Primary structure of the gamma-subunit of the GTP-binding protein from the bovine retina

The complete amino acid sequence of the gamma-subunit of GTP-binding protein from cattle retina has been established. The polypeptide chain consists of 69 amino acid residues and contains an unusual sequence Cys35-Cys36. The molecular mass of the gamma-subunit is 8008,7.     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

TT病毒重组蛋白单克隆抗体的制备

采用杂交瘤技术,获得了4株稳定分泌抗TTV重组蛋白的单克隆抗体杂交瘤细胞株,1株属IgG2bλ链、1株属IgG1κ链、2株属IgG2aκ链。4株杂交瘤细胞培养上清液效价为1：80-1：1280,腹水效价为1：32万-1：160万。     

抗磺胺对甲氧嘧啶单克隆抗体的研制及其特性分析

目的制备针对磺胺对甲氧嘧啶的单克隆抗体,建立对该物质的免疫学检测方法。方法以BSA-磺胺对甲氧嘧啶为免疫原,免疫BALB／c小鼠,取脾细胞与小鼠Sp-2／0骨髓瘤细胞融合后,经筛选和亚克隆,建立杂交瘤细胞株。结果获得2株能稳定分泌抗磺胺对甲氧嘧啶抗体的细胞株。对抗体进行了特性分析,抗体的效价分别为1：400000和1：1630000,抗体类型及亚类都为IgGl。其中,单克隆抗体1H10的亲和力为1．4×109L／mol,利用该抗体采用竞争间接ELISA法检测磺胺对甲氧嘧啶的范围是1025—16μg/mL,最低检测浓度是8μg/mL。单抗1H10与其他6种磺胺药（SMM、SMZ、SM2、SD、SulfaquinoxalineSodium、Sulfametetyrazine）无交叉反应。结论单克隆抗体1H10可用于研制免疫学方法检测磺胺对甲氧嘧啶残留的产品。     

抗酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris)单克隆抗体制备

【背景】基于本实验室已经建立的脂环酸芽孢杆菌检测和鉴定方法工作基础之上,以期建立具有很好的经济价值和实用价值,且更为方便、快捷、准确、特异、灵敏的检测方法。【目的】实现对果汁生产中脂环酸芽孢杆菌从原料到成品的快速检测和鉴定。【方法】采用杂交瘤细胞技术,以Alicyclobacillus acidoterrestris(ATCC49025)免疫BALB/c小鼠,用建立的间接ELISA方法筛选杂交瘤细胞,得到了3株能稳定分泌A.acidocaldarius抗体的杂交瘤细胞株,其中两株为Ig G1亚类,并对其进行生物学特性的鉴定。【结果】得到的两株单抗3F7和9C4是针对不同的抗原位点,且多次传代后稳定性基本保持不变;特异性实验表明两株单抗均不与A.acidocaldarius(NCIMB11725)、Bacillus cereus(ATCC11778)、Bacillus subtilis(ATCC11774)、A.cycloheptanicus(ATCC49029)等发生交叉反应。【结论】3F7和9C4这两株单抗可以进一步用于检测胶体金试纸条的研制。     

Preparation and primary application of monoclonal antibodies against a novel ribosome-inactivating protein Moschatin from pumpkin seeds

Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have been widely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIP recently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin that can selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6, 6F8, 4H10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have been successfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies are IgG1, IgG1, IgG1, IgG1, IgG2a and IgGM. Their affinity constants were determined to be 1.42x10(8), 2.71x10(8), 8.72x10(7), 2.06x10(8), 1.36x10(8) and 1.51x10(8) M(-1) in a sequent order, measured by non-competitive ELISA. The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel, which consisted of a monoclonal antibody 4H10 and Sepharose 4B, was prepared and used to purify Moschatin from pumpkin seeds crude extract.     

Preparation and purification of monoclonal antibodies against chloramphenicol

Monoclonal antibodies (McAbs) against chloramphenicol (CAP) were produced to detect CAP residues, which could be toxic and possesses a potential threat to human health. The CAP-BSA conjugate was obtained by bovine serum albumin (BSA) coupled with CAP, and used to immunize the mice. The splenocytes from the immunized mice were fused with mouse myeloma cells SP2/0 to form hybridoma, which may secrete McAbs against CAP. Hybridomas 1D₁ and 3G₁₂ secreting McAbs against CAP were obtained by screening. Ascites containing McAbs were prepared by injecting 1 x 10⁶ cells of hybridoma 1D₁ and 3G₁₂ into the abdomen of mice. Protein A affinity chromatography was used to purify McAbs against CAP in a single chromatographic step with recovery yield above 80% and purity above 95% and full recovery of antibody activity. Experiments showed that McAb 3G₁₂ was highly specific for CAP and had no cross-reactivity with analogues which have a structure similar to CAP. The IC₅₀ value was 50.8 ng/mL.     

Preparation of monoclonal antibodies against duck hepatitis B core antigen and analysis of the monoclonal antibodies

Mice were immunized against duck hepatitis B virus core (DHBc) particles isolated from the liver of asymptomatic carrier ducks of duck hepatitis B virus (DHBV) by ultracentrifugation. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 12 clones of hybridoma cells secreting antibodies against DHBc (anti-DHBc) were isolated. According to the reactivity to core particles and core peptide obtained from DHBc particles treated with SDS-2ME, the 12 antibodies were classified into two groups. Two monoclonal antibodies reacted against both core particles and core peptide (B-type), the other ten monoclonal antibodies reacted against core particles but did not react against core peptide obtained from DHBc particles treated with SDS-2 ME. (A-type). Solid phase enzyme immuno assay (EIA) using these two types of antibodies could detect core antigenisity not only in the liver homogenate but also in the DHBV infected serum. Sucrose gradient analysis and gel filtration analysis revealed this DHBc antigenisity in the serum is not carried by core particles but carried by core peptide, equivalent to HBe antigen in the serum of Hepatitis B virus (HBV) carrier. This EIA may provide sensitive test monitoring both serum DHBe antigen levels and DHBc antigen levels in the liver during DHBV infection.     

空肠弯曲菌细胞致死性肿胀毒素单克隆抗体的制备与鉴定

【目的】原核表达空肠弯曲菌细胞致死性肿胀毒素B蛋白(CdtB),制备其单克隆抗体(mAb),并研究mAb抗毒性作用。【方法】扩增空肠弯曲菌cdtB基因并将其构建到pET-30a(+)和pGEX-6p-1表达载体,以原核表达的GST-CdtB蛋白为免疫原,应用杂交瘤技术进行细胞融合;采用间接ELISA方法测定细胞上清和mAb腹水效价,Dot-ELISA、Western blot分析mAb特异性,并以CaCo-2和HD-11细胞为模型,鉴定mAb抗毒性能力。【结果】成功构建重组原核表达质粒pET-30a(+)-cdtB和pGEX-6p-1-cdtB,并融合表达rHis-CdtB和rGST-CdtB蛋白。获得5株稳定分泌CdtB抗体的杂交瘤细胞株,分别命名为1F3,1F5,2E4,2E11,2F2。抗体Ig类和亚类检测显示2E11 Ig亚类为IgG2b,其他4株均为IgG1。抗体效价高达1:(1×108)。Dot-ELISA试验表明5株mAb均能与空肠弯曲菌标准株发生特异性反应,与非空肠弯曲菌呈阴性反应;Western blot法分析表明5株mAb均能与纯化蛋白rGST-CdtB有良好的反应性。基于CaCo-2细胞的黏附和侵袭实验表明mAb能显著降低细菌的黏附和侵袭能力(P0.01)。【结论】成功制备了针对空肠弯曲菌CdtB蛋白的mAb。为进一步研究空肠弯曲菌致病机制,以及为研制治疗性类药物奠定了基础。     

Preparation and properties of monoclonal antibodies against Mycobacterium tuberculosis

The above mentioned monoclonal antibodies of idiotype IgG 3/Kappa against TMC 120 and TMC 107 strains of M. tuberculosis were prepared by fusion of mouse myeloma line FO cells with splenocytes BALB/c of mice immunized with complex antigens. These were prepared from the bacterial mass previously inactivated with gamma-rays radiation, and applied without mycobacterial adjuvant agent. Species specificity of both monoclonal antibodies was determined by ELISA testing and dot blot test. The potential use of the mentioned preparations consists in species identifying and taxonomy of mycobacteria, mycobacterial antigen purification and prompt TBC diagnosis (serodiagnosis, identifying of specific antigens in pathological materials.     

Preparation and characterization of monoclonal antibodies against human myeloperoxidase

We established 11 hybridomas producing monoclonal antibodies (MoAbs), designated AM, against human myeloperoxidase (MPO), by immunizing mice with the three forms of MPO (I, II, and III) purified from healthy human polymorphonuclear leukocytes (PMN) and characterized the specificity of the AM MoAbs. Ten of the AM MoAbs reacted similarly to each of the three forms using an enzyme-linked immunosorbent assay. When a cetyltrimethylammonium bromide (CETAB) extract of human PMN was electrophoresed in a CETAB polyacrylamide gel and transferred to a nitrocellulose filter, IgG1 class AM MoAbs immunostained only the MPO band of the proteins of the extract. In addition, the AM MoAbs reacted to two radioactive bands of 94 and 92 kDa in a HL-60 cell lysate labeled with [35S]methionine for 1 h. After a chase period of 24 h, these bands were replaced by four radioactive bands of 64.5, 43, 16.7, and 13.4 kDa, demonstrating that the MoAbs recognize not only mature MPO but also the MPO precursors of 94 and 92 kDa. The data also indicated that the two major bands of 64.5 and 13.4 kDa corresponded to heavy and light chains of mature MPO, respectively, and the additive intermediate bands of 43 and 16.7 kDa were MPO-related proteins. Moreover, AM MoAbs reacted to a similar extent to the deglycosylated form of MPO III with endo-beta-N-acetylglucosaminidase H (Endo-H). Thus, IgG1 class AM MoAbs recognized MPO with high specificity and reacted to the structure which is commonly conserved in the three mature forms of MPO (I, II, and III), MPO precursors, and deglycosylated MPO with Endo-H. AM MoAbs also specifically reacted to PMN and/or monocytes but did not react to lymphocytes when the cell staining method was used.     

Preparation and characterization of monoclonal antibodies against adenylate kinase

Six hybridoma cell lines that can continuously secrete monoclonal antibodies against adenylate kinase (AK) have been produced. The characteristics including the subclass and molecular weight of monoclonal antibodies manufactured by these strains are also determined. Further studies show that the two monoclonal antibodies McAb3D3 and McAMD8 bind easily with AK absorbed on microtitration plates, with affinity constants of 8.4 × 108 M-1 and 9.6 × 108 M-1, while their interactions to AK in solution are much weaker, with affinity constants of 7.0 × 104 M-1 and 3.9×106M-1, respectively. Thus, McAb3D3 and McAMD8 react preferentially to the immobilized AKs. Since pro-teins are often partially denatured when absorbed on microtitration plates, it is suggested that both McAb3D3 and McAMD8 are directed against non-native AK.     

Production and characterization of monoclonal antibodies against bovine pancreatic trypsin inhibitor

Two monoclonal antibodies (MAbs) have been produced, without the use of a supporting carrier, against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a mini-protein composed of 58 amino acids. Both MAbs obtained were found to be IgM. One of them was purified and further characterized. This MAb (ICI) binds to the immunogen with an association constant of 1.6 X 10(6)M-1 at pH 7.4. Competition experiments with trypsin or inactivated trypsin demonstrate that ICI MAb interacts with BPTI at, or near, the proteinase-binding site. ICI MAb binds, with a much lower association constant (approximately 200M-1), to an isoinhibitor (spleen inhibitor II) which differs from BPTI in seven amino-acids; three of these substitutions are at the active site, in the contact area with the proteinase.     

高特异性抗桔霉素单克隆抗体的制备及鉴定

以1, 4-丁二醇二缩水甘油醚(双环氧试剂)为偶联剂,合成桔霉素-蛋白偶联抗原CIT-BSA,将偶联抗原免疫BALB/C小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株稳定分泌抗桔霉素抗体的杂交瘤细胞株H2-F8．该单克隆抗体经过初步鉴定,抗体类型为IgM类,抗体的相对亲和力为4.17×10⁸ L/mol,单抗与黄曲霉毒素B1、赭曲霉毒素A、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮等毒素的交叉反应率均低于0.1%,与红曲色素中的橙色素和红色素的交叉反应率均低于0.01%．在此基础上建立了间接竞争ELISA检测方法,线性范围为0.05～1.0 μg/L,IC₅₀值为0.3 μg/L．结果为快速检测桔霉素的酶联免疫检测方法的建立和检测试剂盒的研制提供技术依据．     

抗重金属汞离子抗体的制备及鉴定

汞、镉、铅等重金属引起的环境污染已在世界范围内造成危害。快速、廉价地监测生境中重金属是减小其对人类及动物危害的先决条件。传统检测方法无法满足高通量的现场检测,建立更快速、更经济的免疫分析法检测汞离子是生产及经济发展的需要。本研究中,报道了汞特异性单克隆抗体的制备与筛选方法和结果。因Hg2+太小以至于不能引起免疫反应,所以用螯合剂(二乙烯三胺五乙酸,DTPA)将金属离子与载体蛋白(匙孔血蓝蛋白,KLH)连接起来。成功合成、鉴定汞复合物抗原后,免疫BALB/c小鼠,通过细胞融合获得了稳定分泌抗体的杂交瘤细胞。用极限稀释法亚克隆,通过ELISA筛选,获得了2株稳定分泌抗汞离子抗体的细胞株(H2H5,H1H8)。小鼠腹腔注射1×107H2H5、H1H8细胞株制备腹水,腹水抗体效价都在1∶51200以上。经鉴定两株杂交瘤均为IgG1亚类,轻链为kappa型且分泌抗体稳定性较好。实验结果为汞离子残留免疫学检测方法的建立提供了技术基础,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。     

Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.     

Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed inE. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoclonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native luciferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.     

幽门螺杆菌尿素酶单克隆抗体的研制及鉴定

应用杂交瘤技术 ,建立稳定分泌抗幽门螺杆菌尿素酶单克隆抗体 (HPU McAb)的细胞系。用纯化幽门螺杆菌尿素酶 (HPU)抗原加福氏佐剂免疫BALB/c小鼠 ,按常规方法进行细胞融合 ,以纯化HPU抗原包被 ,间接ELISA方法筛选 ,并经多次有限稀释法克隆。获得 6株抗HPU的杂交瘤 ,腹水效价达 1∶6 .4× 10 4～ 1∶2 .5 6× 10 5,特异性专一 ,并对其进行体内外连续传代 3个月 ,分泌抗体能力稳定。IgG亚类分型主要为IgG1型。该细胞系能稳定分泌幽门螺杆菌尿素酶单抗。     

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared anti-DDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/1 were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site.