Screening for celiac disease in Down's syndrome patients revealed cases of subtotal villous atrophy without typical for celiac disease HLA-DQ and tissue transglutaminase antibodies

A1M： To investigate the prevalence of celiac disease （CD） as well as CD marker antibodies and susceptibility HLA-DQ haplotypes in 134 karyotyped Down＇s syndrome （DS） patients. METHODS： Immunoglobulin A （IgA） and G （IgG） type anti-gliadin antibodies （AGA）, IgA type anti-tissue transglutaminase （tTG） antibodies （anti-tTG） with antigen of guinea pig and human source were determined by enzyme-linked immunosorbent assay and endomysium antibodies （EHA） by indirect immunofiuoresence test. HLA-DQA1＊0501/DQB1＊0201 （DQ2） was revealed by polymerase chain reaction. Celiac disease was diagnosed by revised ESPGHAN criteria. RESULTS： 41% of DS patients had AGA, 6.0% IgA anti-tTG with guinea pig antigen, and 3.0 % [gA EMA （all positive for anti-tTG with human tTG）. Subtotal villous atrophy was found in 5 out of 9 DS patients who had agreed to small bowel biopsy. One of them had DQA1＊0S01/DQB1＊0201 and anti-tTG and EMA i.e. typical for CD markers （this case also fulfilled the ESPGHAN diagnostic criteria）, but other four lacked these markers. Three non-biopsied DS patients had also most probably CD because DQA1＊0S01/DQB1＊0201 and IgA anti-tTG （EMA） were detected. Thus, the prevalence of CD among our DS patients population is 3.0 % （95 % of confidence interval [CI]： 0.1-5.9 %）. CONCLUSION： We confirm the increased frequency of CD among DS patients. In addition, we have revealed a subgroup of patients with subtotal villous atrophy but without characteristic for CD immunological and genetic markers. Whether these cases represent CD （with atypical immunopathogenesis） or some other immune enteropathy, requires further investigations.

Antibody Phage Library Screening Efficiency Measured by KD Values

An antibody phage library was screened with two target molecules, IFNα-2a and FGFR-GST, and the KD value of each round of panning was measured. It was found that the apparent KD values decreased along with each additional panning round, which indicates the increase of the binding affinity between the phage and the target molecules. This result shows that the KD value is a reliable intrinsic parameter and a new method for screening efficiency detection is thus provided.

Preparation and Identification of Monoclonal Antibodies against Chicken Immunoglobulin G

[ Objective] To prepare monoclonal antibodies against chicken immunoglobulin G （IgG） and improve the diagnostic level of specific antibodies in chickens. [ Method] Chicken IgG was isolated by saturated ammonium sulfate and purified by Sephadex G-200 column chromatography. Then the BALB/c mice were immunized by the chicken IgG, and the spleen cells were fused with mouse myeloma cells SP2/0. Finally, the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] Four hybridoma cell strains secre- ting monoclonal antibodies against chicken IgG were obtained and named as C44, C45, C67 and C68, and their ascites titers in indirect ELISA were 1 ： 640 000, 1 ： 320 000, 1 ： 640 000 and 1 ： 80 000, respectively. The monoclonal antibodies secreted by C44 and C45 could recognize light chains of chicken IgG and those secreted by C,67 and C68 could recognize heavy chains of chicken IgG. They all could not recognize IgG from duck, rabbit and swine. Additionally, the Ig type identification results showed that they all belonged to IgGl. [ Conclusion] Four cell strains of obtained hybridoma can stably produce the monoclonal antibodies against chicken IgG.

Screening Testosterone Residue in the Edible Tissues of Bovine with a Polyclonal Antibody Based Heterologous Immunoassay

Testosterone（17β-hydroxyandrost-4-en-3-one）is a19-carbon steroid hormone with potent androgenic properties.It maintains testicular function and develop secondary male sex characteristics.It also has strong anabolic effects,which initiates increased protein synthesis in muscle and bone[1].In the1950s,the recognition of the growth promoting properties of such a hormone

Comparative Study on 4 EIA Kits for Screening Antibody to Hepatitis C Virus in Pooled Sera

Four enzyme immunoassay (EIA) test kits, 1 Canadian product and 3 Chinese products,were used in the comparative study. Each pool consisted of 5 sera, and the 5 single sera were tested as controls. The tests were carried out according to the instructions, keeping the same dilution of each serum in single and pool samples. It was found that with the Canadian kit,the positive and negative results of opled sera had no difference from that of the controls (P>0. 10). In the case of Chinese Yali and Kehua kits, the positive results of pooled sera showed no difference from the controls (P >0. 10), but the optical density (OD) of negative opls were increased (P < 0. 01 ), though quite distant from the cut-off values. In the case of Changzheng kit, the OD of opitive opls were significantly lower than those of the controls (P < 0. 05 ), and weak positive samples missed the detection. However this problem could be overcome by blocking the microwells beforehand. Our experiment demonstrate that not all EIA test kits are suitable for screening opls for antithey to hepatitis C virus, and that it is important to assess the sensitivity of the EIA kit to be used for this purpose.

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor

Hepatitis B immunoglobulin （HBIG） is important in the management of hepatitis B virus （HBV） infection. Aiming to develop recombinant monoclona# antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen （HBsAg）-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the FIp recombinase-mediated integration （FIp-ln） system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting （FACS）, eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression （FSE）, fluorescent strength of binding （FSB） and relative binding ability （RBA） were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

Current issues and future perspectives of gastric cancer screening

Gastric cancer remains the second leading cause of cancer death worldwide. About half of the incidence of gastric cancer is observed in East Asian countries, which show a higher mortality than other countries. The effectiveness of 3 new gastric cancer screening techniques, namely, upper gastrointestinal endoscopy, serological testing, and &#x0201c;screen and treat&#x0201d; method were extensively reviewed. Moreover, the phases of development for cancer screening were analyzed on the basis of the biomarker development road map. Several observational studies have reported the effectiveness of endoscopic screening in reducing mortality from gastric cancer. On the other hand, serologic testing has mainly been used for targeting the high-risk group for gastric cancer. To date, the effectiveness of new techniques for gastric cancer screening has remained limited. However, endoscopic screening is presently in the last trial phase of development before their introduction to population-based screening. To effectively introduce new techniques for gastric cancer screening in a community, incidence and mortality reduction from gastric cancer must be initially and thoroughly evaluated by conducting reliable studies. In addition to effectiveness evaluation, the balance of benefits and harms must be carefully assessed before introducing these new techniques for population-based screening.

Production and Characterisation of Monoclonal Antibodies against 19-Nortestosterone

Objective To produce anti‐19‐Nortestosterone （NT） monoclonal antibodies and identify their immunological characteristics. Methods Hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate （CAAP） method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant （Kaff）. Results Five hybridoma cell lines, named NT‐1, NT‐2, NT‐3, NT‐4, and NT‐5, were identified and their corresponding mAbs were of the IgG 1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×10 9 L/mol. The titers and IC 50 values of purified ascite fluids were in the range of （0.64–2.56）×10 5 and （0.55–1.0） ng/mL, respectively. Of all the cross‐reacting steroids, α ‐NT was the most reactive with the mAbs at 62% with NT‐1 mAb and 64% with NT‐2 mAb. Negligible cross‐reactivity （0.01%） with other steroids was observed. Conclusion The establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.

Hypothyroidism during pregnancy: Controversy over screening and intervention

Thyroid hormones are critical for foetal neurological development and maternal health. Maternal hypothyroidism during pregnancy is associated with adverse impact on health of the mother as well as the progeny. Reduced thyroid hormone levels predispose the child to develop mental retardation and cognitive delay in early life. In the mother, hypothyroidism during pregnancy is associated with spontaneous abortion, placental abruption, preterm delivery and hypertensive disorders. Therefore, screening and therapeutic intervention is justified to prevent foetal as well as maternal co-morbidities. In view of impact of such a large-scale screening and intervention program on limited healthcare resources, it is debatable if a targeted rather than universal screening program will result in comparable outcomes. In addition, there is an ongoing debate regarding best evidence-based practice for the management of isolated hypothyroxinaemia, subclinical hypothyroidism and euthyroid women with autoimmune hypothyroidism. We have carried out a review of the literature; firstly, to determine whether universal screening for asymptomatic women in early pregnancy would be cost-effective. Secondly, we have retrospectively reviewed the literature to analyse the evidence regarding the impact of therapeutic intervention in women with subclinical hypothyroidism.

Rapid identification of spinal ventral and dorsal roots using a quartz crystal microbalance

The fast and accurate identification of nerve tracts is critical for successful nerve anastomosis. Taking advantage of differences in acetylcholinesterase content between the spinal ventral and dorsal roots, we developed a novel quartz crystal microbalance method to distinguish between these nerves based on acetylcholinesterase antibody reactivity. The acetylcholinesterase antibody was immobilized on the electrode surface of a quartz crystal microbalance and reacted with the acetylcholinesterase in sample solution. The formed antigen and antibody complexes added to the mass of the electrode inducing a change in frequency of the electrode. The spinal ventral and dorsal roots were distinguished by the change in frequency. The ventral and dorsal roots were cut into 1 to 2-mm long segments and then soaked in 250 pL PBS. Acetylcholinesterase antibody was immobilized on the quartz crystal microbalance gold electrode surface. The results revealed that in 10 minutes, both spinal ventral and dorsal roots induced a frequency change; however, the frequency change induced by the ventral roots was notably higher than that induced by the dorsal roots. No change was induced by bovine serum albumin or PBS. These results clearly demonstrate that a quartz crystal microbalance sensor can be used as a rapid, highly sensitive and accurate detection tool for the quick identification of spinal nerve roots intraoperatively.

Prokaryotic Expression and Monoclonal Antibody Preparation of Chicken Major Histocompatibility Complex Class II Molecules

[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha chain of MHC II and exons 3 -6 encoding beta chain of MHC II were performed based on its protein sequences. After BALB/c mice were immunized with the purified fusion proteins, the mouse spleen cells were fused with mouse myeloma cells SP2/0. Then the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] One hybridoma cell strain secreting monoclonal antibody against alpha chain and two strains secreting monoclonal antibody against beta chain were obtained. These three hybridoma cell strains were named as MHC II alpha-4, MHC II betas-2 and MCH II betas-31, respectively. Their titers of ascites in indirect ELISA were 1 ： 256 000, 1 ： 256 000 and 1 ： 1 280 000, respectively. These antibodies could specifically recog- nize MHC II alpha chain or beta chain in western blotting. [ Conclusion] Three obtained hybridoma stains can stably produce the monoclonal antibody against chicken MHC class II molecules.

Influence of obstetric factors on the development of postpartum antithyroid antibodies

Aim: To correlate obstetric data with the appearance of antithyroid antibodies. Methods: A 6 months follow up was performed on 135 healthy women with assessment of TSH, T3, T4 and antithyroid antibodies (anti thyroglobulin and anti peroxidase). Correlation of diverse obstetrical parameters with the appearance of antithyroid antibodies at 2 and 6 months postpartum was determined. Results: Only two parameters were significant during the complete follow up: the newborn weight, which correlated with both antibodies (anti-thyroglobulin and anti-peroxidase) positivity and the maternal height, which exclusively correlated with anti-thyroglobulin positivity. Conclusions: Correlation of maternal height and newborn weight with positive autoantibodies allows to consider a future clinical screening test for this disorder.

孕产妇不规则抗体血清学特征和临床意义分析

目的:明确不规则抗体在孕产妇人群中的血清学特征,探讨其临床意义。方法:选取2017年1月-2022年3月于重庆医科大学附属妇女儿童医院就诊的孕产妇151471例,采用微柱凝胶抗人球蛋白法进行不规则抗体筛查,对部分抗体筛查阳性标本进一步做抗体特异性鉴定。结果:孕产妇不规则抗体筛查阳性率为0.91%(1375/151471),其中0.23%(355/151471)在孕早期检出,0.05%(71/151471)在孕中期检出,0.63%(949/151471)在孕晚期检出,孕晚期抗体阳性率明显高于孕早期和孕中期,且孕晚期新增阳性例数明显高于孕中期新增例数;分析1375例不规则抗体阳性结果的凝集强度,其中凝集强度为弱阳性占比最高,为50.11%(689/1375),可疑阳性占18.69%(257/1375),阳性占31.20%(429/1375),孕中期凝集强度分布与孕早期相比差异无统计学意义,但孕晚期可疑阳性和弱阳性的比例低于孕早期,阳性比例高于孕早期,比较差异有统计学意义(P<0.001)。不规则抗体阳性孕产妇中,妊娠≥2次孕产妇的占比明显高于≤1次者;进行抗体鉴定的60例孕产妇中,Rh系统占25.00%(15/60),Lewis系统占43.33%(26/60),Kidd系统占3.33%(2/60),MNS系统抗体占16.67%(10/60),P1PK系统抗体占1.67%(1/60),自身抗体占1.67%(1/60),4例无法确认特异性占6.67%(4/60);特异性抗体中以抗-Lea(30.00%)最为常见,其次是抗-E(16.67%)和抗-M(16.67%)。结论:不同地区、具有不同遗传背景的孕产妇人群的不规则抗体血清学特征存在差异,掌握本地区孕产妇不规则抗体的特征,对保证孕产妇输血安全、防治新生儿溶血病具有十分重要的意义。

抗-E、抗-c、抗-M、抗-S多重抗体分析

目的对1例存在多重抗体的肾积水伴输尿管结石合并休克老年患者进行抗体特异性鉴定。方法微柱凝胶法进行红细胞不规则抗体筛查和抗体鉴定,酶法,抗体吸收法协助判断患者的抗体特异性;盐水试管法检测患者ABO血型鉴定、Rh血型鉴定、MNS血型。结果患者血型:O、CCDee、NNss,患者体内检测出抗-E、抗-c、抗-M、抗-S联合抗体。结论反复输血可能引起患者存在1种或者多种意外抗体,体内多种或高频率抗体的患者,可能引起鉴定困难和用血延迟。

住院拟输血患者不规则抗体的鉴定与分析

目的:分析71847例拟输血患者不规则抗体的类型和分布特点,探究其对临床安全输血的作用。方法:选取本院2020年1月到2023年10月期间71847例拟输血患者的血液标本,采用微柱凝胶检测卡进行不规则抗体筛查及抗体鉴定。结果:71847例拟输血患者中,共筛选出不规则抗体阳性301例(0.42%),包括同种抗体252例(83.72%);其中Rh系统最高,为179例(59.47%)。Rh系统抗体包括抗E 135例(44.85%)、抗E+c 24例(7.97%)、抗C+e 10例(3.32%)、抗c 6例(1.99%)、抗D 3例(1.00%)和抗D+C 1例(0.33%)。通过对301例不规则抗体进行分析发现,>60岁组的阳性率高于≤60岁组(0.61%vs 0.33%),女性组的阳性率高于男性组(0.50%vs 0.31%),内科组(1.25%)和妇产科组(0.32%)的阳性率均高于外科(0.20%),妊娠/输血史组的阳性率高于无妊娠/输血史组(0.64%vs 0.13%),差异均具有统计学意义(P<0.05)。结论:有必要将RhE纳入血型常规监测,并在输血时尽可能地进行E抗原匹配,这可以很大程度地降低不规则抗体的阳性率,从而达到更安全的输血治疗。

RhC抗原弱表达伴类自身抗-Ce及同种抗-Jkb致配血不合分析

目的:探讨1例患者类自身抗-Ce合并抗-Jkb引起交叉配血不合并分析其RHCE基因弱表达的原因。方法:采用试管法、毛细管离心法对ABO、Rh和Kidd血型抗原进行鉴定。采用盐水、聚凝胺、抗人球蛋白三介质联用多套谱细胞进行抗体筛查及抗体特异性鉴定;采用多重PCR技术对RHCE基因进行测序及单倍体分析并使用SwissModel进行RHCE蛋白建模。结果:该患者血清中检出类自身抗-Ce合并抗-Jkb抗体。RHCE三代单分子测序显示突变组合为c.48G>C、c.150C>T、c.178C>A、c.201A>G、c.203A>G和c.307C>T,在内含子2中存在109 bp插入序列,同时出现内含子5-8大片段丢失,其Rh血型基因型为DCe/DCe,表型为CCDee。结论:基因分型技术可以协助推断患者血清中部分弱表达的RhC、c、E、e的分子机理,以辅助疑难抗体的鉴定,从而保证患者输血安全。

红细胞血型意外抗体筛查专家共识

红细胞血型意外抗体是指正常ABO血型中除抗-A、抗-B之外的血型抗体,其广泛存在于妊娠、输血、移植、注射免疫原性物质或患有自身免疫性疾病的患者中,可造成血型鉴定和交叉配血困难并严重影响输血安全及输血疗效。本共识旨在进一步规范抗体筛查细胞要求、操作规程及临床意义,为临床安全有效 输血提供保障。

NT与血清β-hCG、PAPP-A、ADAM12单独及联合检测对孕早期DS的诊断价值

目的:探讨颈部透明层厚度(NT)与血清人绒毛膜促性腺激素(β-hCG)、妊娠相关蛋白A(PAPP-A)及解整合素金属蛋白酶12(ADAM12)单独及联合检测对孕早期唐氏综合征(DS)的诊断价值。方法:选取2019年1月—2023年1月在厦门大学附属第一医院杏林分院经羊膜穿刺确诊的47例DS孕妇作为DS组,另选取同期49例产前检查正常的孕产妇作为对照组,检测并比较两组NT及血清β-hCG、PAPP-A、ADAM12水平,并采用受试者工作特征(ROC)曲线分析NT与血清β-hCG、PAPP-A、ADAM12单独及联合检测对DS的诊断价值。结果:DS组NT及血清β-hCG水平高于对照组,PAPP-A、ADAM12水平均低于对照组,差异有统计学意义(P<0.05)。ROC曲线分析显示,NT与血清β-hCG、PAPP-A、ADAM12联合检测诊断DS的曲线下面积为0.993,预测敏感度、特异度分别为95.7%、98.0%,均高于上述指标单独检测。结论:NT与血清β-hCG、PAPP-A、ADAM12检测对孕早期DS均具有一定的诊断价值,且联合应用诊断效果更高,可作为孕早期DS筛查的有效指标。

一株大黄鱼虹彩病毒的分离鉴定及多克隆抗体的制备

2021年7月,浙江省象山某养殖场养殖的大黄鱼(Larimichthys crocea)出现类似大黄鱼虹彩病毒引起的疾病。采用鲤上皮瘤细胞培养和病毒主要衣壳蛋白测序分析的方法,从患病的大黄鱼中分离到一株病毒。该病毒接种到鲤上皮瘤细胞(EPC)后出现空斑、脱落的细胞病变症状。根据虹彩病毒MCP和ATPase基因保守序列设计特异性引物对病毒组织样本进行PCR扩增,得到分别为1 367 bp和740 bp的目的基因片段。将MCP基因扩增片段测序,经BLAST对比及系统发育树聚类分析,确定该分离的病毒属虹彩病毒科细胞肿大病毒属。通过蔗糖密度梯度离心纯化,用透射电镜观察该病毒粒子呈正六边形,直径为120~150 nm。用纯化病毒作为抗原免疫小鼠获得抗大黄鱼虹彩病毒的多克隆抗体,效价为1∶7 000;通过SDS-PAGE和Western blotting初步确定3个免疫蛋白。本研究为大黄鱼虹彩病毒纯化提供一种新方法,并初步分离出免疫蛋白,为该病毒相关分子生物学研究、蛋白研究以及疫苗制备等提供理论依据。

同种抗-C、e伴类同种抗-e、Jk b自身抗体的鉴定及输血治疗

目的应用吸收放散和效价积分法对1例抗-C、e同种抗体合并抗-e、Jk b类同种自身抗体进行鉴别诊断。方法采用试管法对ABO、Rh和Kidd血型抗原进行鉴定;盐水、凝聚胺、微柱凝胶3种方法联用2套谱细胞进行抗体筛查及抗体特异性鉴定;多重吸收放散试验和效价积分原则对抗体进一步确认;多重PCR技术对RHCE基因、JK基因进行测序。结果通过血清学分析,该患者血型为A型、Rh分型ccDEE,直抗阳性。结合不同的Rh和Jk表型细胞吸收放散试验以及效价积分原则得出患者血清中含有抗-C、e同种抗体以及抗-e、Jk b类同种自身抗体,红细胞上有抗-e、Jk b类同种自身抗体。基因测序分析RHCE基因第5外显子676位出现G>C,其余外显子没有发生突变,推断Rh分型ccEE型;JK基因中第9外显子838 G/A杂合,Jk血型表型Jk(a+b+),选择交叉配血相合的A型ccDEE、Jk(a+b-)红细胞给患者输注,输血效果良好。结论应用吸收放散试验和效价积分法对患者血清进行抗体鉴定,以及血清学结合分子生物学对患者红细胞表型鉴定,可综合分析及区分出抗体的类型,为患者精准输血提供方案。