ABO血型抗体吸附剂:冻干红细胞膜的初步研究

目的制备冰冻干燥红细胞膜,探索其作为ABO血型抗体吸附剂去除血型抗体、制备通用型红细胞的可行性。方法收集A、B型红细胞膜,作冰冻干燥处理,与O型血浆中血型抗体反应后,检测血浆中抗体效价,评价其对血浆抗体的吸附效果;检测红细胞结构、功能及血浆中凝血因子活性变化,评价冻干红细胞膜与红细胞及凝血因子的兼容性;采用离心的方法去除残留的冻干红细胞膜。结果冰冻干燥红细胞膜上的抗原可有效吸附血型抗体、降低血浆中的血型抗体效价,全血中红细胞的变形指数、ATP含量,与反应前比较差异无统计学意义(P>0.05),反应前后均在正常参考范围内;全血血浆中各种凝血因子活性在吸附前后比较差异亦无统计学意义(P>0.05)。采用离心法可去除冻干红细胞膜,清除率约为77%。结论冰冻干燥的红细胞膜保持了很好的抗原性,作为ABO血型抗体的吸附剂能够有效去除ABO血型抗体;冰冻干燥红细胞膜不影响其中红细胞和凝血因子的理化性状和生物活性,并可通过离心的方法去除。     

红细胞自身抗体

<正> 在免疫血液学中,红细胞自身抗体是指与某人自己红细胞上相应抗原结合的抗体。这种抗体除同自己红细胞起反应外,也可以同其他人的红细胞起反应。因此,它可以表现为非特异性的,但用适当的红细胞作试验时,常显示一定程度的特异性。按自身抗体与红细胞反应时的温度,可分     

100例输血患者血小板特异性抗体调查

<正> 血小板输注对因血小板减少引起的出血有确切的疗效。血小板表面有血小板特异性抗原,又有与红细胞共有的血型抗原,并吸附有较强的 HLA 抗原,临床输全血或输浓缩血小板,一般来自随机供血者,因此多次输血的病人常可产生 HLA 抗体和血小板特异性抗体。国内对输血引起的血小板抗体检测已有报导。Bluberg 等用氯喹处理去除血小板表面吸附的HLA 抗原,以检测伴有 HLA 抗体的血清中的血小板特     

抗-E、抗-c抗体致迟发性溶血性输血反应1例分析

目的 分析Rh血型系统中抗-E、抗-c抗体导致迟发性溶血性输血反应（DHTR）的原因和血型血清学的情况。方法 对1例输血患者,在输血前进行常规定型,发生DHTR后进行各项血液学、生化指标和血型血清学检查。结果 患者红细胞表面缺乏E、c抗原,血清中检出抗-E、抗-c抗体,血液学、生化指标的变化证实发生DTHR。结论 患者由于血型不合的妊娠为初次刺激,输注含回忆反应抗原（E抗原、c抗原）的红细胞,激发免疫应答产生不完全抗体,导致DHTR发生,提示有输血史及妊娠史的患者输血时,必须采用能检测出不完全抗体的酶、抗人球蛋白或更灵敏的方法配血,防止DHTR的发生。     

多血型系统抗体鉴定和交叉配血策略一例抗-f合并抗-M

目的 鉴定一例患者血浆中抗体的特异性并制定交叉配血的策略。方法 采用盐水介质试管法和抗人球蛋白卡(LISS/Coombs卡)做抗体筛选和抗体鉴定,根据谱细胞、酶处理谱细胞及4℃谱细胞反应格局,确定患者血浆中意外抗体的特异性。结果 患者Rh血型为CCDee,MN血型为NN。多种抗体鉴定结果显示,其血浆中存在同种抗体:Rh血型系统抗-f(f:ce复合抗原)合并MNS血型系统抗-M。结论 抗-f仅与具有f抗原(R0、r)的红细胞反应,且能被蛋白水解酶增强;IgG类抗-M在4℃环境的反应性更强。经过输血免疫后,患者产生抗-f和抗-M抗体,交叉配血策略为优先选择无M抗原红细胞进行交叉配血,对配血相合的红细胞成分血进行RhCE抗原检测,选择无f抗原红细胞发血。     

血型抗体减弱伴多凝集红细胞1例

人类血型系统中,体内产生的天然血型抗体相对稳定,红细胞表面的非活性血型抗原同样相对稳定,但在病理情况下,这两种物质会发生改变。现将最近发生在本院的1例血型抗体减弱同时出现多凝集红细胞的病例报道如下。     

多克隆抗RhD抗体的纯化和鉴定

摘要：目的：纯化人血清中多克隆抗RhD抗体,并对其进行鉴定。 方法：收集5例RhD阴性者血清,经不规则抗体筛查和确认后,按等比例混合,分别用RhD(-)A型红细胞和RhD(-)B型红细胞对血清中的抗A、抗B抗体于4 ℃过夜吸收处理;用RhD(+)O型红细胞制备血影细胞,以血影细胞作为抗原,吸附血清中抗RhD抗体,再用低pH值缓冲液洗脱、分离纯化抗RhD抗体,用DEAE-Sephadex A-50层析法提取其中IgG组分;分别用western blot、微柱法鉴定。 结果：5名研究对象血清中均含抗RhD抗体;血清中的抗A和抗B血型抗体被完全吸收,纯化后抗RhD抗体IgG组分效价达到1∶32。 结论：本实验获得了高效价的抗RhD多克隆抗体,为进一步利用噬菌体肽库获得RhD血型抗原表位研究等奠定基础。     

扬州市RhD阴性无偿献血者抗原分布及不规则抗体调查

人类红细胞Rh血型系统发现于20世纪40年代,是人类红细胞血型系统中最具多态性的血型系统。该血型系统重要性仅次于ABO血型系统,可以引起溶血性输血反应和新生儿溶血病[1]。为了进一步提高输血的安全性、有效性和及时性,我们对初筛RhD抗原阴性者再进行阴性确认实验和5种主要抗原及不规则抗体的检测登记。我们对扬州市2008至2009年以来初筛后经确认为抗原阴性的无偿献血者其表型分布和不规则抗体进行了回顾性统计分析。     

重视输血的抗体筛查试验

抗体筛查试验(以下简称抗筛)临床常用于检测样本中是否含有针对红细胞血型抗原的不规则抗体,而不规则抗体的存在是导致临床发生溶血性输血反应的主要原因之一。红细胞血型抗体的分类红细胞血型抗体按其产生的原因可分三类,即天然抗体、免疫性抗体和自身抗体;而按其出现的规律也可分为规则抗体、不规则抗体和特殊类型抗体三类。一、规则抗体规则抗体一般指ABO血型系统的抗体,这类抗体按一定规律出现,如血型为A型的人群存在抗     

用噬菌体呈现技术表达抗人红细胞血型B抗原的单链抗体

本研究的目的是构建表达抗人红细胞血型B抗原5D12杂交瘤细胞的单链抗体(ScFv)。应用重组噬菌体抗体技术,分离与构建5D12 McAb单链抗体基因,将其克隆入噬粒pHEN-1中,转化E.coli TGl,M13KO7辅助噬菌体援救构建5D12噬菌体单链抗体库;用完整红细胞亲和富集法淘选阳性重组噬菌体,重组噬菌体鉴定及序列测定分析;免疫杂交试验检测重组噬菌体单链抗体的特异性抗原活性。经M13K07援救E.coli TGl转化菌中的pHEN-1重组噬菌体,得到滴度为3×10~9pfu/ml噬菌体单链抗体库;免疫杂交试验证实表达产物保留了亲本单抗对人红细胞血型B抗原的特异性亲和力。本研究成功地构建5D12 McAb单链噬菌体抗体库,为进一步研制高特异性和高亲和力的基因工程原核血型检定抗体试剂奠定了基础。     

制备蛋白质芯片的玻璃表面修饰方法比较

目的 探讨用于制备蛋白质芯片的4种玻璃表面修饰方法对蛋白质的固定能力。方法 从蛋白质固定效率和固定蛋白质的反应性2个方面，对戊二醛修饰法、琼脂糖修饰法、巯基修饰法和聚赖氨酸修饰法进行比较。结果 4种修饰方法中聚赖氨酸修饰玻片对蛋白质的固定量较大，比醛基修饰的玻片约高31．5％；与醛基修饰相比，其反应性约高26．6％。结论 聚赖氨酸修饰的玻片较适合于制备蛋白质芯片。     

IMMUNOHEMATOLOGY: Novel antibody screening cells,MUT+Mur kodecytes,created by attaching peptides onto red blood cells

BACKGROUND: Antibody screening and identification panels are generally limited by the natural antigenic phenotypes present in their source donor population. However, the recent ability to attach peptides to the surface of cells has opened up the opportunity to create red blood cells (RBCs) with antigen profiles specifically designed for antibody screening and identification in a target population. STUDY DESIGN AND METHODS: Clinically significant antibodies to variant glycophorins (GPs) such as GP.Mur are more commonly seen in certain Asian populations. Using peptides representative of the MNS antigens MUT and Mur, RBC antibody screening cells were created using KODE cell surface engineering constructs. MUT‐, Mur‐, and MUT+Mur‐modified RBCs, known as kodecytes, were tested against monoclonal reagents and polyclonal sera with specificity for epitopes on GP.Mur‐positive RBCs. RESULTS: Kodecytes retained their normal expression of intrinsic blood group antigens while expressing the new epitopes attached by KODE technology. The MUT, Mur, and MUT+Mur kodecytes, although unreactive with the various monoclonal reagents, were appropriately reactive with polyclonal sera containing antibodies reactive with GP.Mur‐positive RBCs. CONCLUSIONS: This study used selected MUT and Mur peptides and KODE cell surface engineering technology to create MUT+Mur kodecytes suitable for the detection and identification of RBC antibodies in human serum or plasma. This technology has the potential to create a large range of specialized RBCs for antibody screening and identification.     

Serological studies of piperacillin antibodies

BACKGROUND: Penicillin‐induced immune hemolytic anemia (IHA) is associated with immunoglobulin G antipenicillin detected by testing penicillin‐coated red blood cells (RBCs). Antibodies to piperacillin, a semisynthetic penicillin, would be expected to react similarly; however, antipiperacillin can be detected by testing in the presence of the drug. Piperacillin is commonly used in combination with tazobactam, which causes nonimmunologic protein adsorption onto RBCs. In six cases of piperacillin‐induced IHA, reactivity with piperacillin‐coated RBCs was not similar to reactivity of antipenicillin with penicillin‐coated RBCs. STUDY DESIGN AND METHODS: Antipiperacillin was tested against piperacillin‐coated RBCs prepared using different pH buffers. Plasma from blood donors and sera/plasma from patients were tested with piperacillin‐coated, penicillin‐coated, and uncoated RBCs. Hapten inhibition studies were performed using different concentrations of piperacillin. Donors' plasma were tested in the presence of piperacillin; sera from patients with IHA were tested in the presence of tazobactam. RESULTS: Piperacillin required high pH for binding to RBCs. Agglutination of piperacillin‐coated RBCs was observed in 91 percent of donors' and 49 percent of patients' plasma and was inhibited by piperacillin. In contrast to patients with IHA due to piperacillin, donors' plasma tested in the presence of piperacillin did not react. Tazobactam antibodies were not detected. CONCLUSION: A high percentage of donors' and patients' plasma contain an antibody to piperacillin or a chemically related structure detected by testing with piperacillin‐coated RBCs. A diagnosis of piperacillin‐induced IHA should not be made solely on the reactivity of a patient's plasma/serum with piperacillin‐ or piperacillin/tazobactam‐coated RBCs; testing in the presence of piperacillin is more reliable.     

Binding enhancements of antibody functionalized natural and synthetic fibers

Development of low cost biosensing using convenient and environmentally benign materials is important for wide adoption and ultimately improved healthcare and sustainable development. Immobilized antibodies are often incorporated as an essential biorecognition element in point-of-care biosensors but these proteins are costly. We present a strategy of combining convenient and low-cost surface functionalization approaches for increasing the overall binding activity of antibody functionalized natural and synthetic fibers. We demonstrate a simple one-step in situ silica NP growth protocol for increasing the surface area available for functionalization on cotton and polyester fabrics as well as on nanoporous cellulose substrates. Comparing this effect with the widely adopted and low cost plant-based polyphenol coating to enhance antibody immobilization, we find that both approaches can similarly increase overall surface activity, and we illustrate conditions under which the two approaches can produce an additive effect. Furthermore, we introduce co-immobilization of antibodies with a sacrificial “steric helper” protein for further enhancing surface activities. In combination, several hundred percent higher activities compared to physical adsorption can be achieved while maintaining a low amount of antibodies used, thus paving a practical path towards low cost biosensing.

Cotton, nanoporous cellulose and polyester fabric surfaces are functionalized with combinations of in situ grown silica NPs, polyphenol coating, and protein co-immobilization to enhance surface area, antibody binding efficiency, and biosensing.     

异基因造血干细胞移植后并发自身免疫性溶血性贫血患者产生类抗体血清学检测方法及输血策略

目的探讨异基因造血干细胞移植（AHSCT）后免疫介导的自身免疫性溶血性贫血（AIHA）患者交叉配血不合的原因,血浆中不规则抗体筛选试验阳性时,不规则抗体鉴定的血清学检测方法的选择,处理措施及输血策略。 方法一例AHSCT后的3岁男性患儿因重度贫血为改善贫血症状需输注红细胞入住四川省人民医院。通过盐水试管法确定了患儿ABO、Rh血型后进行交叉配血试验发现患儿与多个同型供者血液不相合,考虑到患儿贫血严重,于是采用直接抗球蛋白试验来判断红细胞是否被致敏;通过盐水介质试管法、微柱抗球蛋白法和聚凝胺法进行ABO血型系统以外的不规则抗体筛选试验以确定血浆中有无不规则抗体;根据不规则抗体筛选试验结果选择聚凝胺法来确定抗体的特异性;通过盐水介质试管法、经典抗球蛋白法和聚凝胺法进行交叉配血实验,结合抗体鉴定的结果,综合分析选择合适的供者红细胞输注。 结果本例患儿ABO血型为AB型,Rh分型为CCDee,ABO血型已转变为供者血型。直接抗球蛋白试验强阳性,红细胞被抗体致敏。血浆中检出了不规则抗体,红细胞上放散下来的致敏抗体与血清中检出的不规则抗体均为类抗-Ce自身抗体。选择了避开类抗-Ce抗体的Ce抗原阴性的AB型红细胞输注后血色素升高,3 d后复查血常规Hb为79 g/L,输血有效。 结论任何类型的AHSCT后都有可能发展成AIHA,也就是血浆中可能存在某种自身抗体（类抗体）而破坏自身红细胞,若能够明确患者血清中存在的类抗体,即可避开这种抗体而筛选红细胞无相应抗原的供者,也就避免发生溶血性输血反应以安全输血。     

Antibodies to oxaliplatin, a chemotherapeutic, are found in plasma of healthy blood donors

BACKGROUND: Oxaliplatin is one of the platinum chemotherapeutics that includes cisplatin and carboplatin. Antibodies to all three drugs have caused immune hemolytic anemia (IHA). In an investigation of oxaliplatin‐induced IHA, the negative plasma control agglutinated oxaliplatin‐coated red blood cells (RBCs). Previous preparations of this control had not agglutinated oxaliplatin‐ or cisplatin‐coated RBCs. STUDY DESIGN AND METHODS: Drug‐coated RBCs, prepared by incubating 1/10th volume of RBCs with 1 mg/mL drug in phosphate‐buffered saline for 1 hour at 37°C, were incubated with plasma from random blood donors and patients. Plasma was treated with dithiothreitol to determine the immunoglobulin class. Hapten inhibition was performed by incubating plasma with solutions of oxaliplatin or cisplatin. RESULTS: Nineteen of 121 (16%) donors' plasma samples agglutinated oxaliplatin‐coated RBCs; 7 of 102 (7%) donors' plasma samples agglutinated cisplatin‐coated RBCs. Two of 50 (4%) patients' samples agglutinated oxaliplatin‐coated RBCs. The agglutinin was immunoglobulin M and inhibited by oxaliplatin and cisplatin. CONCLUSION: An agglutinin reactive with oxaliplatin‐coated RBCs was found in 16% of donors' and 4% of patients' samples. Inhibition by oxaliplatin and cisplatin indicates the antibody may be directed to platinum. The presence of this antibody in healthy individuals may be related to the increasing environmental presence of platinum in air and soil as a byproduct of automobile catalytic converters and pharmaceuticals in our water and food chain. This antibody in individuals without IHA suggests that testing untreated and enzyme‐treated RBCs in the presence of a solution of drug may be the best method to investigate IHA caused by drugs in the platinum family.     

The role of the elution in antibody investigations

BACKGROUND: The direct antiglobulin test (DAT) commonly detects immunoglobulin G (IgG) molecules or complement fragments on the red blood cell (RBC) surface. If IgG antibodies are present then elution procedures can be performed to identify the specificity of these antibodies. Our reference laboratory performs elutions on the RBCs of those patients who have received cellular blood products in the past 30 days and have either a newly identified positive DAT with anti-IgG or the agglutination strength is increased over a previous DAT and if ordered by a clinician regardless of transfusion history. This study questioned how frequently elutions contributed novel serologic information under our reference laboratory's current policy or whether elutions should be performed in more selective serologic conditions.
STUDY DESIGN AND METHODS: Recipients whose RBCs underwent eluate testing were identified from the blood bank's database and information about the antecedent DAT and antibody detection test and eluate was recorded.
RESULTS: In total 648 eluates were evaluated and 82 of 648 (12.7%) revealed a novel antibody not present in the serum (an informative eluate). In 2 of 82 informative eluates non–anti-A/B alloantibodies that were not present in the serum were detected: one example each of anti-D and anti-E. Both were associated with a microscopically positive antecedent DAT. The rate of an informative eluate was higher when the antibody detection test was negative.
CONCLUSION: The strength of the DAT does not indicate the likelihood of an informative eluate. Performing an eluate when the antibody detection test is positive has limited value.     

Immobilization of antibody to a plastic surface by toluene 2,4-diisocyanate and its application to radioimmunoassay

A novel procedure to fix antibody on a plastic surface was developed, using a polystyrene tube treated with 0.5% toluene 2,4-diisocyanate (TC) dissolved in carbon tetrachloride. Using 125I-labelled IgG, we found that the rate of immobilization of IgG was significantly higher than that obtained by simple adsorption or coupling by glutaraldehyde in the presence of high concentrations of IgG. The polystyrene tube coated with anti-rabbit gamma-globulin goat serum allowed a simple separation of the tracer bound to the first antibody from the free tracer in a radioimmunoassay of thyroid-stimulating hormone and prolactin, when the coating was done by the TC method but not by the simple adsorption or glutaraldehyde methods.     

Two missense mutations in the CD44 gene encode two new antigens of the Indian blood group system

BACKGROUND: Blood samples were referred over a 10-year period from five patients whose serum samples contained antibodies to unidentified high-incidence antigens. Three patients (A, B, C) were of Moroccan origin and their antibodies and red blood cells (RBCs) were mutually compatible, but incompatible with those of the other two patients (D, E), who were of Pakistani origin. The antibodies and RBCs of D and E were mutually compatible, but incompatible with those of Patients A, B, and C. All the antibodies were detected during pregnancy. STUDY DESIGN AND METHODS: Serologic tests, including the use of enzyme-treated and chemically modified RBCs, suggested a relationship to CD44 (Indian blood group system). The monoclonal antibody immobilization of erythrocyte antigens (MAIEA) assay with monoclonal CD44 antibodies, immunoblotting of RBC membranes, and CD44 gene sequencing were carried out. RESULTS: Positive reactions in the MAIEA assay confirmed that the patients' antibodies are directed at CD44. Immunoblotting with two of the antibodies gave positive reactions of identical size to monoclonal anti-CD44 and failed to react with the RBCs of a CD44-deficient patient. One of the antibodies reacted with purified CD44. Sequencing of Exons 1 to 5 of CD44 revealed 255C>G in Exon 3 for A, B, and C encoding H85Q and 488C>A in Exon 5 for D and E encoding T163K [corrected] CONCLUSION: Two novel CD44 antigens of high incidence have been identified: IN3 (INFI) and IN4 (INJA) in the IN (Indian) blood group system. Lack of IN3 and IN4 results from homozygosity for mutations encoding H85Q and T163R in CD44, respectively.     

Immunoadsorption of IgG onto second antibody covalently attached to Amino-Dylark beads for radioimmunoassays

We present a solid-phase immobilization method for radioligand assays, using an immunoadsorption coating procedure of anti-triiodothyronine rabbit IgG (anti-T3 IgG) onto second antibody (sheep anti-rabbit IgG) covalently bound to Amino-Dylark beads. The second antibody was in excess, compared with the first antibody, thus eliminating reproducibility problems between immunoadsorptions. Beads coated with second antibody can be used to immobilize a variety of antigen-specific first antibodies. The amount of anti-T3 antibody required for solid-phase T3 radioimmunoassay (RIA) was only 10% more, per assay tube, than that utilized in liquid-phase T3 RIA, in which polyethylene glycol solution was the separation reagent; characteristics of assay performance were comparable. The immobilization procedure requires high-titer antisera or antigen-specific IgG and seems advantageous because of the decrease in antibody requirements without significant modification of antibody functionality.