Immunological evaluation of urinary trypsin inhibitors in blood and urine: Role of N- & O-linked glycoproteins

Abstract

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-alpha-I, and I-alpha-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm-Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.

Development of a method for urine bikunin/urinary trypsin inhibitor (UTI) quantitation and structural characterization: Application to type 1 and type 2 diabetes

“…Bikunin determination in urine is mainly performed by enzyme inhibition assays or immunological detection . These methods rely on the use of enzymatic reactions or antibodies for either indirect or direct bikunin quantitation.…”

Two-Dimensional Differential In-Gel Electrophoresis Proteomic Approaches Reveal Urine Candidate Biomarkers in Pediatric Obstructive Sleep Apnea

“…It is postulated that bikunin may play a role in the down-regulation of inflammatory responses; however, the putative increase in bikunin in the urine as inferred from 2D-DIGE will have to await further confirmatory studies (34). Development of a sensitive and reliable ELISA for bikunin is currently underway in our laboratory (35), and should permit more extensive exploration of the validity and reliability of bikunin quantitative assays in the screening of pediatric OSA. a 1 -Microglobulin (A1M) is a 27-kD glycoprotein that is present in various body fluids; its exact biological function is unknown.…”

Application of MALDI/SELDI Mass Spectrometry to Cancer Biomarker Discovery and Validation

“…The eluted proteins were then analyzed with a TOF-MS. A limitation of MSIA is that it is not routinely suitable for analysis of a large number of proteins of interest (Thongboonkerd, 2007). Table 3 (Cadieux et al, 2004;Castanga et al, 2005;Clarke et al, 2003;Dare et al, 2002;Dihazi et al, 2007;Hampel et al, 2001;Hsu et al, 2007;Iwaki et al, 2004;Jahnukainen et al, 2006;Jurgens et al, 2005;Kageyama et al, 2004;Kemna et al, 2007;Khurana et al, 2006;Kiernan et al, 2002Kiernan et al, , 2003Kiernan et al, , 2004Kosanam et al, 2007;Lida et al, 2007;M'koma, 2003;Mosley et al, 2006;Munro et al, 2006;Mushtaq et al, 2007;Nguyen et al, 2005;O'Riordan et al, 2007;Otu et al, 2007;Papale et al, 2007;Pugia et al, 2007;Robijn et al, 2007aRobijn et al, , b, 2008Rogers et al, 2003;Schaub et al, 2004Schaub et al, , 2005Shou et al, 2008;Shui et al, 2008;Traum et al, 2006;Valmu et al, 2006;Vlahou et al, 2001;…”

“…In addition to detecting a novel undescribed amyloid polypeptide, the SELDI TOF-MS approach was also used to simultaneously quantify three peptides that were either in- Castagna et al, 2005;Kemna et al, 2007;Papale et al, 2007;Pugia et al, 2007;Schaub et al, 2004;Traum et al, 2006;Wang et al, 2007Urine 2007 Human MALDI-TOF- MS Hsu et al, 2007;Kosanam et al, 2007;Lida et al, 2007;Wen et al, 2007;Zerefos et al, 2007Zerefos et al, , 2008 …”