Anti-human Hemoglobin Research Articles

Spontaneous Avian Erythroblastosis in a Chicken Confirmed by Immunohistochemical Detection of Hemoglobin in Tumor Cells.

Avian erythroblastosis (AE; erythroid leukosis) was detected in a 78-day-old Japanese native chicken. At necropsy, the liver was enlarged and diffusely dark red in color. Moderate splenomegaly was observed. Histologically, round to polygonal tumor cells were observed only in the blood vessels of the liver and other organs. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded (FFPE) sections to characterize tumor cells. Tumor cells, as well as normal erythrocytes as positive controls, were consistently positive for IHC by using the commercially available anti-human hemoglobin antibody. Exogenous avian leukosis virus (ALV) subgroup B and endogenous ALV-E genes were detected by PCR, although ultrastructural observation revealed no viral particles associated with tumor cells. Results suggest that the commercial anti-human hemoglobin antibody used in the study cross-react to chicken erythrocytes on FFPE sections by IHC and can also be used for definitive diagnosis of the spontaneous case of AE in chickens.

KORELASI ANTARA PERIKSAAN DARAH SAMAR TINJA MENGGUNAKAN ANTI-HEMOGLOBIN MANUSIA DAN PENGAMATAN MIKROSKOPIS

Test for occult blood in faeces is an important part of the early detection of colorectal cancer, gastrointestinal bleeding, and anaemia.Colorectal cancer is the second leading cause of cancer deaths in the United States. Immunochemistry method for detection of humanhaemoglobin in faeces has been developed. The advantages of this method are improving analytical sensitivity and specificity, alsoavoiding the dietary restrictions requirement, compared with benzidine test, and guaiacum test. A study was performed to correlate theresult of fecal occult blood by immunochemistry method using anti-human haemoglobin and microscopic examination of red blood cellsin faeces of outpatients in the Clinical Pathology Laboratory, Dr. Soetomo Hospital, Surabaya. Faeces of fifty one patients tested for fecaloccult blood were examined by immunochemistry method compared with microscopic examination of red blood cells. Comparison ofthe two methods was done by statistical analysis, Mc Nemar test. The correlation was measured using ROC curve. The results showed acorrelation between immunochemistry method and microscopic examination with average red blood cells (RBC) ≤ 2/hpf, p = 0.008; RBC≥ 3/hpf, p = 0.289. ROC curve showed r = 0.941. In conclusion, a significant correlation between positive results of immunochemistrymethod and microscopic examination with average red blood cells ≥ 3/hpf. Further research using larger and more representativesamples should be carried out.

Filaments with Affinity Binding and Wet Strength Can Be Achieved by Spinning Bifunctional Cellulose Nanofibrils.

We demonstrate benzophenone (BP) conjugation via amine-reactive esters onto oxidized cellulosic fibers that were used as precursors, after microfluidization, of photoactive cellulose nanofibrils (CNF). From these fibrils, cellulose I filaments were synthesized by hydrogel spinning in an antisolvent followed by fast biradical UV cross-linking. As a result, the wet BP-CNF filaments retained extensively the original dry strength (a remarkable ∼80% retention). Thus, the principal limitation of these emerging materials was overcome (the wet tensile strength is typically <0.5% of the value measured in dry conditions). Subsequently, antihuman hemoglobin (anti-Hb) antibodies were conjugated onto residual surface carboxyl groups, making the filaments bifunctional for their active groups and properties (wet strength and bioactivity). Optical (surface plasmon resonance) and electroacoustic (quartz crystal microgravimetry) measurements conducted with the bifunctional CNF indicated effective anti-Hb conjugation (2.4 mg m-2), endowing an excellent sensitivity toward Hb targets (1.7 ± 0.12 mg m-2) and negligible nonspecific binding. Thus, the anti-Hb biointerface was deployed on filaments that captured Hb efficiently from aqueous matrices (confocal laser microscopy of FITC-labeled antibodies). Significantly, the anti-Hb biointerface was suitable for regeneration, while its sensitivity and selectivity in affinity binding can be tailored by application of blocking copolymers. The developed bifunctional filaments based on nanocellulose offer great promise in detection and affinity binding built upon 1D systems, which can be engineered into other structures for rational use of material and space.

Scavenging of nitric oxide by hemoglobin in the tunica media of porcine coronary arteries

Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 μmol×min−1×mg−1). The proteins retained on the affinity column were identified as hemoglobin α and β subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue.

Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20years

The effects of various storage conditions on blood identification tests, DNA degradation, and short tandem repeat (STR) typing were evaluated. Bloodstains stored at room temperature, 4 °C, -20 °C, and -80 °C for 20 years; blood samples stored at -20 °C and -80 °C for 20 years; and fresh blood samples were analyzed. Leuco-malachite-green testing, anti-human hemoglobin (Hb) testing (using immunochromatography), and tests for hemoglobin-beta (HBB) mRNA were performed as blood identification tests. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp fragments. STR typing was performed using an AmpFlSTR® Identifiler™ Plus PCR Amplification Kit. All samples were positive in leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in blood samples stored at -20 °C or -80 °C, although this marker was detected in all bloodstains. As indicated by the ratio of 129:41 bp and 305:41 bp DNA fragments, DNA from bloodstains stored at room temperature or 4 °C were significantly degraded compared to DNA from all other samples. STR typing analyses revealed that a portion of the loci was undetected in bloodstains stored at room temperature. Therefore, to prevent DNA degradation during long-term storage, it is recommended that bloodstains and blood be stored at below -20 °C. In addition, because bloodstains are more suitable for detection of blood-specific mRNAs than blood sample, it is desirable that blood is stored as bloodstain for this method.

Prevalence of blood contamination in adult saliva and quantitative measurement of salivary hemoglobin level by using an anti-human hemoglobin monoclonal antibody

Prevalence of blood contamination in adult saliva and quantitative measurement of salivary hemoglobin level by using an anti-human hemoglobin monoclonal antibody

Effects of storage method on DNA degradation in old bloodstain samples

The effects of different storage methods on both DNA degradation and the results of preliminary blood tests were evaluated. Bloodstains stored at room temperature, 4°C, –20°C, and –80°C for 20 years; whole blood samples stored at –20°C and –80°C for 20 years; and fresh whole blood samples were analyzed. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41bp fragments. Preliminary testing included leuco-malachite-green staining, anti-human hemoglobin (Hb) testing by immunochromatography, and the detection of the hemoglobin-beta (HBB) mRNA. Bloodstains stored at room temperature and 4°C were more highly degraded than fresh blood, as indicated by the ratio of 129:41bp DNA fragments and 305:41bp DNA fragments. All samples tested positive using leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in any whole blood samples stored at –20°C or –80°C, although HBB was detected in all bloodstain samples. Therefore, to prevent DNA degradation during long-term storage, it is recommended that blood samples be stored at below –20°C. In addition, stored bloodstains are suitable for preliminary tests of blood, rather than storing blood.

Background coloration of squamous epithelium in esophago-pharyngeal squamous cell carcinoma: what causes the color change?

ObjectivesThis study aims to clarify the cause of background coloration in the epithelia between each dilated intra papillary capillary loop in esophago-pharyngeal squamous cell carcinoma.DesignThis is a single center retrospective study including 124 patients with 160 lesions who underwent esophagogastroduodenoscopy in Nagasaki University Hospital from September 2007 to March 2012; a detailed comparison between endoscopic images and pathology was performed. Immunohistological assessment using anti-human hemoglobin antibody (anti-Hb Ab) was performed to verify the presence of hemoglobin (Hb) component in the cancer cells. Real-time polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) on Hb-β mRNA were performed to assess the production of Hb component within the cancer cells.ResultsA strong positivity for anti-Hb Ab was observed in the squamous cell carcinoma area, whereas non-cancerous mucosa showed no immunopositivity for Hb. The concordance rate between anti-Hb Ab immunoreactivity and the presence of BC was as high as 80.9%. The amount of Hb-β mRNA expression was three times higher in cancer tissues compared with the surrounding non-cancerous mucosa. ISH images showed that the expression exclusively occurred in cancer cells, indicating that Hb is probably produced within cancer cells.ConclusionsThe background coloration observed is partly due to an extravascular component of Hb. RT-PCR and ISH analyses indicate that Hb is produced within cancer cells.

Sa1610 Clinical Impact of Background Coloration (BC) Between IPCLs in Differentiating Esophageal Squamous Cell Carcinoma. What Makes the Color Change?

It has been reported that alteration of intra-papillary capillary loop (IPCL) is important in detecting early esophageal squamous cell carcinoma (SCC). Also, brownish color change in the epithelia between each IPCL that we call “background coloration (BC)” has recently reported as an additional finding to differentiate SCC from non-neoplastic lesion. However, the cause of this phenomenon has not been identified. We applied anti-human Hemoglobin(Hb) antibody immuno-staining for both SCC and non-neoplastic lesions in order to illuminate the reason why cancer lesion can be positive for NBI, which is a specific wave length of Hb.

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe 3O 4, which was synthesized with easy handling feature of Fe 3O 4 by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe 3O 4 magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05–1.5 μg mL −1, with the detection limit of 0.025 μg mL −1. This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c.

On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A 1c level

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA 1c, which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA 1c in blood calibrator samples from other Hbs, e.g. HbA 0, HbA 1a or HbA 1b, were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA 1a and HbA 1b, HbA 1c and HbA 0 fractions were eluted in this order. A linear relationship between HbA 1c levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA 1c. All procedure including antigen–antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA 1c.

Electrochemical detection of HbA 1c, a maker for diabetes, using a flow immunoassay system

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A 1c (HbA 1c) was developed using anti-human hemoglobin (Hb) IgG labeled with ferrocene monocarboxylic acid (Fc–COOH) and boronate-affinity chromatography. An on-chip column packed with boronate-activated agarose beads was used for the separation of HbA 1c from both non-glycated Hb and free antibody. Anti-human Hb IgG conjugated to Fc–COOH (Fc–IgG) was used for the electrochemical detection of HbA 1c. The assay procedure included immunoreactions with Fc–IgG and HbA 1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc–IgG–HbA 1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA 1c–antibody complexes were then trapped onto the column by the affinity of HbA 1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA 1c–antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA 1c amount. A linear correlation between the increase of current and HbA 1c concentration was obtained up to an HbA 1c concentration of 500 μg/ml. The HbA 1c flow immunoassay was successfully achieved using hemolysates. This electrochemical flow immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of glycated proteins including HbA 1c.

Development of capacitance based immunosensors on mixed self-assembled monolayers

The construction and characterisation of a capacitance based immunosensor on mixed mercaptohexadecanoic acid and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-caproyl biotinyl self-assembled monolayers on gold are reported. Immobilisation of IgG antibodies was achieved using the strong non-covalent bond formed between Neutravidin and biotin to tether anti-human haemoglobin goat IgG to the mixed monolayers. Analysis of sensor construction and of the functional immunosensors was characterized via physical techniques, including quartz crystal microbalance, and atomic force microscopy for surface topographical measurement. Sensor assembly and the binding affinity of immobilised antibodies were independently determined using [ 125I]-tagged components. Finally, the electrochemical properties of partially constructed and fully functional immunosensors were analysed by electrochemical impedance spectroscopy in a ferricyanide mediated system. The haemoglobin sensors show a linear response of the imaginary component of impedance ( Z″) across a wide range of concentrations, from 10 −10 (according to Fig. 8, linearity is restricted over the concentration range 10 −8 to 10 −6 M haemoglobin), at the lower range of frequencies examined.

Feasibility of iodine-125 labeled anti-human hemoglobin antibody in the detection of bleeding sites from the large bowel-A preliminary study.

A monoclonal anti-human hemoglobin antibody that cross-reacts with mouse hemoglobin was labeled with Iodine-125 through the Chloramine-T method. The labeled antibody was used in an attempt to recognize bleeding sites from the large bowel in a mouse model, through a non-invasive enema-like study. In vitro experiments after double column chromatography of the labeled antibody and 10% trichloroacetic acid conjugation revealed that about 80% of the radioactivity was incorporated into protein. Inhibition assay containing cold (non-radiolabeled) antibody showed that Iodine-125 radiolabeled antibody preserved its immunoreactivity. Autoradiographs exquisitely demonstrated accumulation of isotope in the corresponding intestinal bleeding areas. These findings suggest that this method can be useful for scintigraphic localization of bleeding sites in the large bowel.

Identification of hemoglobin degradation products in Plasmodium falciparum

Malaria parasites break down human hemoglobin to its constituent amino acids by cysteine and aspartic proteinases. However, no one has previously been able to identify hemoglobin cleavage products in intact parasites. When isolated parasites were subjected to non-denaturing polyacrylamide gels electrophoresis, a unique protein band was found which contains heme and reacts with anti-human hemoglobin antibodies. This protein does not appear to represent oxidized or glycosylated hemoglobin, and is present in isolated parasites but not in the cytosol of infected or uninfected erythrocytes. When this band was eluted and subjected to SDS polyacrylamide gel electrophoresis, three bands were seen on Western blots. The proteins in these bands contain proteins with the N-terminal sequences of α- and β-globin chains but molecular masses of only 13.2–13.4 kDa. These data suggest that hemoglobin α- and β-chains are initially cleaved within the parasite phagolysosome to release peptides of 15–17 and 23–25 amino acids from the C-termini of α- and β-globin chains, respectively. Production of the hemoglobin breakdown products was inhibited by E-64, a cysteine proteinase inhibitor, suggesting the involvement of a cysteine proteinase in an early step of hemoglobin degradation.

Immunohistochemical studies on postmortem lividity

An immunohistochemical investigation of postmortem lividity was performed to illuminate localization of hemoglobin (Hb) and the mechanism of fixed lividity. The fixed lividity was defined as an unfading phenomenon by thumb finger pressure. Skin specimens were taken from 68 autopsy cases 7.5–336 h (2 weeks) postmortem. Localization of Hb of the specimens was examined by a labeled streptabidin biotin (LSAB) method using polyclonal (rabbit antihuman hemoglobin antibody) and monoclonal (mouse anti-human hemoglobin monoclonal antibody) antibodies. Positive staining for Hb was observed in various sites of the skin, i.e. in only intravascular erythrocytes, in vascular walls and perivascular tissue including sweat glands and sebaceous glands, in the dermal connective tissue, and in almost all of skin tissue except the horny layer. The diffusion of Hb into skin tissue was observed in 20 of 41 displaceable lividity cases (49%) and 11 of 27 fixed lividity cases (41%). Compared to displaceable lividity, superficial plexi in fixed lividity were filled with erythrocytes, which were markedly immunodetected. These findings support the hypothesis that the fixation of lividity is not due to diffusion of Hb into skin tissue but hemoconcentration in blood vessels.

High Resolution Light Microscopy and Immunocytochemistry with Glycol Methacrylate Embedded Sections and Immunogold-Silver Staining

AbstractThe immunogold-silver technique was modified so that 1–2 μm thick glycolmethacrylate sections could routinely be used for high resolution immunocytochemistry. A rabbit antihuman hemoglobin antibody was used to demonstrate the distribution of exogenous hemoglobin in mouse tissues. Inclusion of 0.1% glutaraldehyde in the primary formalin fixation improved tissue appearance. Glutaraldehyde treatment after colloidal gold antibody application increased the staining intensity, and gold chloride intensification of the silverenhanced colloidal gold further improved immunosensitivity. Thus, a more accurate analysis of hemoglobin distribution in tissue was obtained. The enhancements were also beneficial for localizing myelin basic protein in brain and for determining the proliferative status of tissue by localization of bromodeoxyuridine incorporation into nuclei.Manual capillary action staining efficiency was demonstrated during all steps in the immunogold-silver staining protocol, greatly simplifying the ...

Detection of fecal blood by colloidal gold agglutination using an anti-human hemoglobin monoclonal antibody

Monoclonal antibodies to human hemoglobin were produced and a colloidal gold agglutination method has been developed for detection of fecal occult blood. Since hemoglobin is composed of the tetramer, α 2 β 2, a single monoclonal antibody-labeled colloidal gold can agglutinate with hemoglobin. The lowest detectable hemoglobin concentration was 0.5 μg/ml. A total of 785 fecal samples were determined using colloidal gold agglutination and compared with latex agglutination. The colloidal gold agglutination detected blood in 75 samples, whereas latex agglutination detected blood in 76 samples, and among them 70 were positive in both methods. Overall agreement between the two methods was 98%.

Assessment of immunochemical occult blood test detectacol for the early detection of colorectal neoplasia

The DETECTACOL test for faecal occult blood uses polyclonal goat anti-human haemoglobin in a radial immunodiffusion assay. Faecal homogenates prepared from three successive bowel actions are tested. Tabled 1 CURRENT STUDY HIGH RISK GROUP 1 J.A.R. Williams, R.Ilunter, D.W.Thomas, M.E.Coles, A.S.Y.Leong, R.L.Walsh, D.C.Iloffman, T.W.liuber, and A.Sen. Aust., N.Z.J.Surg, 1987, 57, 951-957. Participants (no.) 6,323 1,615 Positive rate (%) 5.2 6.1 ADENOMA +ve predictive value (%) 32.1 40.0 Detection rate (per 1000) 11.2 22.3 CANCER +ve predictive value (%) 10.0 14.4 Detection rate (per 1000) 3.5 8.0 1. J.A.R. Williams, R.Ilunter, D.W.Thomas, M.E.Coles, A.S.Y.Leong, R.L.Walsh, D.C.Iloffman, T.W.liuber, and A.Sen. Aust., N.Z.J.Surg, 1987, 57, 951-957. Open table in a new tab These results indicate that DETECTACOL gives a greater detection rate for both bowel cancer and adenomatous polyps in the general community when compared with various studies of screening programmes currently in progress, in which chemical tests for occult blood arc used. The DETECTACOL test for faecal occult blood uses polyclonal goat anti-human haemoglobin in a radial immunodiffusion assay. Faecal homogenates prepared from three successive bowel actions are tested. These results indicate that DETECTACOL gives a greater detection rate for both bowel cancer and adenomatous polyps in the general community when compared with various studies of screening programmes currently in progress, in which chemical tests for occult blood arc used.

Biochemical Characterization of Plasmodium falciparum Hemozoin

Hemozoin, the pigment granule which develops within the blood stage food vacuole of the malaria parasite Plasmodium falciparum, was biochemically characterized. Hemozoin was found to be composed of 65% protein, 16% ferriprotoporphyrin-IX (hematin), 6% carbohydrate, and trace amounts of lipid and nucleic acids. The overwhelming majority of the protein component is a mixture of native and denatured human globin non-covalently associated with the metalloporphyrin. Immunoelectron microscopy, employing anti-human hemoglobin as a probe, identified in situ association of hemoglobin with hemozoin. Hemozoin produced within diabetic blood had a higher proportion of carbohydrate, suggesting that the carbohydrate component comes from non-enzymatic glycosylation of hemoglobin.