Quantitative Measurement of the Novel Human Plasma Protein, IHRP, by Sandwich ELISA.

Abstract

We purified a 120-kDa novel glycoprotein from human plasma and named it IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) because of its sequence similarity to the heavy chains of the inter-alpha-trypsin inhibitor (ITI) family.1) IHRP was certified as the identical protein with PK-120 which was purified as a plasma kallikreinsensitive protein from human plasma.2) The ITI family includes ITI, pre-alpha-trypsin inhibitor and H2-bikunin. [3][4][5] All of them have the unique structures, i.e., bikunin and heavy chain(s) are bridged by a chondroitin sulfate sugar chain. [5][6][7][8] The function of the ITI family is believed to be as a protease inhibitor, however, the extracellular matrix-stabilizing activity of the ITI family is noteworthy. 9,10) In spite of the sequence similarity, IHRP did not form a complex with bikunin because of the deletion of the consensus sequence (DPHFII) for the cleavage of the C-terminal fragment and the attachment to the chondroitin sulfate sugar chain of bikunin.11,12) IHRP was readily cleaved to the N-terminal 85-kDa-fragment and the C-terminal 35-kDa fragment by plasma kallikrein. 1,2) The N-terminal 85-kDa fragment was further cleaved to the N-terminal 57-kDa fragment and to unidentified small fragments. 1,2) In a search of the nucleotide sequence homology of IHRPcDNA, we found that the nucleotide sequence registered as porcine ITI cDNA in LASL-GDB (Gen Bank) data base showed significant homology (83%) to that of IHRP cDNA.11) Since this sequence homology was higher than those (49-57%) of mRNAs among IHRP and ITI family heavy chains, we thought that the registered porcine ITI mRNA, which was detected as the inducible mRNA in the liver after resuscitation from cardiogenic shock, 13) was the species counterpart of human IHRP mRNA rather than porcine ITI family heavy chain mRNA.In this paper, we describe the preparation and the characterization of anti-IHRP mouse monoclonal antibodies and the quantitative measurement of the plasma IHRP concentrations of healthy donors and patients with inflammatory disorders. MATERIALS AND METHODSPreparation of Anti-IHRP Mouse Monoclonal Antibodies Anti-IHRP mouse monoclonal antibodies were prepared according to the method of Galfre et al. 14) A female Balb/c mice was immunized with 50 mg of purified human IHRP 1) as an emulsion with Freund's complete adjuvant. Three weeks after the first immunization, the mice were boosted with 50 mg of IHRP in phosphate-buffered saline (PBS). Three days after the boost injection, the spleen cells of the mice were fused with P3U1 mouse myeloma cells using polyethylene glycol (PEG). The hybridoma cells were selected in HAT medium and the media were screened by ELISA. The positive hybridoma cells were cloned by limiting dilution and the cloned cells (1ϫ10 7 cells/mice) were injected intraperitoneally into female Balb/c mice which had been pre-injected with pristane (0.5 ml/mice) one week earlier. Monoclonal antibodies were purified from the ascites fluids by ammonium precipitation and DEAE-Sephace...

Jacalin Bound Plasma O-Glycoproteome and Reduced Sialylation of Alpha 2-HS Glycoprotein (A2HSG) in Rheumatoid Arthritis Patients

“…Of 18 identified proteins, one up regulated ITIH4 and one down regulated protein A2HSG in plasma of RA patients, were selected for further validation by immunobloting as these are known to be associated with inflammation [23], [24]. Since the enrichment of plasma glycoproteins by jacalin chromatography may not reflect the actual difference in the expression of the protein, the expression levels of A2HSG and ITIH4 were checked in plasma without jacalin enrichment by Western blotting (Figure 2A, C).…”

Quantification of Fragments of Human Serum Inter-α-Trypsin Inhibitor Heavy Chain 4 by a Surface-Enhanced Laser Desorption/Ionization-Based Immunoassay

“…All peptide antibodies above were purified by both protein-A and affinity chromatography (Pierce Biotechnology). Both anti-ITIH4 rabbit antiserum (antiserum) and anti-ITIH4 mouse monoclonal antibody (1A4) were prepared by Dr. N.H. ChoiMiura (3,5 ). Antibody 1A4 recognizes an epitope located on the C-terminal M r 35 000 fragment of ITIH4.…”

“…Inter-␣-trypsin inhibitor heavy chain 4 (ITIH4) 5 is a plasma glycoprotein with a relative molecular mass of 120 000 that is expressed mainly in liver and that acts as an acute-phase protein in several species (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Unlike other members of the inter-␣-trypsin inhibitor family (12,13 ), ITIH4 is present in plasma as a single-chain protein because its COOH terminus lacks the consensus sequence (DPHFII) for bikunin assembly through the glycosaminoglycan bridges (2,3,(12)(13)(14).…”

Site-Specific Glycan Microheterogeneity of Inter-Alpha-Trypsin Inhibitor Heavy Chain H4

“…5 ITIH4 circulates in human blood at a concentration of approximately 100 μg/mL. 6 It is a positive acute phase protein regulated by IL-6 and implicated in liver development and regeneration. 7−11 ITIH4 can be cleaved by plasma kallikrein into two fragments that undergo additional proteolytic processing into an 85 kDa N-terminal fragment and a 35 kDa C-terminal fragment.…”