ELISA for Determination of the Haptoglobin Phenotype

Levy, Nina S.; Levy, Andrew P.

doi:10.1373/clinchem.2004.038547

Cited by 11 publications

(6 citation statements)

References 20 publications

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“…The use of a mAb in this assay has several advantages compared with the single chain antibody used in our previous assay [3], which include greatly improved yields from cell culture and increased stability of the antibody. In addition, the mAb has increased affinity for Hp which allows for coating of the microtiter plates at a concentration of 1 μg/mL mAb compared with 100 μg/mL single chain antibody and use of the secondary, conjugated antibody at a concentration of 0.04 μg/mL instead of 0.8 mg/mL.…”

Section: Discussionmentioning

confidence: 99%

“…An earlier attempt at developing this assay resulted in the isolation of a single chain antibody which was able to accurately distinguish between the three Hp phenotypes in a sandwich ELISA assay [3]. This reagent proved to be unsatisfactory for large-scale analyses due to low yields obtained from bacterial cultures and poor stability of the single chain antibody.…”

Section: Introductionmentioning

confidence: 99%

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An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

Levy

Vardi

Blum

et al. 2013

Clinical Chemistry and Laboratory Medicine (CCLM)

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Background Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. Methods A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. Results Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. Conclusions The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

Levy

Vardi

Blum

et al. 2013

Clinical Chemistry and Laboratory Medicine (CCLM)

View full text Add to dashboard Cite

show abstract

“…Heterozygotes with both Hp 1 and Hp 2 alleles have Hp 1 dimers and Hp 2-1 polymers as well [17]. These proteins can be separated by gel electrophoresis, isoelectric focusing, chromatography, or ELISA [28–31]. A typical diagram of electrophoresis results is shown in Figure 2.…”

Section: Haptoglobin Phenotypingmentioning

confidence: 99%

A Review of Haptoglobin Typing Methods for Disease Association Study and Preventing Anaphylactic Transfusion Reaction

Chang

Kim³

et al. 2013

BioMed Research International

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Haptoglobin, the product of the Hp gene, is a glycoprotein involved in the scavenging of free hemoglobin. Haptoglobin levels increase or decrease in response to various acquired conditions, and they are also influenced by genetic predisposition. There were 2 major alleles, Hp 1 and Hp 2, and 1 minor allele, Hp del. Many researchers have attempted to study the haptoglobin types and their association with disease; however, no definitive conclusions have been reached yet. It is reported that patients who are genetically deficient in haptoglobin are at risk of anaphylaxis against blood components containing haptoglobin. Haptoglobin genotypes also affect the reference intervals of haptoglobin levels. Many studies have attempted to establish simple and accurate typing methods. In this paper, we have broadly reviewed several methods for haptoglobin typing—phenotyping, Southern blotting, conventional PCR, real-time PCR, and loop-mediated isothermal amplification. We discuss their characteristics, clinical applications, and limitations. The phenotyping methods are time consuming and labor intensive and not designed to detect patients harboring Hp del. The rapid and robust haptoglobin genotyping may help in preventing fatal anaphylactic reactions and in establishing the relationships between the haptoglobin phenotypes and diseases.

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“…The Hp-Hb complexes were subsequently detected using sophisticated staining methods such as hemoglobin peroxidase activity and chemiluminescent assays. [12][13][14][15] In addition, methods using spectrophotometer, 16 immunoblotting, 14,17 isoelectric focusing capillary electrophoresis, 18,19 high-pressure gel-permeation chromatography, 20 and even ELISA 21 were developed. There also have been attempts to predict haptoglobin types by detection of free hemoglobin concentrations in serum.…”

Section: Introductionmentioning

confidence: 99%

Classifying Human Haptoglobin Phenotypes on Native‐PAGE Using a Modified Coomassie Brilliant Blue R 250 Staining

Liau

Ueng

Lin

2009

J Chinese Chemical Soc

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There are three types of human Haptoglobin, Hp 1-1, Hp 2-1, and Hp 2-2, each characterized by a distinct combination of the two a chains of the holoprotein. A modified Coomassie Brilliant Blue R 250 (mCBB-R250) staining method for detecting the phenotypes of human blood haptoglobin molecules is presented. Addition of excess hemoglobin to the sample allowed specific formation of different haptoglobin-hemoglobin complexes, which, in turn can be separated into distinctive migration pattern on native-PAGE and stained by CBB-R250. The typing results are consistent with that using Western blotting. In comparison to the existing methods for haptoglobin typing, our method is comparable in accuracy, and is easier to carry out. The results on typing of 29 plasma samples from Taipei Blood Center were also presented.

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ELISA for Determination of the Haptoglobin Phenotype

Cited by 11 publications

References 20 publications

An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

A Review of Haptoglobin Typing Methods for Disease Association Study and Preventing Anaphylactic Transfusion Reaction

Classifying Human Haptoglobin Phenotypes on Native‐PAGE Using a Modified Coomassie Brilliant Blue R 250 Staining

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