Synthetic Peptide Mimotopes of blood Group Antigens for Red Cell Antibody Screening

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 291-291
Author(s):  
Evelyn J A Tait ◽  
Robin Fraser ◽  
Michael Moss ◽  
Stanislaw J Urbaniak

Abstract Background: Antibody screening is performed both routinely in the blood group typing procedures for donors and patients and in more detail as part of special investigations for transfusion-dependent patients such as those suffering from Sickle Cell Disease and Thalassaemia. However, despite the care taken, intrinsic limitations of traditional serological diagnostic tests mean that alloimmunisation of pregnant women and multiply transfused patient may still go undetected, resulting in Hemolytic Disease of the Newborn or Hemolytic Transfusion Reactions, respectively. Furthermore, although the genes encoding the majority of blood group antigens have been characterised, the expression of recombinant gene products and the subsequent determination of protein structure that might lead to novel diagnostic reagents have proved more difficult to achieve. Methods: Phage Display libraries that express random peptide sequences (~1015) on the virion surface were screened using a series of monoclonal antibodies and an anti-RhD polyclonal preparation to identify peptides that mimic epitopes of clinically important blood group antigens. The peptides thus identified, were then synthesised in macroarrays and evaluated using SPOTs (Simple Precise Optimal Test system) in a step towards development of a novel diagnostic antibody-screening assay. Results: The combined approach of phagepeptide display and SPOTs proved powerful. From 490 phage-peptides selected by biopanning, 86 mimotopes bound their cognate antibody in SPOTs assays and represented the clinically important blood group antigens RhD (including epitopes 1.1, 3.1 and 6.3), RhE, Rhe, Fya and Fyb. These peptides ranged in size from 7 to 15 residues and included 7-mers that were constrained at their termini by a di-sulphide bridge. Further SPOTs analyses showed 26 of these phage-peptides (12 RhD, 3 RhE, 1 Rhe, 2 Fya and 8 Fyb) have the appropriate strength of signal and binding specificity for inclusion in any future diagnostic antibody-screening assay. A subset of these peptides has been further tested. These peptides were immobilised on polystyrene microspheres and shown to specifically bind their cognate antibodies in both (1) monoplex gel agglutination immunoassays and (2) microsphere-based, multiplex suspension arrays. Conclusions: We have shown that, regardless of whether or not the mimotopes resemble the original antigen sequence, they bind their cognate antibodies specifically and are therefore genuine mimics of the natural antigenic epitopes. It has also been demonstrated that the context in which a peptide is presented is fundamentally important for antibody recognition. The value of the phage-peptide approach in identifying mimotopes to clinically significant blood group antigens has also been established. Moreover, these peptides could be used in a single, comprehensive screening assay and eliminate many of the problems associated with agglutination assays and may herald the possibility of a synthetic, diagnostic array for routine antibody screening for all patients and donors and patients in the near future.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 557-557 ◽  
Author(s):  
James C. Zimring ◽  
Gregory A. Hair ◽  
Traci E. Chadwick ◽  
Seema S. Deshpande ◽  
Kimberly M. Anderson ◽  
...  

Abstract Background: Transfusion of red blood cells (RBC) into patients with anti-donor RBC antibodies (crossmatch incompatible transfusion) can result in antibody mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival. This phenomenon has now been reported for the major clinically significant blood group antigens, including Rh, Kell, Kidd and Duffy. Although this has been described multiple times in humans, no mechanistic elucidation has been accomplished. In an effort to investigate the mechanism of this process, we describe the first animal model of non-hemolytic antibody induced RBC antigen loss. Methods: mHEL mice express the model antigen Hen Egg Lysozyme (HEL) as a cell surface protein on RBC. Since mHEL mice are on a C57BL/6 background, the mHEL antigen represents a single antigenic difference between donor RBC and recipient mice. Immunizing C57BL/6 mice with HEL/CFA results in the generation of high titer IgG anti-HEL responses rendering the mice crossmatch incompatible with mHEL RBC. This system was utilized to study the effects of transfusing mHEL RBC into crossmatch incompatible recipients. Results: Similar to the antibody induced antigen loss observed in humans, transfusion of donor mHEL RBC into crossmatch incompatible mice results in selective loss of HEL antigen from donor RBC without affecting other blood group antigens or reducing the circulatory lifespan of the donor RBC. In addition, recovered RBC that have lost their antigen have normal morphology. This process is antigen specific and occurs in mice that have received passive injections of anti-HEL antisera. A spleen is not required for antigen loss to occur. However, antigen loss does not occur in animals with a targeted deletion of the FcγIII receptor. Although polyclonal anti-HEL antisera consistently causes antigen loss, and IgG1 and IgG2b are the predominant subclasses of anti-HEL IgG in the antisera, no antigen loss is observed in response to purified monoclonal anti-HEL antibodies of the IgG1 and IgG2b subclass. Conclusion: These studies demonstrate that antibody induced antigen loss is a process that involves interaction of RBC, anti-RBC IgG and FcγIII receptors, thus providing mechanistic insight into the phenomenon of antigen loss during incompatible transfusion. The lack of antigen loss in response to monoclonal anti-HEL IgG1 or IgG2b suggests that antigen loss occurs in response to a minor IgG subtype in antisera, depends upon biological properties of the antibody (such as affinity), or that additional serum cofactors are involved.


2020 ◽  
Vol 9 (2) ◽  
pp. 437
Author(s):  
Shelendra Sharma ◽  
Dharmesh Chandra Sharma ◽  
Sunita Rai ◽  
Anita Arya ◽  
Reena Jain ◽  
...  

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Divjot Singh Lamba ◽  
Ravneet Kaur ◽  
Sabita Basu

Background. Racial differences in blood group antigen distribution are common and may result in striking and interesting findings. These differences in blood group antigen distribution are important due to their influence on the clinical practice of transfusion medicine.Study Design and Methods. This is a prospective study, involving 1000 healthy regular repeat voluntary blood donors associated with the department. The clinically significant minor blood group antigens of these donors were studied.Results. Out of 1000 healthy regular repeat voluntary blood donors, 93% were D positive and 2.8% were K positive. Amongst the Rh antigens, e was the most common (99%), followed by D (93%), C (85.1%), c (62.3%), and E (21.5%). Within the MNS blood group system, antigen frequency was M (88%), N (57.5%), S (57.8%), and s (87.5%). Within the Duffy blood group system, antigen frequency wasFya(87.3%) andFyb(58.3%).Conclusions. This data base will help us to prevent alloimmunisation in young females, pregnant women, and patients who are expected to require repeated transfusions in life by providing them with antigen matched blood. Antigen negative blood can also be made available without delay to already alloimmunized multitransfused patients.


2021 ◽  
Author(s):  
Sudhir S Jadhao ◽  
Candice Davison ◽  
Eileen V. Roulis ◽  
Simon Lee ◽  
Paul Lacaze ◽  
...  

There have been no comprehensive studies of a full range of blood group polymorphisms within the Australian population. The problem is compounded by the absence of any databases carrying genomic information on chronically transfused patients and low frequency blood group antigens in Australia. Here, we use RBCeq, a web server-based blood group genotyping software, to identify unique blood group variants among Australians and compare the variation detected versus global data. Whole genome sequencing data was analysed from for 2796 healthy older Australians from the Medical Genome Reference Bank and compared with data from 1000G phase 3 (1KGP3) databases comprising 661 African, 347 American, 503 European, 504 East Asian, and 489 South Asian participants. There were 688 rare variants detected in this Australian sample population, including nine variants that had clinical associations. Notably, we identified 149 variants that were computationally predicted to be novel and deleterious. No clinically significant rare or novel variants were found associated with the genetically complex ABO blood group system. For the Rh blood group system one novel and 16 rare variants were found. Our detailed blood group profiling results provide a starting point for the creation of an Australian blood group variant database.


2020 ◽  
Vol 7 (2) ◽  
pp. 419 ◽  
Author(s):  
Ansuman Sahu ◽  
Pankaj Parida ◽  
Smita Mahapatra ◽  
Binay Bhusan Sahoo

Background: β-thalassaemia patients receive regular blood transfusion to thrive. Due to antigen disparity between the blood donors and these patients they develop red cell alloantibodies due to alloimmunization.  The objective of this study is to predict the frequency of red cell alloimmunization amongst β-thalassaemia major patients receiving regular blood transfusion.Methods: This study including 106 patients with β-thalassaemia was conducted in the department of Transfusion Medicine, S. C. B. Medical College, Cuttack for a period of 12 months. Alloantibodies to different red cell blood group antigens in multi-transfused thalassaemia patients were detected using the glass bead technology for blood group serology in the present study.Results: Out of 106 β-thalassaemia major patients included in the study, 7.5% of patients developed alloantibodies, all being clinically significant. The alloantibodies were anti-E, anti c, anti e and anti-D. The rate of incidence of these alloantibodies was 3.8%, 1.9%, 0.9% and 0.9% respectively.  There was a significant association between alloantibody formation with number of transfused packed red cells (Mann-Whitney Test: p value = 0.035) and age at first transfusion (p value = 0.001). The factors having no association with alloimmunization to red cell antigens are age and gender.Conclusions: Alloimmunization to various erythrocyte blood group antigens is a common problem in multi-transfused β-thalassaemia patients. There is an association between number of transfused packed red cells and age at first transfusion with alloantibody formation in the study.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2324-2324
Author(s):  
Connie M Westhoff ◽  
Stella T Chou ◽  
Kim Smith-Whitley ◽  
David Friedman

Abstract Abstract 2324 A genomic approach to blood group typing is now possible and high-throughput automated platforms have been developed to test for numerous blood group antigens in a single assay. These methods are reproducible and highly correlated with RBC serologic phenotype. We routinely perform a complete RBC phenotype for clinically significant minor red cell antigens on pre-transfusion samples from patients with sickle cell disease, and we antigen match patients for C, E, and K for transfusion. In this study we compared the historic serologic typing with that predicted from DNA testing for clinically significant antigens in 114 samples from chronically transfused patients with SCD to determine concordance and to evaluate the clinical utility of genotyping for the management of transfusion therapy. Serologic typing was performed by standard methods with licensed commercial reagents. DNA was isolated from WBCs, and minor antigen genotyping was performed with HEA (human erythrocyte antigen) BeadChip (BioArray, Inc). RH genotyping was by a combination of methods including PCR-RFLP, AS-PCR, exon-specific amplification and sequencing, and, for some, Rh-cDNA amplification and sequencing. Comparison of serologic typing with DNA-based testing for thirteen blood group antigens, CcEe, Fya/b, K, Jka/b, MN and Ss, in 114 samples found 8 discrepancies in 1,482 antigens analyzed, for 99.5 % concordance. Discrepancies were in several systems (C, Fy, Ss, and M), and at least one has been confirmed to be a serologic recording error. All are under investigation. DNA-based testing for RH found 54 of 114 patients inherited variant RHD alleles; many also had conventional RHD in trans. Sixteen patients had made anti-D, despite typing as D+. Ten of 35 patients (∼30%) whose RBCs typed as C+ had a hybrid allele encoding variant C antigen. Five had made anti-C, which prompted us to change our protocol so patients with variant C by DNA testing are transfused on a C- protocol. DNA testing found a large amount of diversity in ce-alleles in this population. Seventy-two of 114 patients carried at least one of nine different variant ce-alleles. Ten patients had made anti-e, despite typing as e+, and were homozygous for variant ce-alleles. In total, 49/114 patients with SCD were homozygous for variant RH alleles and were not truly Rh matched for D, C and e antigens by serology. Similar to the way in which HLA typing by DNA has revolutionized bone marrow transplantation by providing a superior alternative to serological testing, we find that minor blood group antigen typing by DNA improves efficiency, reduces cost, and expands antigen matching, especially in the Rh system. Continuing studies are needed to identify more precisely which variant alleles are associated with clinically significant antibody production to improve antigen matching for patients with sickle cell disease. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4279-4282 ◽  
Author(s):  
Christopher A. Tormey ◽  
Gary Stack

Abstract Blood group antigen immunogenicity is a crucial factor in red blood cell alloimmunization. Previous calculated estimates of immunogenicity suffered from several key shortcomings. To address these issues we have (1) introduced a correction factor for antibody persistence rates into traditional immunogenicity calculations, (2) calculated immunogenicities only in men to eliminate pregnancy-related antibodies, and (3) excluded antibodies reactive only at room temperature to minimize the contribution of naturally occurring antibodies. With these corrections, we have calculated the immunogenicities of common blood group antigens using data collected on clinically significant alloantibodies (n = 452) in a male patient population. We observed a 3- to 5-fold increase in immunogenicity for some antigens (ie, Jka, Cw, Lua) and smaller changes in others compared with traditionally calculated estimates. In addition, we have calculated the transfusion-related immunogenicities of antigens traditionally associated with naturally occurring antibodies (eg, anti-Lea, -Leb, -M, and -P1).


Hematology ◽  
2009 ◽  
Vol 2009 (1) ◽  
pp. 171-177 ◽  
Author(s):  
Marion E. Reid

Abstract DNA-based tests are increasingly being used to predict a blood group phenotype to improve transfusion medicine. This is possible because genes encoding 29 of the 30 blood group systems have been cloned and sequenced, and the molecular bases associated with most antigens have been determined. RBCs carrying a particular antigen, if introduced into the circulation of an individual who lacks that antigen (through transfusion or pregnancy), can elicit an immune response. It is the antibody from such an immune response that causes problems in clinical practice and the reason why antigen-negative blood is required for safe transfusion. The classical method of testing for blood group antigens and antibodies is hemagglutination; however, it has certain limitations, some of which can be overcome by testing DNA. Such testing allows conservation of antibodies for confirmation by hemagglutination of predicted antigen-negativity. High-throughput platforms provide a means to test relatively large numbers of donors, thereby opening the door to change the way antigen-negative blood is provided to patients and to prevent immunization. This review summarizes how molecular approaches, in conjunction with conventional hemagglutination, can be applied in transfusion medicine.


2020 ◽  
Vol 295 (52) ◽  
pp. 18426-18435
Author(s):  
Bailey E. McGuire ◽  
Andrew G. Hettle ◽  
Chelsea Vickers ◽  
Dustin T. King ◽  
David J. Vocadlo ◽  
...  

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1–3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure–function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1–3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1–3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.


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