1. A monoclonal antibody specific to the granulocyte-derived elastase-fragment D species of human fibrinogen and fibrin: its application to the measurement of granulocyte-derived elastase digests in plasma
by Isao Kohno, Kimiko Inuzuka, Yumiko Itoh, Kunihiko Nakahara, Yutaka Eguchi, Teruko Sugo, Gilbu Soe, Yoichi Sakata, Hideki Murayama, and Michio Matsuda
Blood
Volume 95(5):
March 1, 2000
©2000 by American Society of Hematology

2. SDS-PAGE analysis of immunoprecipitate
SDS-PAGE analysis of immunoprecipitate.SDS-PAGE analysis was performed under nonreducing conditions for fibrinogen and GE and plasmic digests of fibrinogen and cross-linked fibrin immunoprecipitated with IF-123–conjugated Sepharose 4B.
SDS-PAGE analysis of immunoprecipitate.SDS-PAGE analysis was performed under nonreducing conditions for fibrinogen and GE and plasmic digests of fibrinogen and cross-linked fibrin immunoprecipitated with IF-123–conjugated Sepharose 4B. (A) GE digests. (B) Plasmic digests. In both panels, lanes 1 to 3 represent the applied samples and lanes 4 to 6 the bound proteins to the gels. Lanes 1 and 4, fibrinogen; lanes 2 and 5, fibrinogen digests; and lanes 3 and 6, cross-linked fibrin digests.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

3. Immunoblot analyses.Immunoblot analyses were run under reducing conditions for fibrinogen and its plasmic and GE-digests with an antihuman fibrinogen rabbit antibody (A) and IF-123 (B).
Immunoblot analyses.Immunoblot analyses were run under reducing conditions for fibrinogen and its plasmic and GE-digests with an antihuman fibrinogen rabbit antibody (A) and IF-123 (B). Lane 1, fibrinogen; lane 2, plasmic fragment D1; and lane 3, GE-D.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

4. Reverse-phase HPLC.Separation of the α-remnants of GE-D (A) and its lysyl endopeptidase-digests (B) by reverse-phase HPLC. Only peptide #19 was reactive to IF-123.
Reverse-phase HPLC.Separation of the α-remnants of GE-D (A) and its lysyl endopeptidase-digests (B) by reverse-phase HPLC. Only peptide #19 was reactive to IF-123.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

5. Isao Kohno et al. Blood 2000;95:1721-1728
©2000 by American Society of Hematology

6. Isao Kohno et al. Blood 2000;95:1721-1728
©2000 by American Society of Hematology

7. Localization of the epitope for IF-123
Localization of the epitope for IF-123.The residues constituting the epitope are shaded, and a plasmic cleavage site is shown in the carboxyl terminal 6 residue extension.
Localization of the epitope for IF-123.The residues constituting the epitope are shaded, and a plasmic cleavage site is shown in the carboxyl terminal 6 residue extension.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

8. GE-induced epitope expression
GE-induced epitope expression.Digestion of fibrinogen was performed by proteases released from the activated granulocytes as analyzed by SDS-PAGE (upper panels) and concomitant appearance of the epitope for IF-123 by a sandwich ELISA (lower panels).
GE-induced epitope expression.Digestion of fibrinogen was performed by proteases released from the activated granulocytes as analyzed by SDS-PAGE (upper panels) and concomitant appearance of the epitope for IF-123 by a sandwich ELISA (lower panels). (A) Control without prior treatment of the proteases with any inhibitors. (B) Prior treatment with Z-Gly-Leu-Phe-CH2Cl, a specific inhibitor to cathepsin G, TPCK, and TLCK. (C) Prior treatment with MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a specific inhibitor to GE.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

9. Isao Kohno et al. Blood 2000;95:1721-1728
©2000 by American Society of Hematology

10. Isao Kohno et al. Blood 2000;95:1721-1728
©2000 by American Society of Hematology

11. Measurement of GE-D and GE-XDP added to pooled normal plasma by a sandwich ELISA using IF-123.Plasma was spiked with GE-D (○); GE-XDP (•); plasmic D1(□) or plasmic XDP (▪).
Measurement of GE-D and GE-XDP added to pooled normal plasma by a sandwich ELISA using IF-123.Plasma was spiked with GE-D (○); GE-XDP (•); plasmic D1(□) or plasmic XDP (▪).
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

12. Comparison of levels of GE digests and plasmic digests in plasmas derived from patients with various diseases.They include APL; acute myelocytic leukemia; acute lymphocytic leukemia; malignant lymphoma; solid cancers in the stomach, liver, colon, and lungs;...
Comparison of levels of GE digests and plasmic digests in plasmas derived from patients with various diseases.They include APL; acute myelocytic leukemia; acute lymphocytic leukemia; malignant lymphoma; solid cancers in the stomach, liver, colon, and lungs; sepsis; gestation toxicosis; SLE; and benign gastrointestinal tract diseases such as duodenal ulcer and polyp(s) in the gallbladder and the colon.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

13. SDS-PAGE analyses of immunoprecipitated GE digests and plasmic digests in 3 representative plasma samples.(A) In sample a, GE digests are elevated but plasmic digests are low.
SDS-PAGE analyses of immunoprecipitated GE digests and plasmic digests in 3 representative plasma samples.(A) In sample a, GE digests are elevated but plasmic digests are low. In sample b, both GE and plasmic digests are elevated. In sample c, GE digests are low but plasmic digests are elevated. Individual fragments in the GE digests are termed according to their corresponding fragments assigned for plasmic digests (B) Purified samples are shown as reference.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology

14. GE digests in plasmas derived from patients with various diseases
GE digests in plasmas derived from patients with various diseases.Horizontal bars represent the means. *P < 0.05; **P < 0.001 versus normal control. n.s., not significant.
GE digests in plasmas derived from patients with various diseases.Horizontal bars represent the means. *P < 0.05; **P < 0.001 versus normal control. n.s., not significant.
Isao Kohno et al. Blood 2000;95:
©2000 by American Society of Hematology