Anti-human Hemoglobin Antibody 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.

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.

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.

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.

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 ...

How Long to Abstain from Eating Red Meat before Fecal Occult Blood Tests

Brief Reports1 September 1990How Long to Abstain from Eating Red Meat before Fecal Occult Blood TestsElliot J. Feinberg, MD, William M. Steinberg, MD, Bonnie L. Banks, BA, James P. Henry, BSElliot J. Feinberg, MDSearch for more papers by this author, William M. Steinberg, MDSearch for more papers by this author, Bonnie L. Banks, BASearch for more papers by this author, James P. Henry, BSSearch for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-113-5-403 SectionsAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail ExcerptHemoccult's long recognized use for colon cancer screening (1, 2) has recently been challenged by newer fecal occult blood tests with reputed advantages in sensitivity and specificity.The HemoQuant quantitatively measures the concentration of heme-derived porphyrins (3, 4). The Hemoccult SENSA, a modified guaiac-based test, reportedly detects lower levels of hemoglobin, thereby increasing sensitivity (5). The HemeSelect, using an antihuman hemoglobin antibody, is reported to be highly sensitive and specific (6, 7). Although dietary constituents, especially red meat, can increase the false-positive rate of the Hemoccult (1, 2, 8, 9), Hemoccult SENSA (5), and HemoQuant (4) tests, the comparative duration...

Liver and Kidney Injury After Administration of Hemoglobin Cross-Linked with Bis(3,5-Dibromosalicyl) Fumarate

Human hemoglobin cross-linked between the alpha chains with bis (3,5-dibromosalicyl) fumarate (DBBF-Hb) was exchange transfused in swine and the histomorphologic changes were evaluated. Following exchange, animals were euthanized and tissues were taken for light and electron microscopy at 7.5 hours and days 1, 4, 7, and 15. Consistent hepatocellular and renal epithelial cell changes were seen. Hepatic injury, evident at 7.5 hours as cellular vacuolization, progressed to necrosis and acute inflammatory cell infiltration by days 1 and 4, was resolving by 7 days and was completely resolved by day 15. Cytochemical stains for iron and hemoglobin revealed positive material in Kupffer cells, endothelial cells, and necrotic hepatocytes. Rabbit anti-human hemoglobin antibody staining revealed immunoreactive material diffusely present at days 1 and 4 and limited to solitary hepatocytes by day 15. Kidney injury began as proximal tubular epithelial vacuolization and intraluminal casts progressing to tubular necrosis by 24 hours, with resolution by day 15. Iron and hemoglobin stains demonstrated these materials in the early lesions. Immunocytochemistries demonstrated human hemoglobin that remained as late as day 15. Electron microscopy revealed degeneration and regeneration of epithelial cells. The renal lesions were consistent with hemoglobinuria. The liver lesion was less well defined but was self limited.

第15回日本総合健診医学会学術大会講演抄録 抗ヒトーヘモグロビン抗体を用いた便潜血反応の基礎的検討およびドック受診者における成績について

第15回日本総合健診医学会学術大会講演抄録 抗ヒトーヘモグロビン抗体を用いた便潜血反応の基礎的検討およびドック受診者における成績について

抗ヒトヘモグロビン抗体を用いた免疫学的便潜血反応の検討

消化管出血の検索のため従来から行われている化学的便潜血反応には,ヒトヘモグロビン以外の物質と非特異的反応をおこすという欠点がある.この問題を解決するため抗ヒトヘモグロビン抗体を作製し,二次元両者拡散法(Ouchterlony法)を用いて免疫学的便潜血反応を臨床的に検討した.非消化管疾患107例では9.3%,消化管疾患150例をは48.7%,大腸癌60例では75.0%に陽性を示し,健常者30例では全例で陰性であった.Ouchterlony法は消化管疾患や大腸癌では,化学的便潜血反応であるシオノギスライドB法とほぼ同様の陽性率を示したが,非消化管疾患および健常者では明らかに低率を示し,偽陽性率の改善が可能であった.従って本法は消化管疾患による出血,特に大腸癌からの出血のスクリーニング法として有用と考えられた.

Synthesis of adult-type hemoglobin in human erythremia cell line

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

Synthesis of Adult-Type Hemoglobin in Human Erythremia Cell Line

KMOE -2/05 cells, derived from a patient with acute erythremia, became benzidine-positive after the addition of cytosine arabinoside (CA). Radioimmunoassays using antihuman hemoglobin antibodies revealed an elevated amount of hemoglobin in the CA-exposed cells over that in the control cells (without CA). Isoelectric focusing of the CA-exposed cell lysate formed benzidine-positive foci in the positions of human adult Hb (HbA) and human fetal Hb (HbF). To determine the types of globin synthesized in the CA-exposed cells, globin chains internally labeled with [3H] leucine were purified by carboxy-methyl (CM)-Sephadex column chromatography, immunoadsorption by Sepharose-coupled antihuman Hb antibodies and Sephadex G-100. The labeled globin chains were finally separated by CM-cellulose chromatography in urea. Two distinct peaks of radioactivity were shown in exactly the same fractions as carrier human globin alpha- and beta-chains. These observations indicate that these KMOE -2/05 cells synthesize HbA.

Properties of goat anti-human hemoglobin antibodies fractionated on subunit affinity columns

The antibodies to human hemoglobin A, (HbA 1) in the sera of two goats were fractionated on the basis of their ability or inability to bind to the isolated α and β subunits of the hemoglobin. The fractionation was carried out by a series of affinity chromatography steps and the characterization of the binding of the antibody populations thus obtained to HbA 1, α and β revealed some predominating characteristics common to the two goat sera. In roughly proportional yields, the anti-HbA 1 in the sera were fractionated into three major catergories: antibodies which bind only to HbA 1 and β chains, antibodies which bind only to HbA 1 and α chains and antibodies which bind to HbA 1 and both α and β chains. One goat also had an antibody population that would bind HbA 1 but neither α nor β. There was, in both sera, a larger proportion of α binding control to β binding antibodies and of α−ß non-cross-reacting compared to α−ß cross-reacting antibodies. Upon consideration of the amino acid sequence and three-dimensional structures of both the hemoglobin antigen and the host's own hemoglobin with respect to sequence differences and the exposure and clustering of such differences, the existence and proportions of the diffrent antibody populations can be explained.

Immunochemical and physicochemical properties of haptoglobin, abtihemoglobin antibody, and their complexes with hemoglobin

The immunochemical properties of haptoglobin which binds specifically with hemoglobin were compared with those of antihemoglobin A 1 antibody. Hemoglobin-haptoglobin shows neither precipitation reaction nor complement fixation reaction under conditions in which hemoglobin-antihemoglobin antibody shows these immunological reactions. From these observations, the absence of lattive formation between hemoglobin and haptoglobin was suggested. Antihuman hemoglobin antibody binds deoxygenated human hemoglobin and hemoglobin-haptoglobin complex, both of which human haptoglobin does not. On the other hand, although human haptoglobin binds hemoglobins of horse, rabbit, and frog, antihuman hemoglobin antibody does not. And from the data on circular dichroism spectra of hemoglobin-haptoglobin complex and hemoglobin-antihemoglobin antibody complex, the differences in environmental structure around the heme between hemoglobin-haptoglobin complex and hemoglobin-antihemoglobin antibody complex are clarified which may have some connection with the differences in immunochemical reactions between these two complexes.