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HemogloBind™ Hemoglobin Depletion From Hemolyzed Serum/Plasma

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Product Code: H0145

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Safety Data Sheet

Hemoglobin Removal and Capture

Has a high degree of specificity for hemoglobin, without cross-reacting with other proteins or analytes
Suitable for
- Hemolyzed serum/plasma
- Whole Blood, Dried Blood Cards (DBS) or Erythrocyte lysates
- Tissue Homogenates, compatible with RIPA buffer
Applications in analytical interferences, enzyme monitoring, proteomics
Species Agnostic
Over 30 citations including analysis in cellular thermal shift assay (CETSA), LC-MS proteomics, Hemoglobin derivatives, Western blot, enzyme activity, ELISA
Related product NuGel™HemogloBind™ comes in a dry powder format, compatible with high throughput 96-well automation, see https://www.biotechsupportgroup.com/NuGel-HemogloBind-Hemoglobin-Capture-Reagent-p/np-ho.htm

Click Here To View HemogloBind™ Product Sheet

Click Here For The HemogloBind™ Hemoglobin Depletion Performance On Blood Sheet

Click Here For The HemogloBind™ Hemoglobin Depletion Performance On Dried Blood Spots/Cards

Poly-electrolytes are polymers with repeating units of stationary charges. HemogloBind™ is derived from insoluble elastomeric poly-electrolytes that bind proteins through an empirically derived chemistry combining elements of polymer composition, and cross-linking architecture. As with bio-polymers like DNA and Heparin, governing their reactivity is the spatial presentation of electrostatic groups along a flexible polymer chain.

HemogloBind™ does not cross react with most common serum components, making it an excellent tool in numerous applications. These include analytical protocols where optical interference is problematic. Hemoglobin variants, as in thalassemia, bind with differential affinity towards HemogloBind™, though this has not been fully evaluated. For purification and/or analysis of hemoglobin, a modest elevation in pH will facilitate desorption from the polymer.

Product	Size	Product Code
HemogloBind™	5ml	HO145-50
HemogloBind™	15ml	HO145-15
HemogloBind™	50ml	HO145-50

Hemolyzed Serum

Craig, J. R., et al. "A comparison of the anatomical and gastrointestinal functional development between gilt and sow progeny around birth and weaning." Journal of animal science (2019).

Biotech Support Group reports on an article describing the simplicity and efficiency of their hemoglobin depletion technology for improving ELISA analysis from hemolyzed pig serum. Gilt progeny (GP) often have restricted growth performance and health status in comparison to sow progeny (SP) from birth. To better understand underlying mechanisms, the study aimed to compare differences in growth and development between GP and SP in the first 24 h after birth and in the peri-weaning period. Because serum samples were quite hemolysed after collection and processing, it became necessary to use HemogloBind™ to allow for better detection of IgG by ELISA. The article states “As per the manufacturer’s instructions, 250 μL of Hemoglobind™ was added to 250 μL of hemolyzed serum, vortexed for 30 s before mixing via inversion for 10 min, and then centrifuged at 6,000 g for 2 min at 4°C. Purified serum was then aspirated and assessed in duplicate for IgG concentration … using a commercial ELISA kit (Bethyl Laboratories, Montgomery TX, USA).” The authors conclude their findings suggest that the early development of GP may be delayed compared to SP, and that a number of the anatomical differences between GP and SP that exist after birth are also present at weaning.

Reticulocytes

Nguyen, Anthony T., et al. "UBE2O remodels the proteome during terminal erythroid differentiation." Science 357.6350 (2017): eaan0218.

Biotech Support Group reports on a Science article describing the simplicity and efficiency of their hemoglobin depletion technology for improving LC-MS analysis from lysed reticulocytes.The mechanisms that drive reticulocyte transition to red blood cell in terminally differentiating cells remain largely unclear. During reticulocyte maturation, the proteome is remodeled through the programmed elimination of most generic constituents of the cell, in parallel with abundant synthesis of hemoglobin. The study used multiplexed quantitative proteomics to identify candidate substrates of UBE2O, an E2 (ubiquitin-conjugating) enzyme, in an unbiased and global manner. Because of the overly abundant presence of Hemoglobin, selective depletion of Hemoglobin was necessary. The article states “Reticulocytes were lysed by vortexing for 5 minutes at room temperature… An additional 10 bed vol of Hemoglobind™ suspension was added to the samples, which were then vortexed for another 10 min at room temperature followed by 4 min of centrifugation at 10000 x g. The supernatants, which contain hemoglobin-depleted sample, were … processed for TMT quantification.”. The authors concluded that the ubiquitin-proteasome system is not simply amplified during erythroid maturation, but suggest instead that it is broadly reconfigured to promote remodeling of the reticulocyte proteome. “The large amount of hemoglobin in reticulocytes presents a special challenge for LC-MS quantitative analysis. Here is another article demonstrating that HemogloBind™ can drastically reduce the interference associated with large concentrations of hemoglobin, without compromising the recovery and analysis of the underlying proteome.” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Hemocyte fractions

O’Connell, Grant C., et al. "Monocyte-lymphocyte cross-communication via soluble CD163 directly links innate immune system activation and adaptive immune system suppression following ischemic stroke." Scientific reports 7.1 (2017): 12940

The purpose of this study was to investigate CD163 as a possible effector of stroke-induced adaptive immune system suppression as this is a scavenger receptor expressed on innate immune cell populations. A soluble peptide with lympho-inhibitory properties can be shed from the plasma membrane via the metalloprotease ADAM17. The article states “Total hemocyte fractions were thawed, mixed with NP-40 lysis buffer (Thermo Fisher) at a 1 to 1 ratio, and depleted of hemoglobin via the HemogloBind hemoglobin removal kit (Biotech Support Group, Monmouth Junction, NJ)… ADAM17 activity was measured in 200 ug of total hemocyte lysate”. The authors provide novel evidence that the innate immune system employs protective mechanisms aimed at mitigating the risk of post-stroke autoimmune complications, and that CD163 is a key mediator of this phenomenon..

Perfusates

Laing, Richard W., et al. "The use of an acellular oxygen carrier in a human liver model of normothermic machine perfusion." Transplantation 101.11 (2017): 2746.

The authors present the first experience of using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of Normothermic machine perfusion of the liver (NMP-L). This is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. The article states “All perfusates underwent haemoglobin depletion using Hemoglobind (BioTech Support Group LLC, Monmouth Junction, NJ) as per the manufacturer’s instructions, except using a 1:8 ratio to ensure removal of all free haemoglobin. Perfusate levels of 8-OH-dG were quantified using a competitive ELISA (Abcam) as per the manufacturers protocol” .The authors conclusion is that Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

Hemolysate

Dasauni, Pushpanjali, et al. "Refractive index of blood is a potential qualitative indicator of hemoglobin disorder in humans." Journal of Proteins & Proteomics 9.3 (2018)
Qualitative defects of codons in hemoglobin chains causes hemoglobin disorders such as hemoglobin variants, thalassemia and sickle cell disease. Authors wanted to estimate the concentration of hemoglobin by measuring refractive index and find differences in the refractive index of blood samples. Blood was centrifuged to separate plasma. Hemoglobin is separated from hemolysate using Hemoglobind™ reagent. The refractive index was evaluated in whole blood, plasma, hemolysate and pure Hb.

Cell lysates of reticulocytes

Junqueira, Caroline, et al. "Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax–infected reticulocytes." Nature medicine (2018): 1.

Biotech Support Group reports on a recent research article describing the simplicity and efficiency of their hemoglobin depletion technology for improving Western blot analysis from reticulocytes.The Malaria parasite Plasmodium vivax causes approximately 100 million clinical cases yearly. The basis of protective immunity is poorly understood yet thought to be mediated by antibodies. The authors hypothesized that CD8+ T Cells in patient with P. vivax malaria might become activated by recognizing P. vivax-infected reticulocytes (iRetics), causing them to degranulate. To evaluate the expression levels of proteins involved in antigen presentation in reticulocytes by Western blot, the article states “Cell lysates of reticulocytes…were analyzed by immunoblot…after hemoglobin removal using HemogloBind™ (Biotech Support Group)”. The authors conclude that P. vivax renders reticulocytes granulysin-susceptible and that this unexpected T Cell defense might be mobilized to improve vaccines. “The large amount of hemoglobin in whole blood presents a special challenge for quantitative Western blot analysis. Here is another article demonstrating that HemogloBind™ can reduce the interference associated with large concentrations of hemoglobin, without compromising the recovery and analysis of the protein of interest.” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Whole blood aliquots

Pácal, Lukáš, et al. "Deleterious Effect of Advanced CKD on Glyoxalase System Activity not Limited to Diabetes Aetiology." International Journal of Molecular Sciences 19.5 (2018): 1517.

Biotech Support Group reports on a recent research article describing the describing the simplicity and efficiency of their hemoglobin depletion technology for improving Western blot analysis from whole blood.The authors studied Methylglyoxal production known to increase in diabetic patients. Methylglyoxal is efficiently detoxified by enzyme glyoxalase 1 (GLO1). The aim was to study the effect of diabetic and chronic kidney disease milieu on (a) GLO1 gene expression in peripheral blood mononuclear cells; (b) GLO1 protein levels in whole blood; and (c) GLO1 activity in RBCs in vivo in diabetic vs. non-diabetic subjects with normal or slightly reduced vs. considerably reduced renal function (CKD1-2 vs. CKD3-4). To evaluate the GLO1 protein levels in whole blood by Western blot, t he article states “For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions… ”. The article concludes that chronic kidney disease in advanced stages has prevailing and suppressive effects compared to hyperglycaemia. Chronic kidney disease decreases GLO1 gene expression and protein levels (together with diabetes) without concomitant changes of GLO1 activity.“The large amount of hemoglobin in whole blood presents a special challenge for quantitative Western blot analysis. Here is another article demonstrating that HemogloBind™ can reduce the interference associated with large concentrations of hemoglobin, without compromising the recovery and analysis of the protein of interest.” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

RBC Perfused Livers

Laing, Richard W., et al. "The use of an acellular oxygen carrier in a human liver model of normothermic machine perfusion." Transplantation 101.11 (2017): 2746-2756.

Hemoglobin removal from RBC perfused livers allows measurement of oxygen, metabolism, perfusion parameters in an ischemia reperfusion injury model to evaluate an hemoglobin based oxygen carrier (HBOC) called Hemopure in normothermic machine perfusion of the liver to preserve liver grafts.

Total hemocyte lysates

O'Connell, Grant C., et al. "Monocyte-Lymphocyte Cross-Communication Via Soluble CD163 Directly Links Innate Immune System Activation And Adaptive Immune System Suppression Following Ischemic Stroke." bioRxiv (2017): 144063.

CD163 is linked to peripheral immune response in stroke via the circulating levels of damage associated molecules, hormones and proinflammatory cytokines. Stroke can alter serum neutrophil ADAM17 dependent sCD163 production and lymphocyte proliferation. An ADAM17 activity assay was performed from hemoglobin depleted total hemocyte lysates. Measurement of protein concentrations of hemoglobin depleted total hemocyte lysates and cell culture lysates was obtained by a DC-protein assay.

Serum

Nishida-Aoki, Nao, et al. "Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis." Molecular Therapy 25.1 (2017): 181-191.

This study considers that therapeutic strategies targeting cancer-derived extracellular vesicles (EVs) hold

great promise because of the possibility they reposition microenvironments to accommodate metastasis. The researchers report on a novel strategy of therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model. The article states “Hemoglobin was accumulated with HemogloBind beads (Biotech support group, Monmouth Junction NJ, USA) followed by 0.22 μm filtration. Then, the EVs in the sera were concentrated by ultracentrifugation…”. The authors conclude that therapeutic antibody administration effectively suppresses EV-triggered metastasis and that the elimination of cancer-exosome derived EVs could be a novel strategy for therapy

Bronchoalveolar lavage fluid (BALF)

Maneesh Bhargava, Kevin J. Viken, Sanjoy Dey, Michael S. Steinbach, Baolin Wu, Pratik D. Jagtap, LeeAnn Higgins, Angela Panoskaltsis-Mortari, Daniel J. Weisdorf, Vipin Kumar, Mukta Arora, Peter B. Bitterman, David H. Ingbar, Chris H. Wendt. Proteome Profiling in Lung Injury after Hematopoietic Stem Cell Transplantation. Biology of Blood and Marrow Transplantation 22 (2016) 1383-1390.

The authors consider pulmonary complications due to infection and idiopathic pneumonia syndrome (IPS) in hematopoietic stem cell transplant (HSCT) recipients. For this, a proteomic characterization of global bronchoalveolar lavage fluid (BALF) was studied to identify proteins and pathways that differentiate IPS from infectious lung injury after HSCT. Because the BALF samples appeared tinged with blood, the authors tested HemogloBind™ to determine whether the removal of hemoglobin (in addition to high-abundance proteins) would improve the depth of coverage. The article states “Hemoglobin removal improved protein identification to 845 proteins at 1% global FDR compared with 496 proteins with high-abundance protein depletion alone”. The authors conclude that the protein expression differences provide insights into mechanisms activated in lung injury and suggest potential therapeutic targets to augment lung repair.

Featured Application - Bilirubin Analyses

Parvathi S. Kumar, Haree K. Pallera, Pamela S. Hair, Magdielis Gregory Rivera, Tushar A. Shah, Alice L. Werner,Frank A. Lattanzio, Kenji M. Cunnion, and Neel K. Krishna. Peptide inhibitor of complement C1 modulates acute intravascular hemolysis of mismatched red blood cells in rats.TRANSFUSION Volume 00, May 2016. doi:10.1111/trf.13674.

In brief, the study evaluated the role of the a peptide inhibitor of complement C1 (PIC1) in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios. The authors state "To remove free Hb that may cause optical interference in bilirubin analysis, we treated all the samples with Hb depletion from hemolyzed serum/plasma (HemogloBind, Biotech Support Group). Bilirubin concentration was then measured with a Bilirubin Assay Kit (Sigma-Aldrich, St. Louis, MO)."

Krishna, Neel K., and Kenji Cunnion. "Derivative Peptide Compounds and Methods of Use." U.S. Patent No. 20,160,376,322. 29 Dec. 2016.

The invention describes synthetic peptide compounds and uses for therapy and diagnostics of complement-mediated diseases, such as inflammatory diseases, autoimmune diseases, and microbial infections; and non-complement-mediated diseases, such cystic fibrosis and various acute diseases. The patent states “Due to large amounts of hemolysis in the latter time points and the associated optical interference in bilirubin analysis, all the samples were pre-treated with HemogloBind™ (Biotech, N.J.) prior to analysis with the Bilirubin Assay Kit”

Whole Blood

Chalásová, Katarína, et al. "Transketolase Activity but not Thiamine Membrane Transport Change in Response to Hyperglycaemia and Kidney Dysfunction." Experimental and Clinical Endocrinology & Diabetes (2017).

Biotech Support Group reports on a recent research article describing the simplicity and efficiency of their hemoglobin depletion technology for improving western blot analysis from whole blood protein. Diabetic kidney disease, a common complication of both type 1 and type 2 diabetes, is associated with significant morbidity and mortality, and represents the most common cause of chronic kidney disease. The study hypothesized that protective pentose phosphate pathway action in diabetes might be compromised by limited intracellular availability of an active transketolase cofactor thiamine diphosphate (TDP). To evaluate the levels of thiamine tranporter proteins in whole blood, the article states “For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group) according to manufacturer’s instructions…”. The article concludes that both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while transketolase activity in parallel with intracellular TDP was increased in chronic kidney disease patients with or without diabetes.

Lahut, Suna, et al. "Blood RNA biomarkers in prodromal PARK4 and REM sleep behavior disorder show role of complexin-1 loss for risk of Parkinson's disease." Disease Models & Mechanisms (2017): dmm-028035

Biotech Support Group reports on a recent research article describing the simplicity and efficiency of their hemoglobin depletion technology for improving the signal from whole blood protein immunoblot analysis. In this study, Parkinson’s disease progression is investigated through the accumulation and aggregation of the lipid-binding SNARE complex component alpha-synuclein (SNCA) which underlies vulnerability and defines its stages. The authors studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation), to identify effects of SNCA gain-of-function as potential disease biomarkers. The article states “For protein extraction from the EDTA tubes, 300 μl blood were lysed with equal amount of 1% SDS-RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and one tablet Complete Protease Inhibitor Cocktail (Roche)] and sonicated for 10 sec. The blood lysates were rotated at 4 °C for 30 min and centrifuged at 4 °C for 30 min. The supernatants were depleted in hemoglobin content using a commercial kit (HemogloBind, Biotech) following the manufacturer’s instructions”. After hemoglobin depletion, immunoblot analysis identified that PARK4 blood showed upregulation of alpha-synuclein monomer, with no high molecular weight aggregates. “Because of the large amount of hemoglobin in whole blood, its very exciting to see that HemogloBind™ can reduce the presence of hemoglobin sufficient for such precise immunoblot analysis.” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Thomas H. Snider, Kevin G. McGarry, Michael C. Babin, David A. Jett, Gennady E. Platoff Jr., and David T. Yeung. Acute Toxicity of Phorate Oxon by oral gavage in the Sprague-Dawley rat. Fundam. Toxicol. Sci. Vol. 3, No.5, 195-204, 2016.

The article describes studies of the oral toxicity of Phorate Oxon with emphasis on gender and age-related effects in rats. The authors used HemogloBind™ as part of the protocol to measure cholinesterase activity. The article states “…80 µl aliquots of whole blood were removed, diluted and treated with HemogloBind™ to remove hemoglobin for later assay to detect cholinesterase activity…

Macromolecular Complexes

C Wan, B Borgeson, S Phanse, F Tu, K Drew, G Clark, et al. Panorama of ancient metazoan macromolecular complexes. Nature Volume:525, Pages:339–344 Date published:(17 September 2015). doi:10.1038/nature14877

Two of BSG products, NRicher™ 6 and HemogloBind™, were able to contribute to this rigorous examination of protein complexes. When our products were used as a pretreatment step in the overall workflow, about twice the number of observations and annotations became possible. This further validates that the sub-proteome bias characteristics of NRicher™ 6 can simplify complex proteomes into less complex sub-proteomes with efficiencies suitable for deep functional proteome characterization. Furthermore, this study demonstrated the importance of a key feature implicit to all of our products; that is the maintenance of functional and structural integrity after separations. Without that particular feature, these additional observations would not have been possible.

Human Peripheral Blood Mononuclear Cells (PBMCs)

Rubio-Navarro, Alfonso, et al."Hemoglobin induces monocyte recruitment and CD163-macrophage polarization in abdominal aortic aneurysm."International Journal of Cardiology (2015)."

A research article by authors Rubio-Navarro, Alfonso, et al in the journal International Journal of Cardiology (http://www.internationaljournalofcardiology.com/article/S0167-5273(15)30284-9/fulltext) cites Biotech Support Group’s HemogloBind™ sample preparation reagent to deplete hemoglobin (Hb) from conditioned medium from healthy aortas or abdominal aortic aneurysm. The article states:"Conditioned mediums from AAA were incubated with HemogloBind™ reagent for hemoglobin depletion"

In brief, authors describe high infiltration of CD163 monocytes surrounding micro-vesicles, low expression of CD14+ & CD16- monocytes and high CD163 mRNA/protein expression is a feature of abdominal aortic aneurysm (AAA) molecular pathology. Healthy aorta conditioned medium or complete or hemoglobin-depleted conditioned medium from abdominal aortic aneurysm were mixed with M-CSF macrophages to track CD163 and HLA-DR expression or hemoglobin uptake.

“We are pleased with this data on abdominal aortic aneuryism & hemoglobin proteomics. HemogloBind™ minimizes hemoglobin interference from blood samples and allows research on cardiovascular pathology” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Dried Blood Spot(DBS)/Whole Blood

Hakuna, Lovemore, et al. "A simple assay for glutathione in whole blood."Analyst (2015).

A research article in the journal Analyst ( http://pubs.rsc.org/en/content/articlelanding/2015/an/c5an00345h) cites Biotech Support Group’s HemogloBind™ sample preparation reagent to deplete hemoglobin (Hb) from whole blood samples containing glutathione to minimize interference from Hb in GSH fractions. Using a resorufin-acrylate fluorescent probe, GSH is quantitated in deproteinzed blood plasma and whole blood samples.

The article states:
"Apart from dilution, Hb can be removed using a commercial product, HemogloBind™, which can isolate and remove up to 90% of blood Hb." Glutathione (GSH) is an antioxidant involved on nitric oxide regulation, covalent hemoglobin binding, DNA binding, leukotriene synthesis, protein synthesis and sepsis pathways. GSH is elevated in cancer tissues and proper minimally invasive GSH sample preparation from dried blood samples (DBS) allows research on neurodegenerative diseases, chronic respiratory diseases and diabetes.

Whole Blood

Snider, Thomas H., Christina M. Wilhelm, Michael C. Babin, Gennady E. Platoff Jr, and David T. Yeung. "Assessing the therapeutic efficacy of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig."The Journal of Toxicological Sciences 40, no. 6 (2015): 759-775.

Acetylcholine is an essential neurotransmitter, and inhibitors of cholinesterases(ChEs) are potent toxins. A primary component of anti-organophosphorus therapy is an oxime reactivator to rescue inhibited acetylcholinesterases. For this, clinical signs of toxicity can be measured from blood cholinesterase [Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] activity utilizing a modified Ellman's method. Biotech Support Group’s unique solid-phase polymer for hemoglobin depletion, was used for pretreatment. The article states “Briefly, whole blood samples were treated with HemogloBind™ which interferes with the ChE activity assay due to spectral overlap.”

Brittain, Matthew K., Kevin G. McGarry, Robert A. Moyer, Michael C. Babin, David A. Jett, Gennady E. Platoff, and David T. Yeung. "Efficacy of Recommended Prehospital Human Equivalent Doses of Atropine and Pralidoxime Against the Toxic Effects of Carbamate Poisoning in the Hartley Guinea Pig." International journal of toxicology (2016): 1091581816638086.

The article states “Whole blood samples were processed and analyzed as described by McGarry et al.10 Briefly, whole blood samples were treated with HemogloBind to remove hemoglobin, which interferes with the ChE activity assay due to spectral overlap. To prepare the HemogloBind treated blood samples for ChE activity analysis, samples were diluted 2-fold in assay buffer (1 PBS). Subsequently, samples were diluted an additional 2-fold into the test plate by adding 100 mL of sample to a total volume of 200 mL in each well of a 96-well plate. Cholinesterase activity was assessed using a spectrophotometric assay conducted in a manner similar to Ellman et al,11 as described in the in vitro reactivation section above. The relative AChE activity level for each animal (RAAChE) was defined as the ATC turnover rate in the terminal blood sample divided by that in the same animal’s baseline blood sample. A similar calculation was performed using butyrylthiocholine (BTC) turnover rates to determine RABChE.”

Blood Plasma

Johns, Michael, et al. "SR-135, a peroxynitrite decomposing catalyst, enhances β-cell function and survival in B6D2F1 mice fed a high fat diet." Archives of Biochemistry and Biophysics (2015).

A research article in the journal Archives of Biochemistry & Biophysics( http://www.sciencedirect.com/science/article/pii/S0003986115001988) cites Biotech Support Group's HemogloBind™ sample preparation reagent to deplete hemoglobin (Hb) from lysed red blood cells. Authors cite peroxynitrite decomposing catalysts such as Mn(III) bis(hydroxyphenyl)-dipyrromethene complexes as important molecules in obesity sample preparation & development of anti-diabetic agents. SR-135 and it's analogs are synthesized to decompose peroxynitirie. In addition, authors provide experiment data on prevention of nitration assay, rat islet uptake, quantitation of tyrosine nitration, beta-cell area quantitation, glucose-stimulated insulin secretion and plasma insulin level detection. The article states: "Blood plasma (50 µl) was mixed with HemogloBind (50 µl) to remove hemoglobin from lysed red blood cells."Plasma hemoglobin is depleted using Biotech Support Group's HemogloBind™ to extract hemoglobin from lysed red blood cells. Plasma concentration of high-density lipoprotein (HDL), total cholesterol and triacylglycerol (TAG) is obtained. Precipitation using polyethylene glycol (PEG) of beta-lipoproteins, very low-density lipoprotein (VLDL) and low-density lipoprotein(LDL) from HDL fractions is performed."We are pleased with this research on HemogloBind™ as interference is minimized and concentration of cholesterol proteins is obtained." states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Lung Tissue Specimens & Pulmonary Research (Acute Respiratory Distress Syndrome & Acute Hypoxic Failure)

Bhargava, Maneesh""Proteomic Studies in Acute Hypoxic Respiratory Failure." PhD diss., UNIVERSITY OF MINNESOTA. (2015).

Protein expression in the bronchoalveolar lavage fluid (BALF) from subjects with acute respiratory distress syndrome (ARDS) was evaluated. The BALF samples were processed by desalting, concentration and removal of high abundance proteins. The protein fractions were trypsin digested and labeled with the iTRAQ reagent for mass spectrometry (MS). The thesis states “BALF samples containing at least 1.2 mg of proteins were processed for LCMS/MS…concentrated and desalted using Amicon 3-MWCO filters. Hemoglobin depletion was performed with HemogloBind™ (Biotech Support Group LLC, Monmouth Junction, NJ) per the manufacturer's instructions.”

Red Cell Lysates

Kyoungsook Park, Christopher D. Saudek, and Gerald W. Hart Increased Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals. Diabetes.2010;59(7):1845-50.

Erythrocyte proteins are highly O-GlcNAcylated. In individual with pre-diabetes and diabetes, the level of O-GlcNAcase expressed significantly increases. From serum samples, erythrocyte proteins were extracted and hemoglobin was depleted followed by sonication and centrifugation. From the red blood cell lysates hemoglobin is efficiently depleted using HemogloBind™ from Biotech Support Group. Because HemogloBind™ is engineered for a high degree of selectivity and does not cross react with most common serum components, subsequent analysis of O-GlcNAcylation process in erythrocyte proteins is done by Western blotting using an O-GlcNAc specific antibody. Finally, the study of O-GlcNAcase allows for developing, validating, and qualifying biological markers that are compared with the level of A1C.

Stored Blood Products

Delobel J., Rubin O., Prudent M., Crettaz D., Tissot J.-D., Lion N. Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues. International Journal of Molecular Sciences.2010;11(11):4601-4617

Authors Delobel et al cited Hemoglobind™'s application for hemoglobin depletion from the paper by Alvarez-Llamas et al, A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE. Electrophoresis, 30: 4095–4108. For biomarker discovery from erythrocyte proteome samples of erythrocyte concentrates, platelet concentrates and fresh frozen plasma blood products are used in proteomic analysis. This paper reviews the importance of standardizing sample preparation steps and controlling pre-analytical factors to identify proteins from cytosolic or membrane fractions. After using Hemoglobind™, one dimensional (sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS-PAGE) and 2D electrophoresis are implemented to identify unique proteins from by MALDI-TOF MS analysis.

Red Blood Cells(RBC)/Forensic Research

Danielson, Phillip B. "Isolation of Highly Specific Protein Markers for the Identification of Biological Stains: Adapting Comparative Proteomics to Forensics." (2011).

Biotech Support Group reports on a technical report prepared by the U.S. Department of Justice. The central goal of the project was to isolate and identify candidate protein biomarkers that are highly specific to individual types of biological stains of forensic utility (i.e., saliva, semen, peripheral blood, menstrual blood, vaginal secretions, and urine).

The report is entitled “Isolation of Highly Specific Protein Markers for the Identification of Biological Stains:Adapting Comparative Proteomics to Forensics". The National Criminal Justice Reference Service (NCJRS) has made this federally funded grant final report available electronically at: https://www.ncjrs.gov/App/Publications/abstract.aspx?ID=258706 The report states:

“Serum obtained from menstrual blood samples was typically contaminated with erythrocyte cellular components due to the lysing of fragile red blood cells that are abundant in the endometrial lining during menses. As hemoglobin comprises 32-36% of all the proteins found in red blood cells the serum from menstrual blood samples contained large quantities of hemoglobin which served to mask the detection of less abundant menstrual blood specific proteins. For this reason, hemoglobin was removed from collected serum prior to proteome fractionation through use of HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ). This hemoglobin capture reagent is a solid-phase, non-ionic adsorbent product that binds
specifically to hemoglobin allowing for the removal of 80-90% of hemoglobin from serum or red cell lysates. HemogloBind™ does not cross react with most common serum components, making it suitable for the proteomic applications of this research project.”“Here we find another use for HemogloBind™, further validating our unique surface technology approach, not based on biologicals, as being highly selective and an efficient method for the depletion of hemoglobin” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

Red Blood Cells (RBC) - Blood.

Carvalho, Ana Sofia, Manuel S. Rodriguez, and Rune Matthiesen. "Red Blood Cells in Clinical Proteomics." Serum/Plasma Proteomics: Methods and Protocols (2017): 173-181.

Fractionation into RBC membrane and soluble cytosolic fractions may require hemoglobin depletion. RBC are important for oxygenation, carbon dioxide transport and the release of survival and growth factors. RBC's are linked to cancer pathology, sleep apnea, sickle cell disease, malaria, chronic obstructive pulmonary disease and parasites such as eimeria, babesia, cryptosporidium, toxoplasma, theileria etc. Mass spectrometry of the RBC membrane proteome can be performed after sample preparation.

Cholinesterase Analysis for Evaluating Oxime Therapies.

Christina M. Wilhelm, Thomas H. Snider, Michael C. Babin, David A. Jett, Gennady E. Platoff Jr., David T. Yeung. A comprehensive evaluation of the efficacy of leading oxime therapies in guinea pigs exposed to organophosphorus chemical warfare agents or pesticides. Toxicology and Applied Pharmacology Available online 31 October 2014. doi:10.1016/j.taap.2014.10.009

Acetylcholine is an essential neurotransmitter, and inhibitors of cholinesterases(ChEs) are potent toxins. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection against chemical warfare agents or pesticides. Clinical signs of toxicity were observed for 24 h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellman's method. In the modified Ellman's enzymatic assay for evaluating ChE activity, HemogloBind™, Biotech Support Group's unique solid-phase polymer for hemoglobin depletion, was used for pre-treatment. The article states "Terminal blood samples were collected and processed for all survivors using HemogloBind™".

Barasa, Benjamin, and Monique Slijper. " Challenges for red blood cell biomarker discovery through proteomics." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1844.5 (2014): 1003-1010

Biotech Support Group reports on a recent review article which describes the simplicity and efficiency of their proteomic sample preparation technology for selectively depleting hemoglobin, to help solve the dynamic range problem for comprehensive erythrocyte proteome analysis. The citation is: Barasa, Benjamin, and Monique Slijper. " Challenges for red blood cell biomarker discovery through proteomics." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1844.5 (2014): 1003-1010.In brief, this review describes the many challenges to generate in-depth RBC proteome analysis, such as to obtain pure red blood cells, and to acquire an in-depth proteome, despite the dynamic range problem due to a few highly over-represented RBC proteins – especially hemoglobin which accounts for approximately 97% of the cytosolic mass. The article states "Hemoglobin can also be depleted from an RBC lysate by employing Hemoglobind- [39] or HemoVoid [40] affinity systems. Hemoglobind consist of an elastomeric poly-electrolytic surface that has been optimized to bind Hb from serum samples with high affinity, and can as well be used to remove Hb from RBC lysates [39]. Walpurgis et al. used a complex matrix to deplete the RBC sample for Hb, named HemoVoid, which is made of a library of different ligand combinations, consisting of several kinds of ionic, aromatic, and polymer ligands [40]. Low abundance proteins in the RBC lysate are captured and enriched by the HemoVoid ligand library, while the high abundance proteins such as Hb and CA-I are thought to quickly saturate the system, and they primarily end up in the flow-through. The high abundance proteins in an RBC lysate can thus be easily separated from the low abundance protein fraction. The chosen Hb-depletion approaches by both Alvarez-Llamas et al. [39] and Walpurgis et al. [40] are well compatible with analysis of the RBC protein fractions by 1D or 2D gel electrophoresis, followed by protein identification through mass spectrometry."
"It is worthwhile to note that the authors describe both our strategies for hemoglobin depletion, as the correct choice will vary with the application. With our own experience and with users such as those referenced in this article, we have gained the necessary knowledge to guide our users to the best option for hemoglobin depletion and/or low abundance enrichment" states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

References Acknowledged in the Review
1.[39] G. Alvarez-Llamas, F. de la Cuesta, M.G. Barderas, V.M. Darde, I. Zubiri, C. Caramelo, F. Vivanco A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE Electrophoresis, 30 (2009), pp. 4095–4108.

[40] K. Walpurgis, M. Kohler, A. Thomas, F. Wenzel, H. Geyer, W. Schanzer, M. Thevis Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS Electrophoresis, 33 (2012), pp. 2537–2545

Hikosaka, Keisuke, et al. " Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3 (Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocyte" Journal of Biological Chemistry(2014): jbc-M114.

Authors Hikosaka et al describe research on nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) from red blood cells and its regulation of nicotinamide adenine dinucleotide. The cytoplasmic fraction consisted of hemolyzed samples obtained from the supernatant and ghost for membrane fraction. An alpha cellulose & microcrystalline cellulose column for depleting platelets and leukocytes from heparinized whole blood for an assay of RBC metabolic enzymes is described. Extraction of metabolites from RBCs and NAD related metabolites from whole blood by perchloric acid method for LC-MS/MS measurement is done. Measurement of metabolite levels involved using MS and HPLC, quantitative analysis software and separations using a column. The article states "...Each sample was normalized by hemoglobin concentration at 20 µg/µl, and then hemoglobin was depleted using HemogloBind™".

McGarry, Kevin G., et al. " Evaluation of HemogloBind™ treatment for preparation of samples for cholinesterase analysis." (2013). Advances in Bioscience and Biotechnology, 2013, 4, 1020-1023

In this article, measurement of cholinesterase activity prior to depletion and after removing hemoglobin is performed. A comparison of total cholinesterase activity with Ellman method and after Hemoglobind™ treatment prior to Ellman method did not display a statistical difference in mean ChE activity. Ellman's assay invovled measuring the sum of RBC membrane ChE activity and plasma ChE activity. Total cholinesterase activity of whole blood samples with Hemoglobind™ treatment prior to Ellman method is also consistent. Moreover, the Hemoglobind™ protocol is simpler with one incubation and short, low speed centrifugation.This article further validates our unique surface technology approach, not based on antibodies or engineered bio-ligands, as being highly selective and an efficient method for the depletion of hemoglobin concurrent with the recovery of functional activity.

Alvarez-Llamas, Gloria, Fernando de la Cuesta, Maria G. Barderas, Irene Zubiri, Maria Posada-Ayala, and Fernando Vivanco. " Characterization of Membrane and Cytosolic Proteins of Erythrocytes." In Vascular Proteomics, pp. 71-80. Humana Press, 2013.

Proteomic profiling of erythrocyte proteins to identify novel proteins linked to diseases is an evolving field of clinical proteomics. Cytosolic proteins could contribute to pathology of diseased erythrocytes. Hemoglobin interferes with LC-MS/MS analysis of low abundance cytosolic proteins. Hemoglobin depletion of cytosolic proteins is essential for proteomic sample preparation. Authors Gloria Alvarez-Llamas et al published a chapter in the book Vascular Proteomics titled, Characterization of membrane and cytosolic proteins of erythrocytes, which cites HemogloBind™ from Biotech Support Group for hemoglobin depletion of erythrocyte cells from the cytosolic fraction. A simple method of hemoglobin depletion using HemogloBind™ protocol allows for subsequent downstream proteomic analysis using 2-DE as it reduces major interference of hemoglobin from samples of red blood cells (RBCs) and identifies proteins.

Alvarez-Llamas, G., de la Cuesta, F., Barderas, M. G., Darde, V. M., Zubiri, I., Caramelo, C., Vivanco, F. A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE. Electrophoresis.2009;30:4095-4108

Authors in this study focused on the analysis of human cytosolic and membrane sub-proteomes. Hemoglobin from samples of red blood cells was studied using different strategies for isolation of the membrane and cytosolic fractions to determine the influence it has on proteome profiling by 2-DE and hemoglobin removal. Biotech Support Group's hemoglobin depletion reagent, HemogloBind™ was successfully used to erythrocyte cells. The results showed that Hemoglobind™ does have a high degree of specificity for hemoglobin and minimal interference. Particularly interesting is how authors developed a novel combined strategy based on hypotonic lysis isolation for identification of high molecular weight proteins (i.e. spectrin, ankyrin) by nano-LC coupled to an LTQ-Orbitrap mass spectrometer.

Zihao Wang, Kyoungsook Park, Frank Comer1, Linda C. Hsieh-Wilson, Christopher D. Saudek, Gerald W. Hart. Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus. Diabetes.2008;58, 309-317.

O-GlcNAc actively cycles on erythrocyte, regulates insulin signaling and is a mediator of glucose toxicity. Therefore studying it may reveal potential biomarker for diagnoses of diabetes. Highly efﬁcient enrichment methods based Hemoglobind™ overcome the challenges of low stoichiometry, suppressed ionization efﬁciency in presence of unmodiﬁed peptides, and intrinsic lability in gas phase mass spectrometric methods. In this paper, authors used Hemoglobind™ to study erythrocyte proteins and compared it with their abundance between normal and diabetic samples proteins. Blood samples were obtained from normal and diabetic patients collected into a vial containing EDTA and OGlcNAcase inhibitor PUGNAc. Next the researchers fractionated the blood cells to isolate erythrocytes. After erythrocytes were lysed and centrifuged, the supernatant containing hemoglobin was partially depleted by HemogloBind™ from Biotech Support Group.

Datta, Pradip. Effect of Hemolysis, High Bilirubin, Lipemia, Paraproteins, and System Factors on Therapeutic Drug Monitoring. Handbook of Drug Monitoring Methods.2008; 97-109.

Bilirubin, hemoglobin, lipids, paraproteins are endogenous interferents of immunoassays used in clinical laboratories which affect therapeutic drug monitoring (TDM), drugs of abuse (DAU) testing, and toxicology assays. Hemoglobin interference is caused by its absorption, fluorescence and chemiluminescence properties. Often assays are repeated with different methods or by removing the interferent from the sample. In this book chapter, authors Datta et al cited HemogloBind™ the synthetic solid phase anionic polyelectrolyte from Biotech Support Group for hemoglobin depletion and reduction of matrix effects.

Yuichi Miki, Tomoki Tazawa, Kazuya Hirano, Hideki Matsushima, Shoko Kumamoto, Naotaka Hamasaki, Tomohiro Yamaguchi, Masatoshi Beppu. Clearance of oxidized erythrocytes by macrophages: Involvement of caspases in the generation of clearance signal at band 3 glycoprotein. Biochemical and Biophysical Research Communications.2007; 363(1):57-62

Reduction of erythrocyte clearance by macrophages happens when oxidative stress is decreased either by pretreatment with Hemoglobind™ or enzymes inhibiting caspases causing decreased band 3 aggregation. Band 3 aggregation increased by actions of caspases and was reduced by treatment with caspase inhibitors Z-VAD-fmk or Z-DQMD-fmk (caspase 3 selective) prior to oxidation. Pretreatment of erythrocytes exposed to H2O2 have increased propensity to bind and get phagocyted by macrophages. In this paper authors used anti-band 3 serum to reduce binding and pretreatment of erythrocytes with Hemoglobind™ & polylactosamine-cleaving enzyme.

Sarawathi,et al., Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry, Biochim Biophys Acta. 1999 Feb2; 1426(3):483-90.

Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using Hemoglobind™ gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies.

References

Red Blood Cells

Chen, Yaozhen, et al. "Red blood cells undergo lytic programmed cell death involving NLRP3." Cell (2025).

Hemolyzed Serum Analyses

Neil Adrian P. Ondevilla, Peng-Wen Liu, Wan-Ting Huang, Tzu-Ping Weng, Nan-Yao Lee, Syu-Cing Ma, Jian-Jang Huang, Tak-Wah Wong, Hsien-Chang Chang, A point-of-care electrochemical biosensor for the rapid and sensitive detection of biomarkers in murine models with LPS-induced sepsis , Biosensors and Bioelectronics, Volume 254, 2024, 116202, ISSN 0956-5663, https://doi.org/10.1016/j.bios.2024.116202.

The article states “… Hemolysis is common in blood samples, which can interfere with the detection. Prior to the analysis, the hemolyzed samples were pretreated for 15 min with Hemoglobind™… ”

Nishida-Aoki, Nao, et al. "Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis. " Molecular Therapy 25.1 (2017): 181-191. http://dx.doi.org/10.1016/j.ymthe.2016.10.009

The researchers report on a novel strategy of therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model. The article states “Hemoglobin was accumulated with HemogloBind™ beads… EVs in the sera were concentrated by ultracentrifugation…”.

Red Cell Lysates

Barakat, Amey, et al. "Effects of 2, 3‐DPG knockout on SCD phenotype in Townes SCD model mice ." American Journal of Hematology (2023).

For Western blot analysis, the article states “Red blood cells were lysed by vortexing and hemoglobin was depleted using HemogloBind™.”

Chen, Yaozhen, et al. " https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4626203" Available at SSRN 4626203.

To investigate the downstream signaling of caspase-8 in mature RBCs, label free proteomic analysis was conducted. The article states “RBC lysates (control and complement-treated groups) … Hemoglobin was removed by adding 250 μl of HemogloBind suspension (Biotech Support Group) to the cell lysate.… Subsequently, samples without hemoglobin were analyzed using chromatography-tandem mass spectrometry (LC-MS/MS)”. This analysis identified proteins with both upregulated and downregulated expressions in response to complement activation.

Sae-Lee, Wisath, et al. "The protein organization of a red blood cell. " Cell Reports 40.3 (2022): 111103.

“ For hemolysate preparations, remnant white ghosts were removed by centrifugation at 21,000 ×g for 40 min at 4°C, and the supernatant treated with HemogloBind (Biotech Support Group) in order to bind and remove free Hgb.”

Zhan, Jing, et al. "Silica nanoparticles trigger phosphatidylserine exposure in red blood cells and induce thrombosis risk ." Environmental Pollution (2023): 121591.

For Western blot analysis, the article states “To evaluate the phosphorylated ERK1/2 and total ERK1/2, the protein samples from different groups were mixed with 100 μL of HemogloBind™ suspension (Biotech Support Group LLC, NJ, USA) that could specifically adsorb hemoglobin”.

Hojo-Souza NS, de Azevedo PO, de Castro JT, Teixeira-Carvalho A, Lieberman J, et al. (2020) Contributions of IFN-γ and granulysin to the clearance of Plasmodium yoelii blood stage . PLOS Pathogens 16(9): e1008840. https://doi.org/10.1371/journal.ppat.1008840.

The authors investigated how Plasmodium infection induces MHC-I expression on Retics. To remove interferences associated with Hemoglobin, the article states, “For western blot analysis, erythroblasts pellets were resuspended in RIPA Buffer (Sigma)…. The Retics were treated with HemogloBind …”.

Dziekan, Jerzy Michal, et al. "Cellular thermal shift assay for the identification of drug–target interactions in the Plasmodium falciparum proteome. " Nature Protocols (2020): 1-41.

The cellular thermal shift assay (CETSA) enables proteome-wide target screening for unmodified compounds with undetermined mechanisms of action, providing quantitative evidence about direct drug–protein interactions. The workflow involves treatment of P. falciparum–infected erythrocytes with a compound of interest, heat exposure to denature proteins, soluble protein isolation, enzymatic digestion, peptide labeling with tandem mass tags, offline fractionation, and LC-MS analysis. The article states “The intact-cell CETSA protocol features a HemogloBind-based sample processing step, which provides a relatively fast, reliable and inexpensive method to deplete >90% of hemoglobin from processed intact-cell samples. As a result, it leads to a 40-50% increase in the number of peptide spectrum matches (PSMs) ...”.

Nguyen, Anthony T., et al. "UBE2O remodels the proteome during terminal erythroid differentiation ." Science 357.6350 (2017): eaan0218. This study used multiplexed quantitative proteomics to identify candidate substrates of UBE2O, an E2 (ubiquitin-conjugating) enzyme, in an unbiased and global manner. Because of the overly abundant presence of Hemoglobin, selective depletion of Hemoglobin was necessary. The article states “Reticulocytes were lysed by vortexing for 5 minutes at room temperature… An additional 10 bed vol of Hemoglobind™ suspension was added to the samples, which were then vortexed for another 10 min at room temperature followed by 4 min of centrifugation at 10000 x g. The supernatants, which contain hemoglobin-depleted sample, were … processed for TMT quantification.”.

Whole Blood Lysates

de Boni, Laura, et al. " Aggregation-resistant alpha-synuclein tetramers are reduced in the blood of Parkinson’s patients ." EMBO Molecular Medicine (2024): 1-18. Synucleinopathies such as Parkinson’s disease (PD) are defined by the accumulation and aggregation of the α-synuclein protein in neurons, glia and other tissues. In this study, an in vitro-cross-linking protocol for human EDTA-whole blood was used to determine the relative levels of disordered and higher-ordered multimeric forms of cytosolic α-synuclein in blood. The protocol incorporated HemogloBind™ to remove interference from Hemoglobin.

Das, Amaresh, et al. "Enhanced Recovery and Detection of Highly Infectious Animal Disease Viruses by Virus Capture Using Nanotrap® Microbiome a Particles. " (2024).

This study reports the use of Nanotrap® Microbiome A Particles (NMAPs) to capture and concentrate viruses from diluted suspensions to improve their recovery and sensitivity of detection. Five highly infectious animal disease viruses were used in this study. NMAPs were used to capture spiked viruses from EDTA whole blood (EWB). Virus capture from EWB was partially blocked, most likely by hemoglobin (HMB), which also binds NMAPs and outcompetes the viruses. The interference effect from hemoglobin could be removed by first using

HemogloBind™ (Biotech Support Group; Monmouth Junction, NJ), without interfering with virus capture.

Kaneko, Tomonori, et al. "System-wide hematopoietic and immune signaling aberrations in COVID-19 revealed by deep proteome and phosphoproteome analysis. " Research Square preprint (2021). The author’s goals were to gain systems-level insights into SARS-CoV-2 pathogenesis. For that, they compared the blood proteome and phosphoproteome of ICU patients with or without SARS-CoV-2 infection, and healthy control subjects by quantitative mass spectrometry. To remove the highly abundant amount of Hemoglobin, the article states “Hemoglobin was depleted from PBMC whole cell lysate samples according to HemogloBind^TM manufacturer instruction with modifications.”

Leitner, Dominique F., et al. " Metabolomic, Proteomic, and Transcriptomic Changes in Adults with Epilepsy on Modified Atkins Diet. " Epilepsia (2023).

For Plasma Metabolomics, the article states “Whole blood was thawed on ice and processed to remove hemoglobin by NuGel-HemogloBind according to manufacturer protocol…”

Kaneko, Tomonori, et al. "Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients." (2023). The article states “For Sample processing for proteomics by mass spectrometry,… Hemoglobin was depleted from PBMC whole cell lysate samples according to HemogloBind (Biotech Support Group LLC) manufacturer instruction …” The report shows that COVID-19 PBMC proteome and phosphoproteome undergo dynamic changes during disease progression, and the corresponding protein or phosphoprotein signatures can distinguish longitudinal disease states.

Lahut, Suna, et al. "Blood RNA biomarkers in prodromal PARK4 and REM sleep behavior disorder show role of complexin-1 loss for risk of Parkinson's disease." Disease Models & Mechanisms (2017): dmm-028035. http://dmm.biologists.org/lookup/doi/10.1242/dmm.028035. The authors studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation), to identify effects of SNCA gain-of-function as potential disease biomarkers. The article states “…300 μl blood were lysed with equal amount of 1% SDS-RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and one tablet Complete Protease Inhibitor Cocktail (Roche)] and sonicated for 10 sec. The blood lysates were rotated at 4 °C for 30 min and centrifuged at 4 °C for 30 min. The supernatants were depleted in hemoglobin content using a commercial kit (HemogloBind™) following the manufacturer’s instructions”.

Chalásová, Katarína, et al. "Transketolase Activity but not Thiamine Membrane Transport Change in Response to Hyperglycaemia and Kidney Dysfunction." Experimental and Clinical Endocrinology &

Diabetes (2017). https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0043-115009

Diabetic kidney disease, a common complication of both type 1 and type 2 diabetes, is associated with significant morbidity and mortality, and represents the most common cause of chronic kidney disease. The study hypothesized that protective pentose phosphate pathway action in diabetes might be compromised by limited intracellular availability of an active transketolase cofactor thiamine diphosphate (TDP). To evaluate the levels of thiamine transporter proteins in whole blood, the article states “ For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™…”

Hemoglobin Isolation and Derivative Analysis

Dasauni, Pushpanjali, et al. " Optimization and Identification of Single Mutation in Hemoglobin Variants with 2, 2, 2 Trifluoroethanol Modified Digestion Method and Nano− LC Coupled MALDI MS/MS ." Molecules 27.19 (2022): 6357. “The use of Hemoglobind reduced the time to obtain pure Hb in an easy single–step procedure . Pure Hb protein is required specifically to optimize and standardize methods for diagnostics. … The desorbed Hb was compatible with LC–MS, and other proteomics studies, as we verified, did not show any change in its intact mass either. This one−step affinity purification gave us the utmost purified Hb.”

Igoh, Akihisa, Masanobu Miura, and Satoru Miyaishi. "Animal Species and Blood Identification with Peptide Mass Fingerprinting." Analytical Chemistry (2024). https://doi.org/10.1021/acs.analchem.3c04314

The article states “We have developed the application of commercially available reagents for simply purifying hemoglobin from a wide range of animal species. …Blood, saliva, urine, semen, and sweat (20 μL); filter paper from which sweat was collected; and 0.5, 1, 2, and 5 μL of pooled blood samples were treated with HemogloBind™ (BIOTECH SUPPORT GROUP, NJ, USA) to obtain hemoglobin solutions following manufacturer’s protocol. …Hemoglobin was then dissociated according to the protocol to obtain a purified solution ….”

Yamagishi, Yoshikazu, Hirotaro Iwase, and Yasumitsu Ogra. " Post-Mortem Changes of Methomyl in Blood with Hemoglobin. " Chemical Research in Toxicology. In this study, the researchers considered specific methomyl hemoglobin adducts detected by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF MS). To help isolate Hemoglobin, the article states “Hb was separated with HemogloBind in accordance with the manufacturer’s instructions.” The authors conclude that one Hemoglobin derivative, the W-adduct could be used as a biomarker of methomyl poisoning.

Tissue Lysates, LC-MS Proteomics

Jara, Zaira Palomino, et al. "Distinct Mechanisms of β-Arrestin–Biased Agonist and Blocker of AT1R in Preventing Aortic Aneurysm and Associated Mortality. " Hypertension 80.2 (2023): 385-402.

The article states “Cleaned abdominal aortas were homogenized in T-PER Tissue Protein Extraction reagent…. Abdominal aneurysm samples were treated to remove hemoglobin captured within the vessel wall. NuGel-HemogloBind … was used according to the manufacturer protocol.”

Heather E. McKiernan, Phillip B. Danielson, Catherine O. Brown, Masha Signaevsky, Christian G. Westring and Kevin M. Legg, Developmental Validation of a Multiplex Proteomic Assay for the Identification of Forensically Relevant Biological Fluids, Forensic Science International, (2021)

https://www.sciencedirect.com/science/article/pii/S0379073821002280?via%3Dihub. The aim of this study was to validate a multiplex proteomic assay for the identification of target peptide fragments by multiple reaction monitoring on a triple quadrupole mass spectrometer originating from tissue-specific proteins. The article states “If samples contained excessive quantities of hemolyzed red blood cells, four volumes of HemogloBind™ were added.” The authors conclude that the mass spectrometry-based workflow offers significant advantages compared to existing serological methods.

HemogloBind™, contributed to this rigorous examination of protein complexes. When our products (HemogloBind™ & NRicher™ 6) were used as a pretreatment step in the overall workflow, twice the number of observations and annotations became possible. Furthermore, this study demonstrated the importance of a key feature implicit to all of our products; that is the maintenance of functional and structural integrity after separations.

Species Agnostic – Applications to Different Species

Zhang, X., Li, S., Malik, I. et al. Reprogramming tumour-associated macrophages to outcompete cancer cells. Nature (2023). https://doi.org/10.1038/s41586-023-06256-5 To measure the amino acid content by LC-MS from tumor interstitial fluid, the article states “Samples were brought through a NuGel-HemogloBind (Biotech Support Group) prep prior to extraction to remedy the levels of haemolysis present.

Witchey, Shannah K., et al. "Reproductive and developmental toxicity following exposure to organophosphate ester flame retardants and plasticizers, triphenyl phosphate and isopropylated phenyl phosphate, in Sprague Dawley

rats." Toxicological Sciences (2022). https://doi.org/10.1093/toxsci/kfac135. The investigators used HemogloBind™ to reduce interferences associated with Hemoglobin in whole blood lysates ; the article stating “Blood samples…were analyzed for Acetylcholinesterase and Butyryl cholinesterase activity…”

02102025

Southwell, Rebecca Marie, Kenneth Sherlock, and Matthew Baylis. " Cross-sectional study of British wild deer for evidence of Schmallenberg virus infection. " Veterinary Record (2020). The purpose of this study was to survey wild deer across Great Britain for recent evidence of Schmallenberg virus (SBV). Postmortem blood samples were tested for SBV antibodies. Because of the presence of Hemoglobin interference in many samples, the article states “In order to avoid poor quality samples yielding false ELISA results, 59 samples estimated to have above 50mg/dL and less than 250mg/dL haemoglobin concentration, according to their colour, were selected for treatment with HemogloBind™ (Biotech Support Group, New Jersey, USA).”.

Snider, Thomas H., Christina M. Wilhelm, Michael C. Babin, Gennady E. Platoff Jr, and David T. Yeung. " Assessing the therapeutic efficacy of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig. "The Journal of Toxicological Sciences 40, no. 6 (2015): 759-775.

Clinical signs of cholinesterase inhibitor toxicity can be measured from blood cholinesterase [Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] activity utilizing a modified Ellman's method. Biotech Support Group’s unique solid-phase polymer for hemoglobin depletion, was used for pretreatment. The article states “Briefly, whole blood samples were treated with HemogloBind™ which interferes with the ChE activity assay due to spectral overlap.”

Craig, J. R., et al. "A comparison of the anatomical and gastrointestinal functional development between gilt and sow progeny around birth and weaning. " Journal of animal science (2019). Gilt progeny (GP) often have restricted growth performance and health status in comparison to sow progeny (SP) from birth. To better understand underlying mechanisms, the study aimed to compare differences in growth and development between GP and SP in the first 24 h after birth and in the peri-weaning period. Because serum samples were quite hemolysed after collection and processing, it became necessary to use HemogloBind™ to allow for better detection of IgG by ELISA. The article states “As per the manufacturer’s instructions, 250 μL of Hemoglobind was added to 250 μL of hemolyzed serum…

Parvathi S. Kumar, Haree K. Pallera, Pamela S. Hair, Magdielis Gregory Rivera, Tushar A. Shah, Alice L. Werner,Frank A. Lattanzio, Kenji M. Cunnion, and Neel K. Krishna. Peptide inhibitor of complement C1 modulates acute intravascular hemolysis of mismatched red blood cells in rats. TRANSFUSION Volume 00, May 2016.

doi:10.1111/trf.13674. In brief, the study evaluated the role of a peptide inhibitor of complement C1 (PIC1) in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios. The authors state "To remove free Hb that may cause optical interference in bilirubin analysis, we treated all the samples with Hb depletion from hemolyzed serum/plasma (HemogloBind). Bilirubin concentration was then measured with a Bilirubin Assay Kit (Sigma-Aldrich, St. Louis, MO)."

Urine

Dugbartey, George J., et al. "Static cold storage with mitochondria-targeted hydrogen sulfide donor improves renal graft function in an ex vivo porcine model of controlled donation-after-cardiac-death kidney transplantation. " International Journal of Molecular Sciences 24.18 (2023): 14017.

Urine and arterial blood samples were collected hourly during reperfusion. The article states “Urine protein levels were measured using an IDEXX Urine Analyzer (IDEXX Laboratories, Westbook, ME, USA) following a 1:3 dilution of urine in HemogloBind (Biotech Support Group, Monmouth Junction, NJ, USA) to obtain clearer urine samples after 10 min of vigorous shaking and centrifugation at 12,000× g.”.