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。     

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 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.     

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.     

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.     

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.     

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.     

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 of monoclonal antibodies against planarian organs and the effect of fixatives

Monoclonal antibodies were obtained against all part of the body of the planarian Dugesia japonica japonica except tthe nerve cord, though parts of the enteric canal were also non reactive. The effect of a variety of fixatives (acetone, Bouin, formalin) used on planarian tissues prior to screening with antibodies, was assessed.     

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.     

Mouse monoclonal antibodies against rat estramustine binding protein

Hybridomas producing antibodies against rat prostatic estramustine binding protein (rEMBP) have been obtained by fusion of spleen lymphocytes from Balb/c mice, immunized with rEMBP, and SP2/0 Ag 14 mouse myeloma cells. Anti-rEMBP IgG producing cultures were identified in a solid phase ELISA using alkaline phosphatase conjugated sheep antimouse IgG. Cultures producing specific antibodies were subcloned and expanded. The produced immunoglobulins were characterized according to interaction with subunits of rEMBP. No interaction was found with [3H]estramustine-human estramustine binding protein. Following development of a radioimmunoassay, tissue distribution of rEMBP in the rat was studied. Despite high specificity, shown by selective interaction with rEMBP (F and S subunits), macromolecules interacting with anti-rEMBP were found in high concentrations in the prostrate and also in low concentrations, in other tissues of the male genital tract, and further in the adrenals, pancreas and submaxillary gland. The high specificity also makes it possible to study the F and S subunit selectively. These results show that macromolecules very similar to rEMBP are present in hormone-sensitive tissues in the rat.     

Localization of antigenic sites for monoclonal antibodies against alpha-subunit of Go-protein from the bovine brain]

The properties of monoclonal antibodies (MA) specifically raised against the alpha-subunit of the GTP-binding protein from bovine brain, G0, were studied. The hybridoma clones were found to secrete MA capable to interact with different antigenic sites of G0 alpha. Clone 1D2 MA interacted with the N-terminal domain of G0 alpha. The antigenic sites for clones 3DE. 1H6 and 2E3 MA were localized in the C-terminal domain of the protein molecule. Using clone 1H6 MA, the site of G0 alpha involved in the interaction with the beta gamma complex located in the C-terminal domain of the alpha-subunit, was revealed. It was found that the interaction of the alpha-subunit with the beta gamma complex changed the conformation of the C-terminal fragment of G0 alpha (Mr5000) together with an increase in the alpha-subunit affinity for clone 2E3 MA. It was concluded that the observed conformational changes may be the reason for the increased affinity of the alpha-subunit for the receptor.     

Preparation and characterization of monoclonal antibodies against soybean seed lipoxygenase isoenzymes

Sets of monoclonal antibodies have been prepared using two soybean seed lipoxygenase isoenzymes as the antigens. The antibodies were characterized by ELISA, Western blot analysis, immunoprecipitation, and in kinetic assays. Several antibodies displaying selectivity for the two closely related polypeptides were obtained, while the majority of the antibodies generated were crossreactive. Antibodies specific to the native and denatured forms of the two proteins were also obtained. Two of the monospecific antibodies were shown to immunoprecipitate the appropriate isoenzyme selectively from a mixture. When these antibodies were immobilized on agarose, they were successful in the immunoaffinity purification of the individual isoenzymes. In kinetic experiments certain antibodies were found to influence catalysis upon incubation with lipoxygenase. Antibodies which both inhibited and stimulated catalysis were identified.     

Preparation and use of monoclonal antibodies against seal alkaline phosphatase]

Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively.