Renato de Marco, Isaú H. Noronha, Luiza Zainotti Miguel Fahur Bottino, Alan Andrew Santos dos Silva, Robert Liwski, Maria Gerbase-DeLima
{"title":"通过 EDTA 介导的 Luminex 单抗原珠测定法抗体反应抑制作用揭示的钙依赖性 HLA-DQ 表位。","authors":"Renato de Marco, Isaú H. Noronha, Luiza Zainotti Miguel Fahur Bottino, Alan Andrew Santos dos Silva, Robert Liwski, Maria Gerbase-DeLima","doi":"10.1111/tan.15628","DOIUrl":null,"url":null,"abstract":"<p>Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl<sub>2</sub>). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl<sub>2</sub>. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.</p>","PeriodicalId":13172,"journal":{"name":"HLA","volume":null,"pages":null},"PeriodicalIF":5.9000,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Calcium-dependent HLA-DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay\",\"authors\":\"Renato de Marco, Isaú H. Noronha, Luiza Zainotti Miguel Fahur Bottino, Alan Andrew Santos dos Silva, Robert Liwski, Maria Gerbase-DeLima\",\"doi\":\"10.1111/tan.15628\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl<sub>2</sub>). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl<sub>2</sub>. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.</p>\",\"PeriodicalId\":13172,\"journal\":{\"name\":\"HLA\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":5.9000,\"publicationDate\":\"2024-08-12\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"HLA\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1111/tan.15628\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CELL BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"HLA","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/tan.15628","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
Calcium-dependent HLA-DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay
Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl2). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl2. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.
期刊介绍:
HLA, the journal, publishes articles on various aspects of immunogenetics. These include the immunogenetics of cell surface antigens, the ontogeny and phylogeny of the immune system, the immunogenetics of cell interactions, the functional aspects of cell surface molecules and their natural ligands, and the role of tissue antigens in immune reactions. Additionally, the journal covers experimental and clinical transplantation, the relationships between normal tissue antigens and tumor-associated antigens, the genetic control of immune response and disease susceptibility, and the biochemistry and molecular biology of alloantigens and leukocyte differentiation. Manuscripts on molecules expressed on lymphoid cells, myeloid cells, platelets, and non-lineage-restricted antigens are welcomed. Lastly, the journal focuses on the immunogenetics of histocompatibility antigens in both humans and experimental animals, including their tissue distribution, regulation, and expression in normal and malignant cells, as well as the use of antigens as markers for disease.