BACKGROUND & AIMS: BACKGROUND:An effective malaria transmission-blocking vaccine (TBV) would be a major advance in the current efforts to eliminate and, ultimately, eradicate malaria. Antibodies against Plasmodium falciparum surface protein, Pfs25, are known to block parasite development in the mosquito vector. However, in initial clinical trials the limited immunogenicity of recombinant Pfs25 protein-in-adjuvant vaccines has been a challenge. METHODS:Novel human adenovirus type 5 (Ad5) vectors were used in heterologous prime boost vaccination strategies to augment the immune response against Pfs25. Specifically, an Ad5 vector that directs expression of full-length, membrane-bound Pfs25 was used as a priming immunization followed by a boost with Ad5 viral particles displaying only the Pfs25 epitope targeted by transmission-blocking antibodies 4B7 and 1D2 (Pfs25 aa 122-134) in hypervariable region 5 of the hexon capsid protein. RESULTS:This heterologous prime-boost vaccine strategy induced antibodies that significantly inhibit P. falciparum transmission to mosquitoes in a standard membrane-feeding assay. Further, immunized mice generated a robust anti-Pfs25 antibody response characterized by higher titer, higher relative avidity and a broader IgG subclass profile than observed with a homologous prime-boost with recombinant Pfs25/alum. CONCLUSION:The data suggest that focusing the immune response against defined epitopes displayed on the viral capsid is an effective strategy for transmission-blocking vaccine development.

背景与目标：

BACKGROUND & AIMS: INTRODUCTION:Monoclonal antibody (mAb)-based products are highly specific for a particular antigen. This characteristic feature of the molecules makes them an ideal tool for many applications including cancer diagnosis and therapy. SOURCES OF DATA:We performed comprehensive searches of PubMed, Medline and the Food and Drug Administration website using keywords such as 'therapeutic antibodies' and 'anti-cancer antibodies'. AREAS OF AGREEMENT:Treatment of cancer patients with antibodies when used alone or in combination with chemotherapy and radiotherapy, or conjugated to drugs or radioisotopes, prolongs overall survival in cancer patients. Currently, there are 14 mAb-based drugs that have been approved for the treatment of cancer patients. AREAS OF CONTROVERSY:The response of cancer patients to antibody therapy can be of short duration. Therapeutic antibodies are expensive and may have side effects. There are no reliable predictive biomarkers for sensitivity or resistance to certain therapeutic antibodies. FUTURE FOCUS: There should be additional studies to discover novel therapeutic targets, to develop more effective antibody-based drugs with fewer side effects, to identify more reliable predictive biomarker(s) for response to therapy with antibody-based drugs and to develop alternative strategies (e.g. transgenic plants, transgenic farm animals) for production of large quantities and more affordable batches of therapeutic antibodies. AREAS TIMELY FOR DEVELOPING RESEARCH:A better understanding of cancer biology, the hallmarks of human cancers and the immune system would lead to identification of additional cell surface biomarkers. These in turn would facilitate the development of novel and biosimilar antibody-based drugs and their routine use as 'magic bullets' for the targeted therapy of human cancers.

背景与目标：

BACKGROUND & AIMS: :The potential of bispecific T cell-engaging antibodies is hindered by manufacturing challenges and short serum half-life. We circumvented these limitations by treating mice with in vitro-transcribed pharmacologically optimized, nucleoside-modified mRNA encoding the antibody. We achieved sustained endogenous synthesis of the antibody, which eliminated advanced tumors as effectively as the corresponding purified bispecific antibody. Because manufacturing of pharmaceutical mRNA is fast, this approach could accelerate the clinical development of novel bispecific antibodies.

背景与目标： : 双特异性T细胞结合抗体的潜力受到制造挑战和血清半衰期短的阻碍。我们通过用体外转录的药理优化的核苷修饰的编码抗体的mRNA处理小鼠来规避这些限制。我们实现了抗体的持续内源性合成，与相应的纯化双特异性抗体一样有效地消除了晚期肿瘤。由于药物mRNA的制造速度很快，因此该方法可以加速新型双特异性抗体的临床开发。

BACKGROUND & AIMS: We analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti-Ro, anti-La and anti-histidyl-tRNA synthetase (Jo-1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti-Ro52+ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti-Ro60 and anti-La activity was found in 4% of IIM sera. Anti-Ro52 antibodies were present in 20% of IIM sera. However, in anti-Jo-1+ IIM sera (21%), the frequency of the anti-Ro52 antibodies was found to be much higher (58%). No cross-reactivity between anti-Ro52 and anti-Jo-1 antibodies could be detected in these sera. To learn more about the nature of anti-Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by anti-Ro52+ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long alpha-helical region which contains two potential coiled-coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti-Jo-1+ and anti-Jo-1- IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo-1 in IIM patients is coupled.

背景与目标： 我们分析了112个特发性炎症性肌病 (IIM) 血清中存在抗Ro，抗La和抗组氨酸-tRNA合成酶 (Jo-1) 自身抗体，随后将b细胞表位绘制在anti-Ro52 IIM血清识别的Ro52蛋白上。通过免疫印迹，ELISA和RNA沉淀对血清进行表征。在4% 的IIM血清中发现了anti-Ro60和抗La活性。Anti-Ro52抗体存在于IIM血清的20% 中。然而，在anti-Jo-1 + IIM血清 (21%) 中，发现anti-Ro52抗体的频率要高得多 (58%)。在这些血清中未检测到anti-Ro52和anti-Jo-1抗体之间的交叉反应。为了更多地了解IIM血清中anti-Ro52抗体的性质，我们通过体外翻译的Ro52缺失突变体的免疫沉淀分析了anti-Ro52 IIM血清靶向的Ro52蛋白的主要表位。主要表位被定位在与氨基酸126和252接壤的区域中。蛋白质的这一部分包括一个长的 α 螺旋区域，该区域包含两个潜在的卷曲螺旋结构域以及亮氨酸拉链基序。尽管在anti-Jo-1和anti-Jo-1- IIM血清之间没有观察到Ro52表位识别的差异，但我们的结果表明IIM患者针对Ro52和Jo-1的自身免疫反应是偶联的。

BACKGROUND & AIMS: The growth and vascularization of many tumours has been reported to be associated with the overexpression of the potent mitogenic and angiogenic polypeptide basic fibroblast growth factor (beta-FGF). Consequently, it has been proposed that inhibition of beta-FGF action would prevent the growth of beta-FGF-dependent tumours. In this study, cell culture assays were established to assess the ability of mouse monoclonal DG-2 anti-beta-FGF antibodies to inhibit the mitogenic action of beta-FGF in vitro. Following in vitro characterisation, the monoclonal DG-2 antibodies were used to evaluate the role of beta-FGF in promoting the vascularization and growth of rat chondrosarcoma tumours. The effect the monoclonal anti-B-FGF antibodies had on tumour vascularization and growth in vivo were monitored using a 99m Technetium (99mTc)-labelled red blood cell procedure. The characterization studies confirmed that the DG-2 monoclonal antibody recognised beta-FGF and inhibited its mitogenic action on mouse Balb/c cells and bovine endothelial cells in vitro. When examined in vivo, intralesional administration of mouse monoclonal DG-2 antibody significantly inhibited rat chondrosarcoma growth and vascularization. However when the monoclonal DG-2 antibody was administered intraperitoneally or intravenously no attenuation of rat chondrosarcoma tumour vascularization or growth was observed. This report has confirmed the potential effectiveness of anti-beta-FGF antibodies in the regulation of tumour growth. It has also demonstrated that further studies on the pharmacokinetics of administered antibodies and their mode of delivery are required so that the effectiveness of such anti-growth factor immunotherapy can be assured.

背景与目标： 据报道，许多肿瘤的生长和血管化与有效的有丝分裂和血管生成多肽碱性成纤维细胞生长因子 (β-FGF) 的过表达有关。因此，已经提出抑制 β-FGF作用将阻止 β-FGF依赖性肿瘤的生长。在这项研究中，建立了细胞培养试验以评估小鼠单克隆DG-2抗 β-FGF抗体在体外抑制 β-FGF的促有丝分裂作用的能力。在体外表征之后，使用单克隆DG-2抗体来评估 β-FGF在促进大鼠软骨肉瘤肿瘤的血管化和生长中的作用。使用99m tech (99mTc) 标记的红细胞程序监测单克隆抗B-FGF抗体对体内肿瘤血管化和生长的影响。表征研究证实，DG-2单克隆抗体在体外识别 β-FGF并抑制其对小鼠Balb/c细胞和牛内皮细胞的促有丝分裂作用。当在体内检查时，小鼠单克隆DG-2抗体的病灶内给药可显着抑制大鼠软骨肉瘤的生长和血管形成。然而，当腹膜内或静脉内施用单克隆DG-2抗体时，未观察到大鼠软骨肉瘤肿瘤血管化或生长的减弱。该报告证实了抗 β-FGF抗体在调节肿瘤生长中的潜在有效性。还表明，需要对所给药抗体的药代动力学及其传递方式进行进一步研究，以便可以确保这种抗生长因子免疫疗法的有效性。

BACKGROUND & AIMS: :In order to study the antigenicity of envelope 46 kDa glycoprotein (gp46) of human T-cell leukemia virus type-I (HTLV-1), we have generated monoclonal anti-gp46 antibodies (MAbs), REY-7, REY-11, REY-16, REY-30, MET-2 and MET-3 from rats and mice. Immunoblot and immunofluorescence assays showed that these MAbs recognize gp46 and its related antigens, and specifically stained HTLV-I-bearing cells. All MAbs reacted with a recombinant gp46 antigen, N147, expressing the 147 amino acids in the C-terminal half of gp46. By using various synthetic peptides corresponding to the gp46 sequence, epitopes recognized by REY-7 and MET-3, REY-11 and REY-16, and REY-30 were mapped to regions corresponding to the amino acids 175-199, 253-282 and 288-312, respectively. MET-2 did not react with any of the peptides used. These results indicate that the present MABs are directed against at least 4 distinct epitopes expressed on the C-terminal half of gp46. The binding of these MAbs to gp46 was specifically inhibited by sera from HTLV-I-infected individuals, but none of these MAbs inhibited the cell fusion activity of HTLV-I.

背景与目标： : 为了研究人T细胞白血病病毒I型 (HTLV-1) 的包膜46 kda糖蛋白 (gp46) 的抗原性，我们从大鼠和小鼠中产生了单克隆anti-gp46抗体 (mab)，REY-7，REY-11，REY-16，REY-30，MET-2和MET-3。免疫印迹和免疫荧光测定表明，这些单克隆抗体识别gp46及其相关抗原，并特异性染色的HTLV-I携带细胞。所有mab均与重组gp46抗原N147反应，在gp46的C-末端一半表达147个氨基酸。通过使用对应于gp46序列的各种合成肽，将由REY-7和MET-3、REY-11和REY-16识别的表位和REY-30分别映射到对应于氨基酸175-199、253-282和288-312的区域。MET-2不与使用的任何肽反应。这些结果表明，本发明的mab针对在gp46的C末端一半上表达的至少4个不同的表位。这些单克隆抗体与gp46的结合被htlv-i感染个体的血清特异性抑制，但这些单克隆抗体均未抑制htlv-i的细胞融合活性。

BACKGROUND & AIMS: An enzyme-linked immunosorbent assay for measuring type I collagen degradation products in serum (S-ELISA) was developed. The assay uses a high affinity polyclonal antibody which reacts with an isomerized form of an 8 amino acid sequence of the C-telopeptides of type I collagen (EKAHD-beta-GGR). Cross-reactivity to a nonisomerized synthetic peptide form of the 8 amino acid sequence is less than 0.2%. Values obtained in a group of premenopausal women (age, 33.3 +/- 3.11 years) were 69 +/- 24 ng/ml(n = 22). In a group of early postmenopausal women (age, 51.8 +/- 1.88 years) values obtained were 125 +/- 43 ng/ml (n = 46), which represents an increase of 81% (p < 0.001). Values found in untreated patients with Paget's disease were 234 +/- 95 ng/ml (n = 15), and for primary hyperparathyroidism we found 335 +/- 82 ng/ml (n = 10). Intravenous administration of a bisphosphonate (Pamidronate) to Paget's disease patients for 3 days was reflected in the S-ELISA by a decrease in the values of 55% when compared with values before treatment (n = 15). Following treatment with another bisphosphonate (Alendronate) for 6 months, values were decreased to 48 +/- 19 ng/ml (n = 12), which corresponds to a 62% decrease. Clinical results presented in this context support that the assay is a sensitive and specific index of bone resorption. It may, therefore, prove useful in the follow up of treatment of patients with metabolic bone diseases and in the clinical investigation of osteoporosis.

背景与目标： 开发了一种用于测量血清中I型胶原蛋白降解产物的酶联免疫吸附测定法 (S-ELISA)。该测定使用高亲和力多克隆抗体，该抗体与I型胶原蛋白 (EKAHD-β-GGR) 的C-端肽的8个氨基酸序列的异构化形式反应。对8个氨基酸序列的非异构化合成肽形式的交叉反应性小于0.2%。在一组绝经前妇女 (年龄，33.3 +/- 3.11岁) 中获得的值为69 +/- 24 ng/ml(n = 22)。在一组绝经后早期妇女 (年龄，51.8 +/- 1.88岁) 中，获得的值为125 +/- 43 ng/ml (n = 46)，这表示增加了81% (p <0.001)。在未经治疗的Paget病患者中发现的值为234/- 95 ng/ml (n = 15)，对于原发性甲状旁腺功能亢进，我们发现335/- 82 ng/ml (n = 10)。与治疗前的值相比 (n = 15)，在S-ELISA中反映了将双膦酸盐 (帕米膦酸盐) 静脉给药至Paget病患者3天的55% 值降低。用另一种双膦酸盐 (阿仑膦酸盐) 治疗6个月后，值降低至48 +/- 19 ng/ml (n = 12)，这对应于62% 降低。在这种情况下提出的临床结果支持该测定是骨吸收的敏感且特异性的指标。因此，它可能对代谢性骨病患者的治疗随访和骨质疏松症的临床研究有用。

BACKGROUND & AIMS: :Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin--27 kDa, 50 kDa and 20 kDa--aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the 'anti-27 K' and 'anti-50 K' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the 'anti-50 K' to S-1 decreased upon actin binding, while that of the 'anti-27 K' was not affected by binding of S-1 to F-actin. The 'anti-50 K' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the 'anti-27 K' had no effect. The results indicate that either the epitope of the 'anti-50 K' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.

背景与目标： : 使用两种抗人骨骼肌球蛋白的骨骼肌球蛋白单克隆抗体来研究由兔骨骼肌球蛋白制备的S-1的功能，一级和三级结构之间的相关性。S-1的重链被胰蛋白酶切割成三个片段-27 kDa，50 kDa和20 kDa-从N末端按此顺序排列。第一个抗体的表位被分配给S-1的N-末端1-23个氨基酸段，因为它与S-1的27 kda N-末端胰蛋白酶片段反应，但不与缺少上述氨基酸段的27 kda片段的衍生物反应。将第二个抗体的表位分配到S-1中央50 kda结构域的3 kda N末端区域。该分配基于S-1的蛋白水解和光化学切割以及通过特异性抗体标记其N末端。在抗体与肌球蛋白的旋转阴影复合物的电子显微照片上，可以看到抗体与肌球蛋白头部的结合。显微照片上的测量表明，肌球蛋白的头尾连接与 “抗27 k” 和“ 抗50 k” 表位之间的距离分别为14 nm和17 nm。两种抗体对S-1具有高亲和力。肌动蛋白结合后，“抗-50k” 对S-1的亲和力降低，而“ 抗-27k” 的亲和力不受S-1与F-肌动蛋白结合的影响。抗50 K' 抗体抑制S-1的K + (EDTA) 和肌动蛋白激活的ATPase活性，而抗27 K' 则没有作用。结果表明，“anti-50k” 的表位靠近肌动蛋白或S-1的ATP结合位点，或者在这些位点与表位之间存在以传播构象变化表示的通讯。

BACKGROUND & AIMS: :Serum samples from 224 aboriginal Amazonian Indians were tested for antibodies to HTLV-III/LAV by an indirect immunofluorescence (IF) assay. 9 individuals (4%), 5 of them female, were seropositive by IF and by confirmatory western blotting and radioimmunoprecipitation tests. 3 of the positive sera were collected in 1968. HTLV-III/LAV seropositivity rates varied among the ethnic groups and ranged from 13.3% among the Pemon Indians to 3.3% among the Yanoama tribe. The titres of HTLV-III/LAV antibodies ranged from 1/40 to 1/320. All individuals tested were apparently healthy at the time of the study. None of 211 randomly chosen, healthy blood donors from Venezuelan cities had antibodies to HTLV-III/LAV. The prevalence of specific antibodies among Amazonian Indians suggests the HTLV-III/LAV or a closely related cross-reactive virus may be endemic in this area. The findings also indicate that this virus is indigenous in non-negroid Latin American and negroid tropical populations.

背景与目标： : 通过间接免疫荧光 (IF) 测定法测试来自224土著亚马逊印第安人的血清样品的htlv-iii/LAV抗体。9例 (4%)，其中5例为女性，通过IF以及验证性western印迹和放射免疫沉淀试验呈血清反应阳性。1968年收集3份阳性血清。Htlv-iii/LAV血清阳性率因种族而异，范围从Pemon印第安人的13.3% 到Yanoama部落的3.3%。Htlv-iii/LAV抗体的滴度范围为1/40至1/320。在研究时，所有接受测试的人显然都很健康。来自委内瑞拉城市的211随机选择的健康献血者中没有一个具有针对htlv-iii/LAV的抗体。亚马逊印第安人中特异性抗体的流行表明，htlv-iii/LAV或密切相关的交叉反应病毒可能在该地区流行。研究结果还表明，该病毒在非negroid拉丁美洲和negroid热带人群中是本地的。

BACKGROUND & AIMS: BACKGROUND AND AIMS:Infection with the hepatitis C virus (HVC) is one of the most common viral infections worldwide. Approximately 170 million individuals are infected worldwide. HCV is an important cause of morbidity and mortality. In Mexico, according to the National Health Survey 2000, it is estimated that 70,000 cases exist. We undertook this study to estimate the prevalence of anti-HCV antibodies in patients with association to the risk factors for HCV infection in the lowland (bajio) region. METHODS:There were 2803 individuals 15 years of age or older who were treated at the General Hospital Zone #4 who were included in this study. Following informed consent, the participants were given a questionnaire listing the major risk factors for hepatitis C. If they answered positive to any of these identified factors, a blood sample was taken to determine anti-HCV antibodies via ELISA analysis. RESULTS:Average age in this study was 38.4 ± 13.5 years, and 75.5% were female (n = 2116). Anti-HCV antibodies were isolated in 1.3% of the patients (n = 36). The most commonly identified risk factor among all the participants was a history of previous transfusions (28.8 % of all patients, n = 813 and 41.7%, n = 15 of those with positive HCV antibodies). This was the only statistically significant risk factor identified in this study (p = 0.066). CONCLUSIONS:Mexico is currently considered to have a lower prevalence for HCV in relation to developed countries and other endemic areas. The figures reported are lower than those observed in this study, suggesting that the strategies for detecting HCV in Mexico may be inadequate.

背景与目标：

BACKGROUND & AIMS: BACKGROUND:VAR2CSA is a large polymorphic Plasmodium falciparum protein expressed on infected erythrocytes (IE) that allows their binding in the placenta, thus precipitating placental malaria (PM). The N-terminal part of VAR2CSA that contains the binding site to placental chondroitin sulfate A (CSA) is currently recognized as the most attractive region for vaccine development. An ultimate challenge is to define epitopes in this region that induce a broad cross-reactive adhesion inhibitory antibody response. METHODS:Based on phylogenetic data that identified a dimorphic sequence motif in the VAR2CSA DBL2X, we raised antibodies against the NTS-DBL2X constructs containing one sequence or the other (3D7 and FCR3) and tested their functional properties on P. falciparum isolates from pregnant women and on laboratory-adapted strains. RESULTS:The CSA binding inhibitory capacity of the antibodies induced varied from one parasite isolate to another (range, 10%–100%), but the combined analysis of individual activity highlighted a broader functionality that increased the total number of isolates inhibited. Interestingly, the differential inhibitory effect of the antibodies observed on field isolates resulted in significant inhibition of all field isolates tested, suggesting that optimal inhibitory spectrum on field isolates from pregnant women might be achieved with antibodies targeting limited variants of the N-terminal VAR2CSA. CONCLUSIONS:Our findings indicate that the NTS-DBL2X region of VAR2CSA can elicit strain-transcending anti-adhesion antibodies and suggest that the combination of the two major variants used here could represent the basis for an effective bivalent VAR2CSA-based vaccine.

背景与目标：

BACKGROUND & AIMS: INTRODUCTION:Statins or 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (HMGCR) are among the most commonly prescribed treatment in France. They may be responsible for muscular intolerance with variable severity. They have been recently involved in the occurrence of an acquired inflammatory myopathy associated with anti-HMGCR antibodies. This new type of toxic myopathy remains poorly known by clinicians. OBSERVATION:We report a 61-year-old woman treated with a statin for many years who developed a lower and upper limb disabling myopathy with a rapid unfavourable course despite treatment withdrawal. Clinical history and investigations, especially including an assay for anti-HMGCR antibodies led to the diagnosis of autoimmune necrotizing myopathy with anti-HMGCR antibodies. Subsequent initiation of an immunosuppressive treatment by corticosteroids and methotrexate was effective. CONCLUSION:Statins may unmask or cause an autoimmune necrotizing myopathy associated with the presence of anti-HMGCR antibodies. Their identification is now routinely available. An immunosuppressive treatment is necessary and justified by the autoimmune nature of the disease.

背景与目标：

BACKGROUND & AIMS: :We investigated immunoglobulin G (IgG) subclass antibody responses to Plasmodium falciparum merozoite surface protein 1 (MSP-1) and MSP-2 in 112 malaria-exposed subjects in Brazil. IgG3 polarization was primarily epitope driven, being little affected by cumulative or current exposure to malaria and not affected by a subject's age and Fcgamma receptor IIA genotype.

背景与目标： : 我们调查了巴西112名疟疾暴露受试者对恶性疟原虫裂殖子表面蛋白1 (MSP-1) 和MSP-2的免疫球蛋白G (IgG) 亚类抗体反应。IgG3极化主要是表位驱动的，几乎不受累积或当前暴露于疟疾的影响，并且不受受试者年龄和Fcgamma受体IIA基因型的影响。

BACKGROUND & AIMS: :Diagnosis of eukaryotic parasitic infection using antibody-based tests such as ELISAs (enzyme-linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three-dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods. To address this problem, a structural approach was used to develop monoclonal antibodies (mAbs) that detect asparaginyl-tRNA synthetase (AsnRS) secreted from B. malayi. B. malayi and human AsnRS amino acid sequences were aligned to identify regions that are relatively unconserved, and a 1.9 A crystallographic structure of B. malayi AsnRS was used to identify peptidyl regions that are surface accessible and available for antibody binding. Sequery and SSA (Superpositional Structural Analysis) software was used to analyze which of these peptides was most likely to maintain its native conformation as a synthetic peptide, and its predicted helical structure was confirmed by NMR. A 22-residue peptide was synthesized to produce murine mAbs. Four IgG(1) mAbs were identified that recognized the synthetic peptide and the full-length parasite AsnRS, but not human AsnRS. The specificity and affinity of mAbs was confirmed by Western blot, immunohistochemistry, surface plasmon resonance, and enzyme inhibition assays. These results support the success of structural modeling to choose peptides for raising selective antibodies that bind to the native protein.

背景与目标： : 使用基于抗体的测试 (例如ELISAs (酶联免疫吸附测定)) 诊断真核寄生虫感染通常是有问题的，因为需要区分同源宿主和病原体蛋白，并确保针对肽产生的抗体也会结合肽在其三维蛋白质结构的背景下。由线虫Brugia malayi引起的丝虫病是一种重要的世界性热带疾病，其中寄生虫在白天从血液中消失，从而阻碍了标准的显微镜诊断方法。为了解决这个问题，使用了一种结构方法来开发单克隆抗体 (mab)，该单克隆抗体可检测马来亚芽胞杆菌分泌的天冬酰基-tRNA合成酶 (AsnRS)。对马来亚杆菌和人AsnRS氨基酸序列进行比对以鉴定相对不保守的区域，并且使用马来亚杆菌AsnRS的1.9晶体结构来鉴定表面可及且可用于抗体结合的肽基区域。Sequery和SSA (叠加结构分析) 软件用于分析这些肽中的哪一种最有可能保持其天然构象为合成肽，并通过NMR确认了其预测的螺旋结构。合成了22个残基的肽以产生鼠mab。鉴定出四种IgG(1) mab，它们识别合成肽和全长寄生虫AsnRS，但不识别人类AsnRS。通过蛋白质印迹，免疫组织化学，表面等离子体共振和酶抑制试验证实了mab的特异性和亲和力。这些结果支持结构建模的成功，以选择用于产生与天然蛋白质结合的选择性抗体的肽。

BACKGROUND & AIMS: OBJECTIVES:Anti-phospholipid antibodies have been recognized to play a role in vascular thrombosis and pregnancy morbidity. They were first thought to be directed to phospholipids, but it is now known that the majority of pathogenic antibodies recognizes epitopes on phospholipid-binding plasma proteins such as beta2-glycoprotein I (beta2GPI) or possibly also annexin A5 (ANXA5). The mechanism of their prothrombotic action is still not completely understood. The aim of the present study was to observe the effect of antibodies against ANXA5 (aANXA5) and antibodies against beta2GPI (abeta2GPI) on the binding of ANXA5 to the negatively charged phospholipid membrane. METHODS:Giant phospholipid vesicles (GPVs) were used as a simple model of the membrane surface. GPVs composed of phosphatidylserine and phosphatidylcholine were produced in an aqueous medium. A single GPV was transferred to the solution containing ANXA5 conjugated with Alexa Fluor 488 (FANXA5) and (i) aANXA5 or abeta2GPI and (ii) different concentrations of abeta2GPI together with beta2GPI. The emission of the fluorescent light from the GPV surface, as the result of FANXA5 binding, was measured. RESULTS:Beta2GPI together with abeta2GPI reduced the binding of FANXA5 to GPVs. On the contrary, aANXA5 enhanced the binding of ANXA5 to the GPV surface. CONCLUSIONS:Our results point to the competition between FANXA5 and complexes of beta2GPI-abeta2GPI for the same binding sites and therefore support the hypothesis of the disruption of the ANXA5 protective shield on procoagulant phospholipid surface. The influence of increased cell surface ANXA5 concentration in the presence of aANXA5 on coagulation needs to be further studied.

背景与目标：