Cryopreservation of hybridomas

Summary A method for the long-term storage of hybridoma cells in liquid nitrogen is described. Hybridoma cells are harvested in logarithmic phase of growth and suspensed in freeze medium containing dimethylsulfoxide. Using a programmable freezer, the cells are frozen very slowly (1°C/min) and then transferred rapidly to liquid nitrogen refrigerators. Consequently, the hybridoma cells can be stored for long periods without substantial loss of viability and with minimal biochemical changes.     

A simple,rapid method for freezing hybridomas

Summary A simple, rapid method for freezing hybridomas in bio-freeze vials and in situ in 96-well microculture cluster plates is described. The method offers a convenient means of preserving hybridomas and a more manageable system for distributing the workload necessitated by the exponential increase in the number of wells after fusion or cloning or both.     

IgA hybridomas: A method for generation in high numbers

An immunization regimen has been developed which yields a high frequency of hybridomas producing IgA isotype, antigen-specific antibody when spleen cells from immunized mice are fused with non-immunoglobulin secreting murine myeloma cells. Germfree BALB/c mice were carrier-primed with sheep erythrocytes (SRBC) by gastric intubation (GI) for 2 consecutive days followed 1 week later by GI with trinitrophenyl (TNP)-haptenated SRBC. After 7 days, spleen cells were fused with non-immunoglobulin secreting myeloma cells (X63-Ag8.653), and 2–3 weeks later, culture wells were scored for hybrid clones. Of 240 culture wells plated, 157 wells (65.4%) exhibited clones producing anti-TNP antibodies as determined by enzyme-linked immunosorbent assay. A total of 50 specific cell lines were established, of which 27 clones (54%) produced IgA isotype anti-TNP antibodies, while the anti-TNP antibodies produced by the remaining 23 clones were approximately equally distributed between the IgM and IgG isotypes. The IgA and IgM monoclonal antibodies were more effective in hemagglutinating TNP-SRBC than were IgG isotype antibodies. This study describes a method for production of a high number of antigen-specific IgA hybridomas which will allow production of IgA monoclonal antibodies to important antigens on mucosally-associated pathogens, and thus allow elucidation of functions of IgA antibody at mucosal surfaces.     

Growth of rat-mouse hybridomas in nude mice and nude rats

Athymic (nude) mice and rats were inoculated intraperitoneally with rat-mouse hybridoma cells secreting monoclonal antibodies to rat MHC class I antigens in order to improve the yield of antibodies. Pristane priming and subsequent intraperitoneal injection of the hybridoma cells in to nude mice yielded ascites which contained antibody in high concentration (10-15 mg/ml). Complete Freund's adjuvant, mineral oil, pristane or antibody-antigen complexes were used to induce ascites in nude rats, but only pristane priming did so consistently. The hybridoma cells in the ascitic fluid failed to secrete antibody, although they contained intracellular antibody. However, when the pristane-primed nude rats received 250-500 rads of total body radiation prior to injection with the hybridoma cells, they produced large amounts of antibody. When the nude rats were splenectomized and injected with the hybridoma cells, they also produced antibody in high titers. There was no in vitro inhibition of antibody formation by the hybridoma cells cultured in medium containing 15% serum from nude rats, but co-culture of the hybridoma cells with splenic lymphocytes from normal or nude rats markedly inhibited antibody production. These results indicate that the defect in antibody secretion by the hybridoma cells in the ascites of nude rats is due to the presence of radiation-sensitive suppressor cells in the spleen.     

Cell separation using positive immunoselective techniques

Positive immunoselection is the direct selection and recovery of cells which express a given specificity from among a heterogeneous group of contaminating cells. A variety of methods are available to effect such separations. The principles of affinity chromatography, using solid-phase matrices or cellular immunoadsorbents, are extensively used. Liquid-phase positive immunoselection can also be performed using either a fluorescence-activated cell sorter or by using 'cellular engineering' to protect a cell from an otherwise noxious environment. The enzyme catalase coupled to specific antibody has been used for this purpose and renders cells resistant to hydrogen peroxide. The various positive immunoselection techniques available are reviewed and evaluated in the following report.     

The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas

Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50-100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM). Similar effects were elicited by MCM from 3 continuous macrophage lines. The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction. Conditioning by the P388D1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide. By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s). Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum. The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM. The results indicate that MCM has advantages as an alternative to 'feeder cells' and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations. The nature of the soluble factor(s) in MCM which promote these effects remains unknown.     

Rescue of human monoclonal antibody production from an EBV-transformed B cell line by fusion to a human-mouse hybridoma

A human-mouse cell line that is hypoxanthine-aminopterin-thymidine sensitive and ouabain resistant was derived from a fusion between human B lymphocytes and a mouse myeloma line. This new mutant, when fused to a relatively unstable EBV-transformed B cell secreting a human monoclonal anti-A (red blood cell antigen) antibody, resulted in stable hybridomas capable of long term production of the specific human monoclonal antibody. Furthermore, some of the hybrid clones secreted antibody in far greater titer than the original EBV cell line. We conclude that fusion to this human-mouse line is an efficient approach to the production of human monoclonal alloantibodies and an effective method of 'rescuing' secretion of desired antibody from EBV cell lines.     

Rat monoclonal antibodies. III. A simple method for facilitation of hybridoma cell growth in vivo

Hybridoma cells that did not grow when injected subcutaneously or intraperitoneally in histocompatible or Rnu/Rnu rats were injected intravenously into histocompatible recipients. Eight of the 9 cell lines injected in this way grew in several organs of the recipient 3-7 weeks later. Hybridoma cells proliferated mainly in the liver. When the liver homogenate of these animals was injected intraperitoneally into histocompatible recipients, hybridoma cells grew readily giving rise to ascites containing the expected monoclonal antibody for 6 of the 9 cell lines.     

鼠抗人IL－5单克隆抗体研制及初步应用探讨

Ｔ细胞杂交瘤来源的人ＩＬ—５免疫小鼠。用鼠鼠杂交瘤技术获得两株分沁抗人ＩＬ—５特异性单抗（Ｓ１７和Ｓ６６）的杂交瘤，并对Ｓ１７单抗的特性进行了探讨。实验结果表明；两株单抗皆为ＩｇＧ１亚类；Ｓ１７单抗能显著抑制ＭＴＨｈ５Ｒ细胞的增殖，不能竞争性抑制ＮＣ１７单抗与人ＩＬ—５的结合。利用Ｓ１７单抗作为包被抗体建立的检测人ＩＬ—５ＥＬＩＳＡ法具有较高的敏感性，提示其在临床上检测病人血中ＩＬ—５浓度应用的可能性。     

Magnetic bead selection of hybridomas producing pesticide antibodies

Standardized immunoassays for environmental monitoring require monoclonal antibodies (MAbs) with defined selectivities and affinities. An important step in the production of MAbs is the development of efficient screening procedures to identify suitable cell lines. We demonstrate the usefulness of immunomagnetic cell separation for the screening of hybridomas. It is based on the expression of surface receptors resembling the secreted antibodies on the membrane surface of hybridoma cells. Producers are tagged by the target molecule covalently linked to magnetic beads and subsequently removed by permanent magnet. Several examples are given surpassing conventional techniques for the screening of s‐triazine antibodies.     

A rapid solution-phase screening technique for hybridoma culture supernatants using radiolabeled antigen and a solid-phase immunoadsorbent

The solid-phase immunoassay commonly used to screen hybridoma supernatants at times gave falsely positive results. A solution-phase screening technique, which uses radiolabeled antigen and a solid-phase immunoadsorbent, is described. This technique overcomes the problem of false positives and can be easily adopted for other soluble antigens.     

Enhancement of limiting dilution in cloning mouse myeloma-spleen hybridomas by human low density lipoproteins

The limiting dilution technique is a critical step in the cloning of hybridomas for the preparation of monoclonal antibodies. We have found that culture medium supplemented with human plasma low density lipoproteins (LDL) markedly enhanced the yield of hybridoma clones derived from P3 X 63 Ag or FO mouse myeloma cell lines upon limiting dilution. Such enhancement was dependent on the concentration of LDL employed, being optimal at 1-10 micrograms/ml. At LDL concentrations greater than 20 micrograms/ml, the increase in yield of hybrid clones was not significant. The mechanism by which LDL enhances the yield of hybrid clones was partially elucidated by the demonstration that LDL could increase the DNA synthesis of hybridomas as assessed by [3H]thymidine incorporation. The data suggest that LDL play a role in the proliferation of hybridomas. It also indicates that LDL can be utilized for limiting dilution to increase the yield of desired clones. Since LDL is one of the most abundant lipoprotein fractions (approximately 500 micrograms/ml) in human plasma and the isolation procedure is simple, hybridoma culture medium supplemented with human LDL will prove to be a valuable reagent for investigators currently employing monoclonal antibody technology.     

A simple method for ranking the affinities of monoclonal antibodies

Measurement of the binding of constant trace amounts of labelled antigen by increasing dilutions of culture supernatant allows the ranking of monoclonal antibodies in the order of their affinity for the antigen. The theoretical basis for this method is discussed and it is illustrated with data from a set of anti-alphafoetoprotein monoclonal antibodies. Hybridomas secreting antibodies of desired affinity for immunoassay, histochemistry or antigen purification can thus be selected at an early stage after fusion.     

甲病毒属系列单克隆抗体的研制及特性鉴定

本文报道应用电融合法和PEG法建立了11种甲病毒(SIN,SF,CHIK_1,CHIK_2,EEE,WEE,MAY,GET,RR,SAG,M-1)单克隆抗体杂交瘤细胞系。分别对各种McAb进行了多项鉴定。特异性试验结果表明,有亚组特异性的和种特异性的McAb。对于今后该类疾病的流行病学调查和临床诊断,以及有关毒株的鉴定具有一定实用价值。     

人—人杂交瘤免疫球蛋白分泌稳定性的研究

对三株人—人杂交瘤细胞株的免疫球蛋白分泌稳定性作了研究。研究中注意到用有限稀释法多次克隆的杂交瘤细胞(C_(10),13H_2)与未经克隆的细胞株(F_3)在体外培养和扩增中,其细胞形态大小,染色体数和抗体分泌能力有不同程度的变化。研究证实杂交瘤球蛋白分泌稳定性与染色体丢失或不分泌群体的过度生长有关。我们用HAT筛选培养液进行再处理,注意到C_(10)这一株细胞的四倍体细胞数明显增加,抗体分泌量也有所增加。结果提示对杂交瘤细胞及早克隆,或适时用HAT进行重筛选,对稳定分泌群体可能有一定的效果。     

鼠抗人IL－5单克隆抗体研制及初步应用探讨

Ｔ细胞杂交瘤来源的人ＩＬ—５免疫小鼠。用鼠鼠杂交瘤技术获得两株分沁抗人ＩＬ—５特异性单抗（Ｓ１７和Ｓ６６）的杂交瘤，并对Ｓ１７单抗的特性进行了探讨。实验结果表明；两株单抗皆为ＩｇＧ１亚类；Ｓ１７单抗能显著抑制ＭＴＨｈ５Ｒ细胞的增殖，不能竞争性抑制ＮＣ１７单抗与人ＩＬ—５的结合。利用Ｓ１７单抗作为包被抗体建立的检测人ＩＬ—５ＥＬＩＳＡ法具有较高的敏感性，提示其在临床上检测病人血中ＩＬ—５浓度应用的可能性。     

抗创伤弧菌单克隆抗体杂交瘤细胞系的建立及特性鉴定

目的:制备高效、特异的抗创伤弧菌的单克隆抗体(mAb),并进行特性鉴定。方法:用创伤弧菌菌体蛋白抗原免疫小鼠,利用杂交瘤细胞融合技术,制备抗创伤弧菌的杂交瘤细胞株。用ELISA法及Western blot等筛选、鉴定其与创伤弧菌溶血素蛋白(vvhA)及其他重要海洋细菌的交叉反应性和效价。结果:共获得5株抗创伤弧菌的mAb,鉴定结果表明,5株mAb均具有良好的特异性和免疫反应性。结论:获得5株抗创伤弧菌的特异性mAb,为建立创伤弧菌快速检测试剂盒提供了重要制剂。     

适于杂交瘤细胞长期大量繁殖的SCI－8910培基的研制

单抗的免疫导向治疗已成为国内外肿瘤研究的热点之一。目前杂交瘤分泌单抗的来源一部分是小鼠腹水，另一部分培养于含血清的培养基内，这对单抗纯化带来困难，对免疫导向治疗也带来许多不利条件，如牛的ＩｇＧ、鼠的病毒等的污染。因此，我们着手研制一种能适应于杂交瘤长期培养的无血清培基，定名为ＳＣＩ－８９１０。现已进行了９株杂交瘤细胞的长期培养，同时用该培基在１．５Ｌ的Ｃｅｌｌｉｇｅｒ（ＮＢＳＵＳＡ）搅拌生物反应器中长期培养鼠－鼠杂交瘤细胞２Ｆ７（分泌抗人小细胞肺癌单抗）获得成功，经检测单抗纯度达到一定要求，产品在８ｍｇ／Ｌ以上。结果提示，该无血清培基制备的ＭＣＡｂ用于免疫导向治疗的可行性及大规模生产的可能性。