.

2. Urtrasensitive Enzyme Immunoassay for Haptens.
Currently available immunoassay methods for haptens may be divided into two groups, competitive and noncompetitive immunoassay methods. However, the detection limit of haptens by competitive immunoassay is at femtomole or higher levels in most case. Therefore, the blood concentration of subjects, which is around pmol/ml levels, could not be measured directly by competitive immunoassay, and extraction and concentration using several ml of plasma were required. By contrast, in noncompetitive two-site immunoassay, the detection limit of haptens is attomole levels. We developed a new technique for the measurement of haptens with high sensitivity, employing a noncompetitive two-site binding method that utilizes two kinds of antibodies. In addition, an immune-complex transfer method (ICT-EIA) was employed to improve sensitivity even further. As a result, a-atrial natriuretic peptide, adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and ghrelin in plasma of healthy subjects could be first measured directly by noncompetitive two-site enzyme immunoassay without extraction and concentration.

3. Ultrasensitive Enzyme Immunoassay for Antibodies.
As described for antigens and haptens, noncompetitive immunoassay systems potentially provide higher sensitivities for antibodies than competitive ones. In the most widely used conventional enzyme immunoassay (so-called enzyme-linked immunosorbent assay, ELISA) for antibodies, the sensitivity is seriously limited by the nonspecific binding of nonspecific immunoglobulins and probably other substance(s) in the samples. We developed a new technique for the measurement of antibodies with high sensitivity, employing a noncompetitive two-site binding method, the immune-complex transfer enzyme immunoassay (ICT-EIA). The detections of antibodies for HIV-1 p17, p24 and reverse transcriptase (RT) was 3,000 to 100,000-fold more sensitive than conventional methods such as gelatin particle agglutination, ELISA and western blotting. The use of more than 1 μl of whole saliva was recommended for reliable diagnosis of t HIV-1 infections, whereas 1 μl of serum was sufficient for the purpose. As a result, in HIV-1 seroconversion serum panels, antibody IgG to p17 was detected as early as or earlier than the detection of HIV-1 RNA by RT-PCR in all panels. Furthermore, the immune complex transfer enzyme immunoassay was developed for simultaneous detection of p24 antigen and antibody IgGs to RT and p17 in a single assay tube, and the window period (8 weeks, although widely variable), during which diagnosis of HIV-1 infection is not possible due to the absence of detectable antibodies to HIV-1, was shortened by 2 weeks. On the other hand, Autoantibody anti-thyroglobulin IgG was detected not only in all patients with Graves' disease and chronic thyroiditis but also in all healthy subjects, whereas anti-thyroglobulin antibodies were detected only in approximately 60% of patients and in a few percent of healthy subjects by the conventional ELISA and hemagglutination test
In future, we would like to develop ultrasensitive enzyme immunoassays, which have been shortened and simplified with even further improvement of the sensitivity not only to antigens but also to antibodies.

4. Recently, in order to diagnose the functional deterioration of pancreatic beta-cells earlier and more precisely, we developed a detection method that detects pancreatic islet-associated autoantibodies (insulin autoantibodies, GAD antibodies, IA-2 antibodies etc.) with high sensitivity. Furthermore, since there is a risk of being overlooked by single antibody detection, we are developing a novel high-sensitivity multi-antibody detection method (simultaneous detection of insulin autoantibody, GAD antibody, IA-2 antibody) and verified it using a cohort sample.
On the other hand, we focused on diabetic nephropathy and are developing a diagnostic method for early diabetic nephropathy by discriminating the molecular pattern of adiponectin in urine.

Selected Papers
1. Hashida, S., Hashinaka K. and Ishikawa, E. Ultrasensitive enzyme immunoassay. In: El-gewely, M.R. ed. Biotechnology Annual Review Volume 1 (1995) pp403-451, Elsevier Science Publishers B.V., Amsterdam.
2. Hashida, S., Ishikawa, E., Nakagawa, K., Ohtaki, S., Ichioka, T. and Nakajima, K. Demonstration of human growth hormone in normal urine by a highly specific and sensitive sandwich enzyme immunoassay. Anal. Lett. 18 (B13): 1623-1634 (1985)
3. Hashida, S. and Ishikawa, E. Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay. J. Biochem. 108: 960-964 (1990)
4. Hashida, S., Tanaka, K., Yamamoto, N., Uno, T., Yamaguchi, K. and Ishikawa, E. Detection of one attomole of [Arg8]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). J. Biochem. 110: 486-492 (1991)
5. Hashida, S., Hashinaka, K., Nishikata, I., Oka, S., Shimada, K., Saito, A., Takamizawa, A., Shinagawa, H. and Ishikawa, E. Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay. J. Virol. Methods 62: 43-53 (1996)
6. Hashida S., Kitamura K., Nagatomo Y., Shibata Y., Imamura T., Yamada K., Fujimoto S., Kato J., Morishita K. and Eto T. Development of an ultra-sensitive enzyme immunoassay for human proadrenomedullin N-terminal 20 peptide (PAMP) and direct measurement of two molecular forms of PAMP in plasma from healthy subjects and patients with cardiovascular disease. Clin. Biochem. 37: 14-21 (2004).
7. Obata T, Yokota I, Yokoyama K, Okamoto E, Kanezaki Y, Tanaka Y, Maegawa H, Teshigawara K, Hirota F, Yuasa T, Kishi K, Hattori A, Hashida S, Masuda K, Matsumoto M, Matsumoto T, Kashiwagi A, Ebina Y.: Soluble insulin receptor ectodomain is elevated in the plasma of patients with diabetes mellitus.
Diabetes. 56: 2028-2035 (2007)
8. Umehara A, Nishioka M, Obata T, Ebina Y, Shiota H, Hashida S: A novel ultra-sensitive enzyme immunoassay for soluble human insulin receptor ectodomain and its measurement in urine from healthy subjects and patients with diabetes mellitus. Clin Biochem 42, 1468-1475. (2009)
9. Numata S., Umehara A., Katakami H., Inoue S. and Hashida S: Development of a novel and ultra-sensitive enzyme immunoassay for human insulin autoantibodies. Clin Biochem 45: 1086-91 (2012)
10. Hashida S., Miyazawa Y., Hirajima Y., Umehara A, Yamamoto M., Numata S. Development of ultra-sensitive enzyme immunoassay for insulin and application to evaluation of the diabetic risk by urine in the morning. In Eltayb Abuelzein ed. Trends in Immunolabelled and Related Techniues. (2012) pp83-100, In Teck d.o.o, Croatia. (ISBN; 978-953-51-0570-1)
11. Numata S., Umehara A., Katakami H., Inoue S. and Hashida S: Development of a novel ultra-sensitive enzyme immunoassay for human GAD65 antibody. Annals Clinical Biochemistry, 53:495-503 (2016)
12. Hamada, Junpei, Onuma, Hiroshi, Ochi, Fumihiro, Hirai, Hiroki, Takemoto, Koji, Miyoshi, Akiko, Matsushita, Manami, Kadota, Yuko, Ohashi, Jun, Kawamura, Ryoichi, Takata, Yasunori, Nishida, Wataru, Hashida, Seiichi, Ishii, Eiichi, Osawa, Haruhiko. Endoplasmic reticulum stress induced by tunicamycin increases resistin mRNA through the PERK-ATF4-CHOP pathway in THP-1 human monocytes. Journal of Diabetes Investigation 7: 312–323 (2016)
13. Yuasa T, Amo-Shiinoki K, Ishikura S, Takahara M, Matsuoka T, Kaneto H, Kuroda A, Matsuhisa M, Hashida S.: Sequential cleavage of insulin receptor by calpain 2 and γ-secretase impairs insulin signalling. Diabetologia.59:2711-2721 (2016).
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