Comparison of the action of pristan and Freund's incomplete adjuvant on the development of ascites in mice--the recipients of hybridoma cells

Production of immunodiagnostic preparations based on monoclonal antibodies requires large amounts of such antibodies. Most frequently preparative quantities of monoclonal antibodies are provided by ascitic fluid from hybridoma-carrying mice. Prior to intraperitoneal administration of hybridoma cells mice are subjected to stimulation with pristan, a mineral oil component. The authors showed that the Freund's incomplete adjuvant (FIA) may be as well used for this purpose. The effect of pristan and the FIA on ascites development in mice with hybridomas producing monoclonal antibodies to the Ia-like antigens of man was studied. The stimulants were administered in amounts of 0.5 ml per a mouse. It was shown that irrespective of the stimulant injection time and the number of the administered hybridoma cells the amount of the ascitic fluid formed in female mice BALB/c stimulated with the FIA was 1.5-3 times higher as that in the animals stimulated with Pristane, the antibody titer in the ascitic fluid being unchanged.     

Monoclonal antibody specific for yeast elongation factor 3

Hybridomas have been prepared by fusing mouse myeloma (P3 X 63 Ag8) cells with spleen cells of mice immunized with a yeast fraction enriched with respect to non-ribosomal translational components. Cloned hybridoma lines were grown in the form of ascites tumors, and the monoclonal antibodies produced were purified from the ascites fluid by chromatography on DEAE-Affi-Gel Blue. One of the antibodies, from a hybridoma cell line designated as PSH-1, inhibited the translation of natural mRNA and poly(U) and polysomal chain elongation in a cell-free protein-synthesizing system from yeast. Resolution and partial purification of the elongation factors indicated that the monoclonal antibody from PSH-1 did not interact with EF-1 or EF-2 but reacted with and inactivated EF-3, the 125 000 molecular weight additional elongation factor specifically required with yeast ribosomes. The EF-3 purified from the cytosol by immunoaffinity chromatography was comparable to that prepared by ion-exchange chromatography. Evidence was obtained which indicated that EF-3 was essential for the translation of natural mRNA as well as poly(U), was associated with polysomes but not ribosomal subunits, and was required for every cycle in the elongation phase of protein synthesis.     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

Lupus anticoagulant and antiplatelet properties of human hybridoma autoantibodies

The clinical association of lupus anticoagulant antibodies with thrombocytopenia and thrombosis was the rationale for investigating the in vitro reactivity of these human hybridoma lupus anticoagulant antibodies with platelets. Fifty human hybridoma antibodies from 13 patients with systemic lupus erythematosus, 2 women with multiple spontaneous abortions, and 4 normal individuals were analyzed for lupus anticoagulant, antiplatelet, anti-DNA, and antiphospholipid reactivities. Of the hybridoma antibodies studied, 25 had lupus anticoagulant activity, 21 had antiplatelet reactivity, and 7 of these antibodies had both lupus anticoagulant and antiplatelet properties. No correlation was found between lupus anticoagulant antibody activity and antiplatelet, anti-denatured DNA, anticardiolipin, anti-egg phosphatidylethanolamine, antiphosphatidylserine, antiphosphatidylinositol, and antiphosphatidylcholine reactions. In contrast, antiplatelet activity was strongly correlated with antiphosphatidylethanolamine (rho = 0.761, p less than 0.001), anticardiolipin (rho = 0.748, p less than 0.001), and anti-dDNA (rho = 0.745, p less than 0.001) reactivities. Pretreatment of platelets with deoxyribonuclease, ribonuclease, trypsin, or phospholipases A2 and C resulted in different effects on the binding of individual hybridoma antibodies to platelets, suggesting that antiplatelet antibodies may recognize different epitopes on the platelet membrane. Our data demonstrate that most hybridoma lupus anticoagulant antibodies did not bind directly to platelets in vitro. This suggests that additional serum factors may be required in vivo to explain the association of these antibodies with thrombocytopenia and thrombosis.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An (H-2k/H-2d)F1 sarcoma cell line was subjected to immunoselection using ascites fluid from a mouse growing a hybridoma secreting an anti H-2Kk antibody.One hundred random clones were picked from the surviving population and screened by direct cytolysis using the hybridoma antibody or alloantisera against H-2Kk and H-2Dk. Fifty-nine clones were resistant to all three antisera, indicating that they no longer expressed the entire H-2k haplotype. Thirty-two were resistant to the ascites and to the anti H-2Kk alloantiserum, but sensitive to the anti H-2Dk serum, indicating that they had lost H-2Kk antigen, but retained H-2Dk. Nine clones were sensitive to the alloantisera, but resistant to the hybridoma, indicating that, though they retained the product(s) recognized by the alloantiserum against H-2Kk, they had lost the site(s) that bound the hybridoma antibody. Quantitative absorption assays using lymph-node cells from young BALB.K (H-2Kk) mice as targets show that one representative clone from the last group absorbs the anti H-2Kk activity in the alloantiserum. This implies that the sensitivity of the variant clone to the alloantiserum is not due to contaminating anti C-type virus antibodies in the serum. The possible implications of these data are discussed.     

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)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,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。     

Purification of Monoclonal Antibodies Using High Performance Liquid Chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Conditions for forming ascitic tumors in hybridoma cultivation in vivo

The conditions of the formation of ascitic cells in BALB/c mice injected with hybridoma cells were studied. All the hybridomas under study, producing monoclonal antibodies to viral antigens, induced the formation of ascitic tumors when introduced into the abdominal cavity of BALB/c mice pretreated with sensitizing agents. In the mice pretreated with pristane hybridoma cells took at a rate of 43-80% and in the mice pretreated with Freund's complete adjuvant, 31-70%. Angara oil and perfume oil, as well as Bayol F, were less effective. The time of the formation of ascites was inversely proportional to the dose of the injected cells, while the volume of ascitic fluid depended rather on the type of hybridoma and not on the dose of the injected cells. The study showed that the use of physiological saline or culture medium without serum for washing the abdominal cavity of mice after withdrawing ascites permitted the additional collection of 2.6-13.7 million hybrid cells, as well as a considerable amount of immunoglobulins.     

Purification of monoclonal antibodies using high performance liquid chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

Isolation of monoclonal antibodies against avian oncornaviral protein p19

For the production of monoclonal antibodies against pp60src and the gag precursor protein Pr76gag, the spleens of mice bearing tumors that had been induced by avian sarcoma virus Schmidt-Ruppin D-transformed cells were used. One hybridoma culture produced antibodies that were directed against the p19 portion of the gag precursor. However, no antibodies directed against pp60src could be detected in any of the hybridoma supernatants. The anti-p19-producing hybridoma culture was cloned twice in soft agar, and a stable clone was used for the production of high-titer ascites fluid in mice. The monoclonal antibodies belonged to the immunoglobulin G subclass 2b. The antibodies precipitated Pr76gag and the processed virion-associated p19, as well as the 75,000-molecular-weight gag fusion protein from avian erythroblastosis virus-transformed bone marrow cells. Also, viral ribonucleoprotein complexes were specifically precipitable, indicating that they contain p19 molecules.     

Monoclonal antibody production in murine ascites. II. Production characteristics

OBJECTIVE: To characterize monoclonal antibody production parameters of five hybridoma cell lines in murine ascites for correlation with clinicopathologic changes in mice. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally. Ascites fluid was collected a maximum of three times by abdominal paracentesis; volume was measured and antibody concentration was determined by ELISA for each sample. RESULTS: Trends differed among cell lines when comparing ascites volumes and antibody concentrations over time from the first to the third tap. Antibody production was greatest at tap 1 for Groups 2B11 and 2C6D9; tap 2 for Group 3C9; and tap 3 for Groups RMK and 3D6. Total antibody production ranged from 422.90 to 996.64 mg; total ascites fluid volume ranged from 74.2 to 115.7 ml; and mean antibody concentration for taps 1, 2, and 3 ranged from 2.50 to 15.03 mg/ml among cell lines. CONCLUSION: Production characteristics were significantly different among hybridoma cell lines. Determination of production characteristics of hybridomas and correlation with clinicopathologic changes in mice may be valuable in making recommendations for managing mice with ascites.     

Monoclonal antibodies against ovalbumin

Fusion of cells of the NS-1 mouse myeloma line with spleen cells from $\text{BALB}\text{c}$ mice immunized against ovalbumin produced hybrid cells which continuously secrete antibodies specific for ovalbumin. One of these cells was used to establish a cloned line. Studies of its antibody obtained either from ascites fluid or from medium from hybridoma cultures showed high titer and specificity against ovalbumin using the double antibody technique with rabbit anti-mouse immunobeads; the antibody proved to be of the IgG₁ (kappa) subclass and type.     

天然属性抗LDL及oxLDL IgM亚类抗体的制备与鉴定

目的：制备天然属性抗低密度脂蛋白（LDL）及抗氧化低密度脂蛋白（oxLDL）IgM亚类抗体。方法：给予Babl/c小鼠高胆固醇饮食,4周后取脾细胞直接与SP2/0细胞融合,以纯化的LDL及oxLDL为抗原,对阳性杂交瘤细胞生长孔进行间接ELISA筛选。鉴定杂交瘤上清的免疫球蛋白亚类,进而采用免疫沉淀和免疫印迹法对获得的抗体进行免疫学反应性鉴定。结果：杂交瘤细胞分泌的抗LDL及抗oxLDL的天然抗体通过ELISA法被筛选出来,可以与LDL或oxLDL发生高亲和力结合,经过4次克隆化,最终获得2株稳定分泌天然抗LDL的抗体,命名为5G8和2H7,及1株稳定抗oxLDL的抗体,命名为3A6,3株抗体均属于IgM亚类,无交叉反应,可以满足免疫印迹、免疫沉淀等实验要求。结论：成功制备了抗LDL及抗oxLDL IgM亚类抗体,为研究天然抗体在体内脂质代谢和相关心脑血管疾病如动脉粥样硬化等发生发展中的作用提供了重要的研究工具。     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

人生长分化因子15单克隆抗体制备、鉴定及药用价值初探

目的:制备稳定分泌抗人生长分化因子15(GDF15)单克隆抗体(m Ab)的杂交瘤细胞系,并对其分泌的m Ab进行鉴定。方法:根据人GDF15氨基酸序列特征,设计合成了8条能够免疫产生GDF15特异性抗体的抗原多肽,与VLP载体偶联后,免疫雌性BALB/c小鼠,利用杂交瘤技术制备鼠源抗人GDF15的m Ab,用间接ELISA检测m Ab腹水效价。结果:获得针对7个抗原多肽的12株稳定分泌抗人GDF15的杂交瘤细胞系,腹水m Ab效价可达1×104~1×109。结论:获得了针对不同抗原多肽的抗人GDF15的特异性m Ab,为进一步研发以GDF15为靶点的单克隆抗体抗肿瘤药物奠定了基础。     

Properties and applications of new monoclonal antibodies raised against calf DNA polymerase alpha

A panel of 12 hybridoma cell lines secreting monoclonal antibodies against alpha-polymerase were prepared by fusion of mouse myeloma cells and spleen cells of a rat immunized with homogeneous calf thymus alpha-polymerase. Hybridomas were selected and cloned on the basis of immunobinding to pure alpha-polymerase in solid phase radioimmunoassay. Antibodies secreted by these cells eventually were purified in milligram quantities from ascites fluids. These antibodies, all of the rat immunoglobulin M class, cross-reacted with alpha-polymerases from calf and monkey cells as revealed by immunobinding in radioimmunoassay and by immunoprecipitation of DNA polymerase activity. The antibodies were not capable of neutralizing the enzyme activity. With the methods described these antibodies may be used to immunoprecipitate alpha-polymerase from crude extracts of mammalian cells and to measure levels of the enzyme protein.