Some biological characteristics of hybridomas and parental myelomas cultivated in serum-free medium for long-term period

It has been developed in this laboratory that a serum-free medium, designated asDMI,is adaptive for long-term culture,freezing and resurgence in liquid nitrogen,and cloningof hybridoma and parental myelomas as well as for cell fusions.In this report,it is describedthat the myeloma cells grown in DMI for more than 3 months maintained their biological charac-teristics such as induction of aseites and subcutaneous somatic tumor in BALB/c mice and toler-ance toward 8-azaguanine,ete..However,the ability of the tumor cells to attach to glass wallwas decreased.The selecting assay of these cells in HAT medium(medium containing hypoxan-thine,aminopterin and thymidine)showed that the death time in DMI was similar to that in theconventional 15％ newborn calf serum-supplemented medium(RPS15).The hybridoma cellsadapted in DMI secrete monoclonal antibodies in quantities comparable to those produced inRPS15.

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells

Hybridoma cells display an increase in antibody productivity following exposure to hypertonic conditions. However, the underlying mechanism is not well understood. In the present study, we hypothesize that the nuclear factor of activated T cells 5 （NFAT5）/tonicity enhancer binding protein （TonEBP） functions to increase the antibody productivity of hybridoma cells. NFAT5 is an osmosensitive mammalian transcription factor. However, its ubiquitous expression in various organs that are not bathed in hypertonic milieu suggests that NFAT5 may also regulate cell growth and function under isotonic conditions. In this study, we examined the expression of NFAT5 in hybridoma cells by Western blot analysis, and found that it increased significantly in hypertonic medium. To further define the function of NFAT5 in hybridoma cells, RNA interference technique was used to downregulate the expression of NFAT5 in SGB-8 cells （a hybridoma cell line）. In isotonic medium, antibody productivity ofhybridoma cells was reduced by downregulation of NFAT5 while cell proliferation was not influenced. The results presented here demonstrate that NFAT5 not only plays an important role in osmotic stress response pathway in hybridoma cells but also is essential for optimal antibody productivity.

Relationship between chromosome behaviour and stability of human-mouse and human-(human-mouse)hvbridomas

Chromosomes in human-mouse and human-(human-mouse)hybridomas wereanalysed by G-banding methods.It was found that most human chromosomes,exceptNo.13 and X,Y,were retained.The frequencies of chromosomes No.1,3,4,5,6,17,19,21 and 22 were higher than those of other chromosomes in each hybridoma clone.The myeloma cell lines X63-Ag8.653 and SHM-D33 were also analysed.The morphologyof marker chromosomes was apparently different between hybridomas.There were 7 kindsof marker chromosomes in human-mouse hybridomas and 16 kinds of markerchromosomes in human-(human-mouse)hybridomas.Clones that retained humanchromosome No.1 were more stable and clones that did not retain human chromosomeNo.14 were still capable of secreting human immunoglobulin.Clones that retained humanchromosome No.2 did not secret human k light chain McAb while clones that retainedhuman chromosomes No.2 and No.22 only secreted λ light chain.

Monoclonal antibody technology applied to the detection of <i>Cryptosporidium parvum</i>oocysts in human and cattle fecal samples

With the discovery that the coccidian parasite Cryptosporidium sp. can cause severe symptoms in humans, many diagnostic techniques were quickly implemented such as microscopic visualization, immunofluorescence and PCR. Currently, there is no effective drug treatment and none of the current diagnostic methods is 100% accurate. In this study, a BALB/C mouse was subcutaneously immunized with Cryptosporidium parvum oocysts extract. The spleen was removed and the splenocytes were fused with SP2/0 myeloma cells in order to obtain hybridoma cells secreting antibodies specific to C. parvum antigens. Human and cattle fecal samples previously characterized by microscopy [Ziehl-Neelsen staining (ZN) and Lugol] and PCR for the presence of C. parvum and Giardia duodenalis, were analyzed by indirect immunofluorescence, using the developed hybridomas supernatants. The study shows that the selected hybridomas supernatants identify C. parvum oocysts in fecal samples in correlation with C. parvum oocysts identified using ZN/PCR. No false positive results were obtained and the two best supernatants gave 20% -30% of false negative results. No cross reaction with G. duodenalis was observed. By comparing our results with those obtained with an immunofluorescence commercial kit, it suggests the potential use of the monoclonal antibodies present in two of the hybridomas supernatants as a detection tool of C. parvum. With a reliability of 80.8% and 73.1% versus ZN and PCR methods for IFI, compared with a reliability of 76.9% and 92.3% versus ZN and PCR for commercial DIF kit, the supernatant 4.1D5 seems to be the most promising subject to further study its usefulness for C. parvum detection.

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.

PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST THE EXTRACELLULAR DOMAIN OF INTEGRIN a6 SUBUNIT-THE SPECIFIC LAMININ RECEPTOR

Objective: To prepare monoclonal antibody (McAb) against the Integrin α6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin α6 extracellular domain (GST-IAGED) was expressed in E.coli. JM109 and used for immunizing BALB/C mice. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium. ELISA and immunocytochemistry staining were used to select hybridomas. Results: One strain of hybridoma cells that secreted specific monoclonal antibody against integrin α6 extracellular domain was indentified. The immunoglobulin subclass of the McAb was IgG1. Conclusion: The McAb against the extracellular domain of integrin α6 was successfully prepared by using GST-IA6ED fusion protein expressed by E.Coli. And the McAb had positive reaction with human hepatocarcinoma cells-BEL-7402.

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.

人脑肌酸激酶单克隆抗体的制备及鉴定

用事故死亡者脑组织中提纯的人脑肌酸激酶（ＣＫ－ＢＢ）对ＢＡＬＢ／Ｃ小鼠进行免疫，应用杂交瘤技术将免疫小鼠脾细胞分别与２种骨髓瘤细胞（ＮＳ－１，６５３）融合，筛选得到８株能稳定分泌ＩｇＭ抗体的杂交瘤细胞。对分泌抗体经酶联免疫电泳转移试验（Ｗｅｓｔ－Ｂｌｏｔ）进行了鉴定，其中４种抗体能特异地与ＣＫ－ＢＢ的ＳＤＳ－ＰＡＧＥ电泳带结合。提示这些单克隆抗体有可能应用于肌酸激酶的结构功能研究和临床检验中。

Establishment of anti-thyrotropin monoclonal antibody hybridoma cell lines with extract of human pituitary gland

To get the hybridoma cell lines secreting anti-thyrotropin monoclonal antibodies with high affinity and specificity. Methods: BALB/c mice were immunized with extract of human pituitaries. The spleen cells of one immunized mouse were fused with mouse myeloma cells in polyethylene glycol and the positive clones were subcloned 3 times. Results: Two hybridoma cell lines which secrete anti-thyrotropin monoclonal antibodies with high affinity and specificity have been collected. The antibodies were of the IgG1 subclass and their maximum binding with thyrotropin was 60% and 45. 1% respectively. Using competitive binding assay,the antibodies were found to direct against different epitopes of human thyrotropin. Conclusion: The extract of human pituitaries could be used to produce monoclonal anti-pituitary hormone antibodies. The two anti-thyrotropin monoclonal antibodies produced in this study could be used in the establishment of a sensitive measurement of human thyrotropin.

Development of H5 subtype-specific monoclonal antibodies (MAb) and MAb-based assays for rapid detection of H5 avian influenza

Avian influenza (AI) virology surveillance is the most important method to monitor AI virus (AIV) in poultry so as to effectively prevent and control AI outbreaks. Monoclonal antibodies (MAb)-based assays are highly sensitive and specific for AIV detection, and much practical and economic for test-in-field or onsite. Many such assays have been developed and are still in developing since the H5N1 highly pathogenic AI (HPAI) outbreaks occurred in South East Asia in 2003. A MAb-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed in our lab during late 1990s and early 2000s. Meanwhile, AIV H7 and H5 subtype specific-MAbs have been successfully developed in our laboratory to enhance the Dot-ELISA and other MAb-based assays for AIV detection. Production and purification of the H7 and H5 MAbs were made to provide essential reagents for Dot-ELISA and other immunoassays, and the current development of a novel Biosensor technique for rapid detection of AIV from clinical and field specimens.

Serological Detection of Tomato Pepino mosaic virus in Morocco

Pepino mosaic virus （PepMV）, monopartite RNA virus, 6,500 pb, belonging to Flexiviridae and Potexvirus group, is highly infectious and easily transmissible. Its economic impact is major for the tomato producer＇s countries. Prevention, based on early virus detection is the only effective control measure. Monoclonal antibodies appeared to be very useful tool. The authors used for the production of monoclonal antibodies hybridomas technique, by fusing spleen cells of immunized BALB/c mice to PepMV and SP2/O cancerous cells. The aim of this work is to produce hybridomas producers of Mab that could be used for ELISA in Morocco. At the same time, these efforts will serve to decrease expenses of producers concerning phytosanitory control. We obtained 16 hybridomas lines producers of Mab specific for PepMV. They were tested for efficiencies in ELISA and two lines were retained for production of Mab on large scale （1B 11-G 10 and 5A l-G5）. Isotyping of these two lines showed that they are belonging to IgG 1 class and easily purified by affinity chromatography in agarose column by protein A. The conjugation of these two antibodies to alkaline phosphatase has been verified by DAS-ELISA. These antibodies will enable to diagnose the disease from infected tomato plants, integrating several serological tests to control it and target the actions of struggles.

THE DEVELOPMENT OF MONOCLONAL ANTIBODY AGAINST rhTNF AND ITS CURATIVE EFFECTSON E.Coli INFECTED MICE

Fifteen hybridoma cell lines producing monoclonal antiboclies (McAb) against recombinant human tu-mor necrosis factor a (rhTNFa) have been established by fusing SP 2/0 cells with spleen cells from aBALB/c mouse immunized with rhTNFa. Following J M Davis’s Works, semi-solid medium was usedfor initial cloning. Five of them were studied further. Their main chromosome- numbers range were 96 to105, all of them were IgG1 subclass. The affinities of these McAbs were estimated to be 1. 25 ×108 mol/L, 1. 12×108 mol/L, 2. 34×108 mol/L, 8. 55 × 107 mol/L, 1. 04×108 mol/L, respectively.Two groups of mice challenging with E Coli (107 organisms), one group treated with 2mg/kg anti-TNF monoclonal antibody, the other did not. There was a higher survival rate in treated group, the serumTNF level was significantly lower too, and the untreated mice had severe pathologic changes in vlscera.

巴泰病毒单克隆抗体的制备及鉴定

巴泰病毒(Batai virus,BATV)是一种人兽共患病毒,近年在全球广泛流行。我国也有人和动物感染的相关报道,而目前对BATV的研究仅局限于分子检测和全基因测序,还没有关于BATV单克隆抗体的相关研究,但制备特异性强、活性好的单克隆抗体也是防控BATV的关键。为了制备BATV单克隆抗体,本研究利用纯化的BATV作为抗原免疫雌性BALB/c小鼠,制备鼠源单克隆抗体。通过免疫小鼠后断尾取血,间接ELISA测其血清效价后,将SP2/0细胞与小鼠的脾细胞融合。ELISA方法筛选阳性细胞株,并进行亚克隆后制备单抗腹水,并利用辛酸-硫酸铵沉淀法纯化腹水。结果获得7株单克隆抗体,包括5株IgG型和2株IgM型。其中制备的3E2单克隆抗体效价最高为1∶32 000。经间接ELISA、间接免疫荧光和Western Blot检测表明,制备的3E2单抗具有较好的纯度、活性和特异性,为BATV快速检测方法的建立及致病机制研究奠定了基础。

Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles

Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect > 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.

MONOCLONAL ANTIBODIES FOR DETECTING BACTERIOCIN M2 FROM STRAIN M2 OF Pseudomonas solanacearum

Plant wilt bacteria (P. solanacearum)have a wide host range and cause wilt diseases of more than 200 species from 33 families. Because of no suitable resistant materials to wilt diseases, Fan Yun-liu et al. first proposed that this disease would be controlled with the genetic engineering of bacteriocin. Since bacteriocin M2 from strain M2 of P.

Immunolocation of antisperm monoclonal antibody 6B10 and corresponding antigen

An antisperm monoclonal antibody 6B10 was produced by hybridoma technique of the isotype IgG. The monoclonal antibody was purified by means of ammonium sulfate precipitation and protein A-Sepharose C1-4B affinity chromatography. SDS polyacrylamide gel electrophoresis was used to evaluate the purity of the antibody. Evaluation of the sperm acrosomal status was determined by chlortetracycline (CTC) staining. It was found that monoclonal antibody 6B10 can inhibit the sperm acrosome reaction induced by progesterone. The corresponding antigen recognized by monoclonal antibody 6B10 was located on the plasma membrane of the sperm acrosome by indirect immunofluorescent microscopy and immunoelectronmicroscopy. Sperm protein was extracted by 1 % Triton X-100. The molecular weight of the antigen is 50 ku, detected by Western blot. The antigen is a key protein in the sperm acrosome reaction and may be the receptor of progesterone on the sperm acrosome. It may either be developed as a candidate contraceptive vaccine or be used as a tool in pest/rodent management.

STUDIES ON A SPECIFIC ELECTROFUSION METHOD TO PRODUCE McAbs

The positive rate of hybridomas is low in routine McAb(monoclonal antibody)preparation because of non-selective cell fusion,thus bringing about a lot of heavy work load in screening. Meanwhile, it is not easy to obtain McAbs with high affinities to the administered antigen. These years, M.S. Lo et al. and Don M. Wojchowski et al. established two specific elec-