Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Tseng, Chi Feng; Huang, Hsing Yi; Yang, Yixia; Mao, Simon J.T.

doi:10.1016/j.pep.2003.09.006

Cited by 30 publications

(42 citation statements)

References 27 publications

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“…The plasma of each Hp phenotype was purified using chromatography on a mAb-based affinity column followed by a high-performance liquate chromatography procedure [16]. The plasma sample (2 mL) was added into the antibody affinity column.…”

Section: Methodsmentioning

confidence: 99%

Elevated Plasma Haptoglobin Level as a Potential Marker for Poor Prognosis in Acute Cerebral Infarction

Zhang

Xiang

2018

Eur Neurol

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Background: Relationship between peripheral circulation concentration of haptoglobin (Hp) and risk of cerebrovascular disease has not been well investigated so far. The aim of the study was to reveal the prognostic significance of Hp on acute cerebral infarction. Methods: A total of 200 patients with acute cerebral infarction and 200 controls were included. Research data were collected from their medical records. Blood samples were obtained on admission. Hp phenotyping was performed using a native polyacrylamide gel electrophoresis. Plasma Hp levels were determined using enzyme-linked immunosorbent assays. The patients were followed up yearly, and endpoint was overall death. Results: Plasma levels of Hp were significantly elevated in the patients than in the controls (p < 0.001). Three-year overall death in the follow-up period was associated with the high plasma level of Hp (Hazard ratio [HR] 2.33, 95% CI 1.74–3.12). But, 3-year overall death was not related to Hp phenotyping (Hp 2–1 vs. 1–1: HR 1.07, 95% CI 0.66–1.73; Hp 2–2 vs. 1–1: HR 1.08, 95% CI 0.67–1.74). Conclusion: Peripheral circulation concentration of Hp might be an independent prognostic factor for acute cerebral infarction. But there was no relationship between Hp phenotyping and prognosis in this disease.

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Section: Methodsmentioning

confidence: 99%

Elevated Plasma Haptoglobin Level as a Potential Marker for Poor Prognosis in Acute Cerebral Infarction

Zhang

Xiang

2018

Eur Neurol

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“…After SDS-and native-PAGE, gels were transferred to PVDF membranes according to a standard procedure as previously described [15]. After the transfer process, PVDF membranes were blocked with blocking buffer containing 5 % skimmed milk (w/v), 0.1 % Tween 20 in PBS (pH 7.4) for 1 h at room temperature.…”

Section: Western Blottingmentioning

confidence: 99%

“…Three phenotypes (Hp1-1, Hp2-2 and Hp2-1) of Hp have been identified in humans. It has been reported that the Hp phenotype identified in rats is similar to Hp1-1 phenotype in human and had a heterotetrameric (α2β2) structure [10]. The structural and functional differences between Hp phenotypes in humans have important biological and clinical consequences.…”

Section: Introductionmentioning

confidence: 97%

Age-related changes in haptoglobin phenotypes, some non-enzymatic antioxidants and electrophoretic profiles of serum proteins in rats

Çiftçi

Yarım²,

Çenesiz³

et al. 2012

Turk J Bioch

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Aim: Determination of age-related changes in levels of some non-enzymatic antioxidants such as serum haptoglobin, haemoglobin and albumin, serum haptoglobin phenotype and serum protein profile was aimed with this study. Methods and results: Fifty Sprague-Dawley male rats were divided equally into five groups as 1 (one-month old), 2 (six-months old), 3 (twelve-months old), 4 (eighteen-months old) and 5 (twenty-four-months old). The serum protein concentrations were measured by autoanalyzer. A decrease was determined in the concentrations of the total protein and albumin related to age. However, no clear decrease in the serum globulin concentrations occurred. Serum haptoglobin concentrations were measured by ELISA and an increase was detected with age. Serum haemoglobin concentrations were increased up to 3 months old, but decreased from 3 to 24 months old of age. The electrophoretic profiles of serum total protein were determined by native-PAGE. No differences were observed in total serum profiles between age groups. To determine serum haptoglobin profiles, western blot was performed and haptoglobin patterns were designated as "aa" (single band) or "ab" (two bands) patterns. The "ab" pattern was more frequently observed in older rats. Also, 15 protein bands ranged between 19 to 244 kDa, were detected by SDS-PAGE. After SDS-PAGE, western blot were performed and three haptoglobin bands (10, 37 and 47 kDa) were detected. Conclusion:The age-related changes in total serum protein, albumin, haemoglobin and haptoglobin concentrations as well as the prevalent haptoglobin phenotypes may be considered as indicators in some age-related diseases.

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“…We therefore used a previously described HPLC-based size-exclusion chromatography procedure [24] for the isolation of deer Hp. However, this procedure is only suitable for isolating the Hps with a homogeneous structure, and is not suitable for human Hp 2-2 or 2-1 [22]. One minor disadvantage of the method was the contamination of the isolated Hp by a trace amount of hemoglobin (Fig.…”

Section: Isolation Of Deer Native Hpmentioning

confidence: 99%

“…Native human Hp was isolated from plasma using an immunoaffinity column followed by size-exclusion chromatography on an HPLC system using previously described procedures [22].…”

Section: Preparation Of Mouse Mab and Human Hpmentioning

confidence: 99%

A unique tetrameric structure of deer plasma haptoglobin – an evolutionary advantage in the Hp 2‐2 phenotype with homogeneous structure

et al. 2008

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Haptoglobin (Hp) is an acute-phase protein (responsive to infection and inflammation) that is present in the plasma of all mammals [1][2][3][4]. A recent study has found that Hp also exists in lower vertebrates (bony fish) but not in frog and chicken [5]. The most frequently reported biological functions of the protein are to capture released hemoglobin during excessive hemolysis [6] and to scavenge free radicals during oxidative Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by a2-b subunits or (a2-b) n (where n ‡ 3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the a-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (a-b) 4 , although the locations of )SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 m urea under reducing conditions (143 mm b-mercaptoethanol), followed by renaturation, sustained the formation of (a-b) 4 , suggesting that the Hp tetramers are not randomly assembled. Interestingly, an a-chain monoclonal antibody (W1), known to recognize both human and deer a-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer a-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.Abbreviations Hp, haptoglobin; b-ME, b-mercaptoethanol.

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Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Cited by 30 publications

References 27 publications

Elevated Plasma Haptoglobin Level as a Potential Marker for Poor Prognosis in Acute Cerebral Infarction

Elevated Plasma Haptoglobin Level as a Potential Marker for Poor Prognosis in Acute Cerebral Infarction

Age-related changes in haptoglobin phenotypes, some non-enzymatic antioxidants and electrophoretic profiles of serum proteins in rats

A unique tetrameric structure of deer plasma haptoglobin – an evolutionary advantage in the Hp 2‐2 phenotype with homogeneous structure

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