anti-B Monoclonal Antibodies Research Articles

Development of Blood Donation Activities in Kyrgyzstan and General Implications for Blood Donation

Objectives: This article is aiming to give an essential informative principle about the development of blood donation activities in Kyrgyzstan. Background: The sequence of blood donation procedure steps before donation, during donation, and after donation was shown and discussed. Methods/Materials: Rhesus factor determination in donors was achieved by “COLICO” method which is based on the use of monoclonal antibodies solution. Results: According to the blood donation activity of donors in Osh, Kyrgyzstan for the 2018 year, it was found that gratuitous donors who donated blood in 6 months were 269, in 9 months were 366, and in 1 year were 499 people. Relative donors who donated blood in 6 months were 3480, in 9 months were 5100, and in 1 year were 6701 people. Active donors donated blood in 6 months were 12, in 9 months were 14 and in 1 year were 17 people. Conclusion: No agglutination was observed in any of the plates for the first blood group; agglutination was observed with anti-A monoclonal antibody solution for the second blood group; agglutination was observed with anti-B monoclonal antibody solution for the third blood group; agglutination was observed with both of anti-A and anti-B monoclonal antibody solutions for the fourth blood group.

THE APPLICATION OF MONOCLONAL REAGENTS IN FORENSIC PRACTICE

Anti-A and anti-B monoclonal antibodies (MCA) may be used in the analysis of liquid blood, spots of blood and saliva traces in order to detect A and B blood group antigens using common methods of evidence investigation. The use of MCA and anti-A and anti-B antigens in the absorbtion-elution reaction during the analysis of minute saliva traces enhances the possibility of establishing the origin of the saliva at the expense of non-secretor. Key words: monoclonal antibodies (MCA), group antigens, research methods, liquid blood, spots of blood, saliva.

Cirrhosis and Autoimmune Liver Disease

Autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC) constitute the most frequently observed forms of autoimmune liver diseases. Each of these autoimmune liver diseases might present with cirrhosis at diagnosis, and a significant proportion of patients develop cirrhosis during follow-up. This manuscript provides a review that addresses how to monitor and manage patients with cirrhosis secondary to autoimmune liver diseases. For patients with PBC, the farnesoid X receptor (FXR) agonist, obeticholic acid (OCA), is the first approved drug since ursodeoxycholic acid (UDCA) and is licensed for non-responders to or those intolerant of UDCA. Bezafibrate has been shown to be effective in non-responders to UDCA, but is not licensed for this clinical indication at present. For patients with AIH, rituximab, a monoclonal antibody against the protein CD20 is a potential option for patients with suboptimal response to corticosteroids. New treatment options are currently being investigated for AIH and PSC that include the anti-B cell-activating factor receptor monoclonal antibodies, nor-UDCA, amongst others, but the efficacy in patients with cirrhosis has not been fully established. Cirrhosis is present in 30–50% of patients at the time of diagnosis of AIH, in 20% of patients with PBC, and in 35% of patients with PSC. Therefore, cirrhosis constitutes one of the main complications of autoimmune liver disease and continues to develop at a frequency of approximately 3–6% per year. Patients with compensated AIH cirrhosis and histological inflammatory activity benefit of corticosteroid treatment. Patients with PBC and compensated cirrhosis should receive UDCA. OCA should be considered in non-responders but used with very close monitoring in patients with decompensated cirrhosis. There is no effective treatment option available for patients suffering from PSC. Liver transplantation is indicated for patients who have progressed to decompensated cirrhosis, and those with intractable symptoms or hepatocellular carcinoma within transplant criteria. Standard monitoring for cirrhosis-related complications is highly recommended.

IMMUNOGENETIC AND PHARMACOCHEMICAL CHARACTERIZATION OF THE ABO SYSTEM GLYCOPROTEIN PROPERTIES AS CRITERIA OF INDIVIDUAL SENSITIVITY TO ANTITUMOR AGENT BORTEZOMIB IN THE PLASMA CELL MYELOMA PATIENTS.

Experimental study of the effect profile of bortezomib in the plasma cell myeloma (PCM) patients depend- ing on a specific phenotype carrier state and a pharmacochemical characteristics of ABO system glycoproteins. The research was conducted on the 104 PCM patients, including the Chornobyl NPP acci- dent survivors (n = 49) and 65 study subjects in the comparison group. Immunogenetic criteria for positive response to the applied treatment protocols were issued according to the duration of remission, absence of infectious com- plications, and evidence of chronic renal failure as a disease complication. Possibility of glycoproteins A and B participation in the formation of human biological individuality at a level of protein-protein interaction with antineoplastic drug bortezomib, which is widely used in cancer management prac- tice, in particular in the PCM treatment is considered. The glycoprotein B was shown being a selective target for borte- zomib, slowing down the recognition and interaction of antigen B with monoclonal anti-B antibody, while the agglu- tination period lengthens at that by 66 %. Assumption that the formation of bortezomib complex with glycoprotein B provides a background for interaction with the key reaction of proteasome 26S inhibition, which to some extent con- tributes to the drug effect retardation was confirmed through the quantum-chemical calculations. Equilibrium is shift- ed toward the main reaction leading to a higher drug efficacy in patients with blood groups O (I) and A (II). Since the complexation occurs predominantly in alkaline medium the administration of drugs with alkaline reaction should be restricted for at least round the clock after administration of bortezomib according to its half-life in plasma in patients with B (III) blood group and chronic renal failure.

Study on creating the hybridoma for producing a monoclonal antibody that causes agglutination of the red blood cell B group

ABO incompatibility is a potential lethal barrier in tranfusion therapy. ABO blood cell grouping for all of the donors and potential recipients is the unique way for ensuring the highest safety for potential recipients in blood transfusion. ABO blood cell grouping must be performed by both of the serum sample method and erythrocytes sample method. Nowadays, when applying the serum sample method, the monoclonal antibodies are commonly used to identify the antigens on the surface of the red blood cells. In this study, for the first time in Vietnam, the hybridoma technology was successefully developed and screened hybrid cells for producing monoclonal antibody specifically agglutinated with red blood cells B group. After being isolated from spleen and iliac lympho node of the human red blood cells B group immuned BALB/c mice, the lymphocytes B was fused with myeloma sp2/0. As the results, 10 single hybrid cell lines B4D6C6, B4D6E2, B4D10C9, B4D10B6, B4D10D5, B4D10D6, B4D10E4, B4H6C5, B8F6B6, B8F6D4 have been screened by a specific agglutination with sample red blood cells group B. Among them, the hybrid cell line B4D10C9 was the best secreting anti-B monoclonal antibody into culture, that presented by antibody titer reached 1/256, hence it was selected for further studies. The ELISA method helped to isotype the anti-B monoclonal antibody which was produced from B4D10C9 hybridoma. As a result, the anti-B monoclonal antibody contained IgM heavy chains and kappa light chains. The IgM antibody could be able to agglutinate red blood cells 25 times more than IgG antibody. The success of screening B4D10C9 hybridoma producing IgM monoclonal antibody is the most significant result of this study.

Incomplete Antibodies May Reduce ABO Cross-Match Incompatibility: A Pilot Study.

Objective:Any erythrocyte transfusion among humans having type A or B blood groups is impossible due to antibodies causing fatal transfusion complications. A cross-match test is performed to prevent immune transfusion complications before transfusion. Our hypothesis is that the fragment antibody (Fab) part of the antibody (incomplete antibody) may be used to prevent an immune stimulus related to the complete antibody. Therefore, we designed a pilot study to evaluate the effectiveness of these incomplete antibodies using cross-match tests.Materials and Methods:Pepsin enzyme and staphylococcal protein A columns were used to cut anti-A and anti-B monoclonal antibodies and purify their Fab (2) fragments, respectively. An Rh-positive erythrocyte suspension with purified anti-A Fab (2) solution and B Rh-positive erythrocyte suspension with purified anti-B Fab (2) solution were combined correspondingly. Cross-match tests were performed by tube and gel centrifugation methods. The agglutination levels due to the anti-A and anti-B Fab (2) antibodies and their effects on the agglutination normally observed with complete antibodies were then measured.Results:No agglutination for the purified incomplete anti-A Fab (2) with A Rh+ erythrocyte and anti-B Fab (2) with B Rh+ erythrocyte combinations was observed in the tube cross-match tests. These agglutination levels were 1+ in two wells in the gel centrifugation cross-match tests. Fab (2)-treated erythrocytes were also resistant to the agglutination that normally occurs with complete antibodies.Conclusion:We determined that the Fab (2) fragments of antibodies may not only be used to obtain a mild or negative reaction when compared to complete antibodies, but they might also be used for decreasing ABO incompatibility. Incomplete antibodies might be a therapeutic option in autoimmune hemolytic anemia and they may also be used in solid organ or hematopoietic stem cell transplantation. Therefore, we have planned an in vivo study to prove these in vitro findings.

STUDY ON MIXING ANTI-A MONOCLONAL ANTIBODY AND ANTI-B MONOCLONAL ANTIBODY TO MAKE ANTI-AB SAVE AS ABO BLOOD GROUPING REAGENT

So, it needs to balance the ratio of anti-A monoclonal antibody and anti-B monoclonal antibody in the mixing so that the possibility of agglutination is the best. In this paper, anti-A monoclonal antibody (titer is 1/256) and anti-B monoclonal antibody (titer is 1/256) was used. The best results were obtained at one volume anti A monoclonal antibody is mixed one volume anti-B monoclonal antibody. The anti-A,B antibody titer was 1/128 for red blood group A and it was 1/128 for red blood group B. The intensity of agglutination reached 3+ for both red blood group A and B.

Establishment of a procedure for producing the anti-b monoclonal antibody from B4C10D9 hybridoma cell line

There are two the most important blood group system in blood transfusion, that is the ABO system and the Rh system. The anti-A or anti-B antibodies in the blood of recipient cause strongly agglutination the red blood cells of donor bearing A or B antigens on their surface respectively. So, all donated blood and received blood must be typed group for the ABO system to be safe in blood transfusion. To blood grouping, used of know antisera reacts with the whole blood samples to determine antigen on the surface of red blood cell or use of know red blood cells reacts with the individual's serum to determine antibody. The serology method is widely used, antisera are usually monoclonal antibodies secreted by hybridoma cells. Our previous studies showed that the B4C10D9 hybridoma cell line generated anti-B monoclonal antibody was produced by hybridoma technology. This antibody causes specificity agglutination the red blood cells caring B antigen on their surface. It is classed as IgM antibody with it heavy chains are µ and κ light chains. The aim of the study was to produce the anti-B monoclonal antibody form B4C10D9 hybridoma cells line serve as a part of ABO blood grouping reagent. Based on results of experiments, procedure for producing the anti-B monoclonal antibody from B4C10D9 hybridoma cell line has also been established with following steps. At first, refresh the frozen B4C10D9 hybridoma cells and the density of B4C10D9 hybridoma cells is 4.0 x 104 cells/ml at 24 hours post-inoculation. Then the B4C10D9 hybridoma cells are splitted 1:5 every 48 hours for 2 times to obtain the cell biomass. In the next step, the hybridoma cells are splitted into 125 cm2 flasks with the initial cell density of 105 cells/ml. The B4C10D9 hybridoma cells density reach 9.9 x 106 cells/ml at 48 hours post-inoculation. After 144 hours of post-inoculation, collect culture medium and concentrate the anti-B monoclonal antibody by precipitation with saturated ammonium sulfate at a final concentration of 50%. If resuspending volume equals initial volume, then the anti-B monoclonal antibody titer reaches 1/256 and the antibody reaction intensity will be 3+. And if the obtained antibody solution volume equals one fifth of the original volume, then the anti-B monoclonal antibody titer reaches 1/1024, and the antibody reaction intensity will be 4+. Citation: Establishment of a procedure for producing the anti-b monoclonal antibody from B4C10D9 hybridoma cell line. Tap chi Sinh hoc, 39(3): 342-348. DOI: 10.15625/0866-7160/v39n3.10765. *Corresponding author: tnhai@ibt.ac.vn Received 20 August 2017, accepted 20 September 2017

Clinical presentation, management and outcomes of influenza in Africa: systematic review, 2009-2014

Background: This paper aims to review the effectiveness of diagnostic and management of influenza in Africa, specifically mortality, treatment and outcomes. Methods & Materials: In two times, we searched the online databases PubMed™ and Scopus™ for articles and abstracts published in English and French between January 2003 and December 2014, with the following terms :(influenza OR flu) AND (clinical) AND (management OR outcomes) AND (Africa) at the first time, and online databases of internationals conferences for the abstract who do not meet the consent of the editor of scientific journals at the second time. Cross- sectional, longitudinal studies and randomized clinical trial on influenza were selected when clinical, management and outcomes were reported. Results: Patients with influenza were more likely to present with fever as initial and main symptom, followed by shortness of breathing, cough, muscle & joint pain, sore throat, hemoptysis, dyspnea, and gastrointestinal complaints (vomiting and diarrhea), pneumonic infiltrations in the chest. For the diagnostic nasal secretions were collected in patients presenting with flu syndrome and follow-up by laboratory identification of viruses was performed by the ELISA technique using anti-A and anti-B monoclonal antibodies (immunocapture) and by isolation on MDCK cells, quantitative real-time polymerase chain reaction (qRT-PCR) assay of the upper respiratory tract is used increasingly to diagnose lower respiratory tract infections. Amongst samples analyzed for influenza; 1–45% had laboratory-confirmed influenza infections; including influenza virus A (H3N2) type, A (H1N1) type, A (H5N1) type and influenza virus B. All confirmed cases received oseltamivir in any setting. Among groups known to be at high risk of influenza-associated complications, Included age<5 years, asthma, cardiac disease, pregnancy, diabetes mellitus, active pulmonary tuberculosis and chronic malnutrition. Mortality rate was 28 – 68.4%. Female sex, age>15 years, and receiving the first dose of oseltamivir>2 days after illness onset, non vaccination against the virus the circulating influenza, cardiovascular complications and ventilatory associated pneumonia were identified as mortality predicting factors. Conclusion: The classic presentation of influenza in Africa is most often confused with malaria, low technical platforms limit the detection of virus in the samples. The progressive creation of influenza sentinel surveillance system will improve care in Africa.

Serum ABO Blood Typing by Surface Plasmon Resonance Technique

Aim of this work is to develop a surface plasmon resonance (SPR) technique for detection of anti-A and anti-B which are the antibodies specific to ABO blood group in serum based on solid phase immobilization of antigens on the surface. Synthetic antigens A and B were immobilized on the carboxymethyldextran (CMD) surface to measure the antibodies. The immobilized synthetic antigen surface were tested function by injection of monoclonal anti-A and anti-B. Total 20 plasma samples at 1:10 dilution were measured by SPR techniuqe and the result were concordant to standard agglutination technique. The cost of blood typing could be economized by high throughput and surface regeneration on SPR technique.

Blood group antigen studies using CdTe quantum dots and flow cytometry.

New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs]) as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC) antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I) lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion, this methodology with high sensibility and specificity can be applied to study a variety of RBC antigens, and, as a quantitative tool, can help in achieving a better comprehension of the antigen expression patterns on RBC membranes.

Genetical study of mutation in maternal-fetal ABO incompatibility.

This study looked into a family involving a rare mother-child ABO blood type inconsistency and explored its genetic and molecular basis. In the family, the mother had type AB blood and the father was blood type B and they gave birth to a baby of blood type O. Their blood types were phenotypically identified by using different techniques, including micro-column gel test, immune inhibition test, absorption and elution tests. The sequences of all 7 exons of ABO allele from the core family members were determined by using PCR and clone-based sequencing. The loci of mutated gene were compared against normal human genes. The result showed that the mother's erythrocytes were agglutinable with monoclonal anti-A antibody (2+) and had agglutination reaction with anti-B antibody (4+). The mother's serum registered agglutination action with standard blood type A cells. The findings showed an ABO inconsistency. When domestic antibodies were used, the mother's erythrocytes yielded agglutination reaction with humanized anti-B serum (4+) and anti-B monoclonal antibody but were non-agglutinable with humanized anti-A serum and anti-A monoclonal antibody. Upon absorption and elution, the titer of anit-A antibody was 128 both before and after the absorption test, with no significant difference found between pre- and post-absorption values. Our results confirmed that the mother's allelic gene was type B and contained type A. The father's blood type was type B, and son's blood type was type O. Clone-based sequencing revealed that the mother carried a heterozygous gene of B101.01 (ntA640→G)/O01, which contained an M214→V mutation that could express a weak expression of antigen A, resulting in blood type AB. However, their son did not have the M214→V mutation, which yielded a false ABO-inconsistency between him and his mother. We were led to conclude that type B gene with a M214→V mutation can encode both antigen B and weak antigen B that can lead to false ABO-inconsistencies.

Functional characterization of botulinum neurotoxin serotype H as a hybrid of known serotypes F and A (BoNT F/A).

A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.

Variation of Both Blood Group H and ABO Glycans Is Associated with VWF Levels

Abstract 3314ABO(H) is a carbohydrate blood group system primarily expressed on red cells, blood vessels, and mucosal surfaces. ABO blood group is known to influence von Willebrand Factor (VWF) levels, with low VWF associated with blood group O and increased diagnosis of von Willebrand Disease. Conversely, non-O blood group is associated with high VWF levels, and both non-O blood group and high VWF are associated with thrombotic vascular disease. We sought to characterize VWF levels relative to ABH glycan phenotypes in 1129 individuals from a healthy sibling cohort, the Genes and Blood Clotting Cohort (GABC). VWF:Ag levels were determined by AlphaLISA (Perkin Elmer) in platelet poor plasma. ABH forensic techniques were adapted to samples of RBC-rich, frozen buffy coat in the study repository. In brief, buffy coat fractions were diluted in TBS, applied to nitrocellulose, and A and B glycans detected using murine monoclonal anti-A (Immucor) or murine monoclonal anti-B (Immucor) followed by and streptavidin-conjugated donkey anti-mouse IgG HRP (Jackson ImmunoResearch). A biotinylated Ulex europaeus agglutinin (UEA lectin, Vector Labs) was used to detect H, followed by streptavidin-HRP. Blots were imaged using ImageQuant (GE) and scored semi-quantitatively for glycan density by two blinded independent observers using reference buffy coats from normal blood donors.ABO blood group frequencies were similar to that of the U.S. population (O=40%, A=42%, B=12%, AB=6%). Also consistent with previous reports, VWF:Ag levels varied significantly between ABO blood groups (O<A<B=AB, see Table 1). Within blood group A, we observed variation in A glycan density (scored 1+ to 3+). Lower A glycan density (similar to the A2 reference) correlated significantly (p<0.01) with lower VWF:Ag levels (see Table 1), and also appeared to have higher detectable H antigen. We also observed wide variation in H glycan density (scored 1+ to 5+). Overall, detection of A and B glycan density patterns were inversely related to H glycan, consistent with glycosylation of H by the ABO enzyme. Interestingly, higher H density correlated significantly (p<0.01) with lower VWF:Ag levels, even within blood group O (see Table 2).In summary, ABH glycan variation may impact VWF in a more complex fashion than previously suspected. Although these data are limited to semi-quantitative analyses by the heterogeneous nature of RBC-rich buffy coat, our findings are consistent with expectations for major ABO blood group frequencies and show that variation of RBC rich-buffy coat ABH glycan density within and between ABO blood groups correlates with VWF:Ag level. This work also suggests a previously unsuspected association between VWF and variation in H antigen, which we hypothesize may be due to variation in the FUT genes or other loci affecting H glcosylation patterns. Further work to characterize the complexity of the ABH carbohydrate system and its genetic determinants is warranted to better understand the impact of ABH subtypes on VWF and vascular phenotypes.Table 1ABO Blood Groups, Buffy Coat A Glycan Density, and VWF:Ag LevelNumber of SubjectsABO Blood GroupVWF:Ag (+/−1SD)VWF Difference (Ovs.Avs.Bvs.AB)*Difference from Group O*454O90+/−35473A119+/−45p<0.01p<0.01138B130+/−50p<0.01p<0.0164AB128+/−52NSp<0.01Number of SubjectsA Glycan DensityAvg H Glycan DensityVWF:Ag (+/−1SD)VWF Difference (A1+vs.2+vs.3+)*Difference from A 1+*681+3.598+/−401952+2.4123+/−44p<0.01p<0.012103+2.0123+/−45NSp<0.01*Each section above: One-way ANOVA; post-hoc Tukey's Least Significant Difference.Table 2Buffy Coat H Glycan Density and VWF:Ag LevelH Glycan Density in All SubjectsNumber of SubjectsH Glycan DensityVWF:Ag (+/−1SD)VWF Difference (H1vs.2vs.3vs.4vs.5)*Difference from H 1+*521+135+/−462572+130+/−53NSNS2603+117+/−44p<0.01p<0.013074+99+/−37p<0.01p<0.012525+88+/−32p<0.05p<0.01H Glycan Density in Blood Group O SubjectsNumber of SubjectsH Glycan Density**VWF:Ag (+/−1SD)VWF Difference (H3+vs.4+vs.5+)*Difference from H 3+*293+115+/−572074+92+/−33p<0.01p<0.012145+85+/−30NSp<0.01*One-way ANOVA; post-hoc: Tukey's Least Significant Difference.**No blood group O individuals scored <3+ H glycan density. Disclosures:No relevant conflicts of interest to declare.

Discrepancies Between ABO Genotype and ABO Glycan Phenotypes

Abstract 274The ABO(H) carbohydrate blood group system is highly polymorphic in humans, with >200 ABO alleles described to date. The ABO gene encodes the glycosyltransferase, ABO, which has two main isoforms which generate blood group A or blood group B. Blood group O is the result of a non-functional ABO glycosyltransferase. ABO blood group is commonly imputed in genome-wide association studies where ABO is associated with phenotypes such as von Willebrand Factor level or myocardial infarction. We sought to determine the accuracy of a common ABO genotyping algorithm in a repository of 1092 individuals from a healthy sibling cohort study, the Genes and Blood Clotting Cohort (GABC). We utilized three published single nucleotide variants (SNVs) to assign ABO blood group (A2, B, and O) relative to the A1 reference allele: rs8176704 (G/A) to annotate A2, rs8176749 (C/T) to annotate B, and rs687289 (A/G) to annotate O. We detected ABO glycans in red blood cell-rich buffy coat samples using an adaptation of published ABO forensic dried blood spot techniques. Briefly, samples were diluted and applied to a nitrocellulose membrane, blotted with murine monoclonal anti-A (Immucor) or murine monoclonal anti-B (Immucor) antibodies followed by and streptavidin-conjugated donkey anti-mouse IgG HRP secondary (Jackson ImmunoResearch). Blots were developed, imaged using ImageQuant 350 (GE), and scored semi-quantitatively by two blinded independent observers relative to red blood cell-rich buffy coat reference panels from normal blood donors.Comparison of ABO imputed genotype to detected ABO glycans in red blood cell-rich buffy coat identified discrepancies in the predicted major ABO blood group assignment (i.e. blood group O, A, B, or AB) in 4.4% of individuals. Interestingly, A glycan density was generally low in individuals with a discrepant A phenotype, suggesting that these individuals harbor a less robust blood group A enzyme variant. There was no common pattern for genotype-phenotype discrepancies. Multiple types of errors (both presence and absence of ABO glycans) were observed, and divergent patterns of ABO glycans were observed between homozygotes for ABO SNVs, suggesting multiple ABO alleles or other genetic modifiers underlie the discordant calls. Interrogation of the Exome Variant Server (http://evs.gs.washington.edu/EVS/) found one-third of ABO nonsynonymous polymorphisms in the exome data set are novel, indicating a large proportion of the genetic variation at ABO has not been previously described. Furthermore, the majority of nonsynonymous variants in the EVS database for the genes encoding H antigen (the ABO acceptor structure), FUT1 and FUT2, were also novel.In summary, ABO(H) is more variable genetically than previously suspected. A commonly applied ABO SNV genotyping algorithm resulted in 4.4% frequency of major ABO blood group glycan discrepancy as detected in red blood cell-rich buffy coats, likely due in large part to the underlying genetic diversity at ABO. Further work is needed to deeply characterize the genetic diversity at ABO, FUT1, and FUT2 and correlate the spectrum of allelic variants with ABO(H) carbohydrate phenotypes. Such high resolution ABO and H classification would likely advance our understanding of the complex physiologic processes influenced by ABH blood groups, including disorders of hemostasis and thrombosis, vascular disease, infectious disease susceptibility, complications of pregnancy, and allogeneic exposures. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Indigenously developed monoclonal antibody specific for human blood group B

Objective: The quality blood grouping reagents is clearly an important factor for blood transfusion and diagnostics and many international standard Anti-B reagents are available for blood grouping in India and there is a need of indigenously developed, cost effective potent Anti-B secreting monoclonal antibody which can be used as a standard blood grouping reagent. To develop indigenous, cost effective standard Anti-B reagent which can be used in the scale up system to ensures constant supply which will ultimately be cost-effective if produced in-house. Method: Murine anti-B monoclonal antibodies have been produced by hybridoma fusion with specificity for human blood group B following the immunization of BALB/c mice with group B cells. Results: Three hybridoma clones were developed with specificity to human B blood group. Out of three hybridoma clones two of them, show potent titer, avidity, and sensitivity to be used as blood typing reagent. One clone, 3D5D7G2 reacted well in screening tests with an avidity of &lt; 4 seconds with B cell. Conclusion: This IgM antibody reacted with 100% accuracy when tested with more than 2500 samples of A, B, O blood groups and satisfies all the laid down criteria under WHO guidelines to be labeled as an Anti-B reagent. The suitability of these antibodies as blood grouping reagents is discussed.

Skin biopsy as a routine diagnostic tool for primary Sjögren’s syndrome

Sjogren’s syndrome is an autoimmune disease characterized by disruption of epithelial cells and lymphoplasmocytic infiltration of exocrine glands throughout the body, mainly salivary and lacrimal glands. The lymphocytic infiltrates in the minor salivary gland biopsy and the presence of autoantibodies are the gold standards for diagnosis. However, skin dryness is often underestimated. In addition, skin biopsy of patients with Sjogren’s syndrome may have lymphocytic infiltrates, and some patients with nonconclusive minor salivary glands may present infiltrates in the skin. We propose to add the question of skin dryness to the symptoms questionnaire designed for patients presenting clinical features of Sjogren’s syndrome and to perform skin biopsy as a routine tool in the diagnosis of Sjogren’s syndrome, especially in patients with nonconclusive minor salivary glands. The emergence of new and potentially active treatments, most notably anti-B lymphocyte monoclonal antibodies, may conduct to the use of skin b...

Quantifying cell binding kinetics mediated by surface-bound blood type B antigen to immobilized antibodies

Cell adhesion is crucial to many biological processes, such as inflammatory responses, tumor metastasis and thrombosis formation. Recently a commercial surface plasmon resonance (SPR)-based BIAcore biosensor has been extended to determine cell binding mediated by surface-bound biomolecular interactions. How such cell binding is quantitatively governed by kinetic rates and regulating factors, however, has been poorly understood. Here we developed a novel assay to determine the binding kinetics of surface-bound biomolecular interactions using a commercial BIAcore 3000 biosensor. Human red blood cells (RBCs) presenting blood group B antigen and CM5 chip bearing immobilized anti-B monoclonal antibody (mAb) were used to obtain the time courses of response unit, or sensorgrams, when flowing RBCs over the chip surface. A cellular kinetic model was proposed to correlate the sensorgrams with kinetic rates. Impacts of regulating factors, such as cell concentration, flow duration and rate, antibody-presenting level, as well as pH value and osmotic pressure of suspending medium were tested systematically, which imparted the confidence that the approach can be applied to kinetic measurements of cell adhesion mediated by surface-bound biomolecular interactions. These results provided a new insight into quantifying cell binding using a commercial SPR-based BIAcore biosensor.

Neonatal alloimmune thrombocytopenia associated with maternal‐fetal incompatibility for blood group B

Blood group A and B antigens are expressed only weakly on platelets (PLTs) of most individuals but are very strongly expressed on PLTs from approximately 1 percent of normal subjects (Type II high expressers). The implications of this trait for transfusion medicine are undefined. A family was studied in which two Group B infants were born with neonatal thrombocytopenia, whereas a third infant whose blood group was A(2) had a normal PLT count at birth. Serologic studies demonstrated a maternal antibody that reacted strongly with PLTs from the father and the two group B children in flow cytometry and with GPIIb/IIIa from their PLTs in solid-phase assays. No PLT-specific antibodies were detected in maternal serum sample, but it contained a high-titer immunoglobulin G antibody specific for blood group B. All PLT-reactive antibody in the mother's serum was removed by absorption with pooled, washed group A and B red cells (RBCs). Studies with monoclonal anti-B and measurement of serum B-glycosyltransferase activity showed that the father and both group B children were Type II high expressers of blood group B. The findings indicate that high-titer blood group antibodies acquired from the mother can cause thrombocytopenia in infants possessing the Type II high-expresser phenotype despite competition for antibody binding by blood group antigens expressed on RBCs and other tissues.

Identification of peptide mimetics of xenoreactive α-Gal antigenic epitope by phage display

The prevention of hyperacute rejection (HAR) triggered by interaction between the human natural antibody and xenoreactive antigenic epitope (Gal-α1, 3Gal) present on pig cells is the key to success in pig-to-human xenotransplantation. The phage display technology offers an effective strategy for screening peptides which can interact with the anti-Gal antibody to block α-Gal antigen binding site. Two peptide libraries, linear 7 peptide library and C7C library, were panned on the anti-B monoclonal antibody which has the characteristic of binding to the α-Gal antigenic epitope. After four rounds of panning, 22 positive phage clones were selected. Highly homologous sequence PT and STL existed among these selected peptides. Stachyose competitive ELISAs revealed that these peptides specifically bound to α-Gal antigen binding site. Eight peptide mimics of α-Gal antigenic epitope could inhibit the agglutination of pig red blood cells mediated by human sera in a dose-dependent manner. These results demonstrated that the selected peptides can mimic the conformational structure of α-Gal antigenic epitope and have the therapeutic potential in xenotransplantation.