烟草花叶病毒单克隆抗体杂交瘤细胞株的建立

用TMV免疫的BALB/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,获得10株能稳定传代並分泌抗烟草花叶病毒(TMV)单克隆抗体(McAb)的杂交瘤细胞株,其中T73McAb滴度最高,ELISA滴度高达1/655,360,能被TMV兔免疫血清所阻断,能中和TMV的感染性。但与其它植物病毒无交叉反应,与TMV不同毒株均有反应。     

De novo design and Rosetta-based assessment of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike receptor binding domain (RBD)

The continued emergence of new SARS-CoV-2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high-affinity Ab variable regions (Fv) through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn-2.0. Subsequently, we carried out computational affinity maturation of the designed variable regions through amino acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9-mer library of “human Abs” based on a human string content score. We generated 106 different antibody designs and reported in detail on the top five that trade-off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn-2.0 using a Rosetta-based approach. We used Rosetta SnugDock for local docking of the designs to evaluate their potential to bind the spike RBD and performed “forward folding” with DeepAb to assess their potential to fold into the designed structures. Ultimately, our results identified one designed Ab variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of Abs that can quickly be adapted to target spike epitopes of emerging SARS-CoV-2 variants or other antigenic targets.     

胆囊收缩素卵黄抗体在大鼠十二指肠的吸收

为了探讨CCK卵黄抗体能否被动物消化道所吸收 ,我们应用SDS PAGE电泳、ELISA和Westernblot方法 ,检测灌胃CCK卵黄抗体后SD大鼠十二指肠静脉血液中CCK卵黄抗体的免疫活性及存在形式。实验采用1 4只雄性成年SD大鼠 ,随机分成试验组和对照组 ,分别灌胃 1 5 0mg/mlCCK卵黄抗体粉混悬液和生理盐水 (2ml/只 )。安装亚急性十二指肠静脉插管 ,在灌胃后第 2、 3、 4h采集十二指肠静脉血液 ,分离血浆。SDS PAGE结果显示 ,CCK卵黄抗体分子量为 2 0 0kD。ELISA研究表明 ,试验组第 2、 3h的血浆中 ,均检测到CCK卵黄抗体效价 ,效价达 1∶1 2 8以上。同时 ,Westernblot分析发现 ,试验组第 2、 3h血浆中存在完整分子形式的卵黄抗体。研究结果提示 ,CCK卵黄抗体能被SD大鼠十二指肠段吸收或部分吸收进入血液 ,发挥其免疫学活性     

应用抗体捕捉ELISA法测定病毒特异性IgM抗体

本文以测定麻疹IgM抗体为模型,研究了应用抗体捕捉酶联免疫吸附法(Antibody capture ELISA简称ACELISA)测定病毒性疾病特异性IgM抗体的实验条件,结果表明;用于包被的抗μ链抗体、抗原,检测抗体的剂量对所测得的标本OD值都有不同程度的影响,其中以抗原的影响最大,不同株单克隆抗体(McAb)作检测抗体效果也有差别,最佳株与多克隆抗体(PcAb)相似,以本方法测定麻疹IgM抗体的敏感性和特异性很高,并不受类风湿因子的干扰。     

Development of an enzyme-immunoassay (EIA) for the measurement of follicle-stimulating-hormone (FSH) in callitrichid primates using a monoclonal antibody against the human-FSH-β-subunit

Despite the importance of Callitrichid primates in both biomedical and conservation research, practical and reliable immunoassays for the measurement of follicle-stimulating hormone (FSH) have not yet been described. A panel of monoclonal antibodies against specific peptide fragments within either the alpha or beta subunit of human FSH was evaluated for their ability to recognize FSH from Callitrichid and other New World primates. One of these, monoclonal antibody 46.3h6.b7 raised against human FSH, was selected due to its ability to recognize marmoset monkey FSH and its low crossreactivity with other gonadotrophins. The antibody formed the basis of an enzymeimmunoassay using a highly purified human urinary FSH (Metrodin®, Serono) preparation coupled to biotin as label and unmodified as standard. After 24 h incubation, antibody bound label was visualized by addition of streptavidin-peroxidase followed by the appropriate substrate. Parallelism was obtained between the standard and dilutions of pituitary extracts, urine and plasma from the common marmoset (Callithrix jacchus) as well as from two tamarin species (Saguinus fuscicollis and S. oedipus) and one squirrel monkey (Saimiri sciureus). Profiles of plasma and urinary FSH during the follicular phase are shown for two individual marmosets. The ability to measure FSH in Callitrichidae provides new opportunities for studies of the reproductive biology of these New World primate species. Am. J. Primatol. 41:179–193, 1997. © 1997 Wiley-Liss, Inc.     

B3(ds-scFv)靶向超抗原的制备及活性鉴定

To construct the expression vector of a recombinant toxin composed of a disulfide stable single-chain antibody from mAbB3 and SEA(D227A),the binding ability and cytotoxicity of the purified renatured products against the B3 positive carcinoma cells was examined. The VH and VL fragments of the mAbB3 were ligated by overlap PCR, the PCR product was cloned to the pET22b expression vector, then the SEA fragment was inserted into the B3dsscFv-pET22b expression vector which was digested by the same restriction enzymes. The expression plasmid was identified by restriction endonucleases digestion and transformed into E.coli BL21(DE3) followed by IPTG induction. The inclusion body was purified through SP-Sepharose cation exchange column after denaturing and refolding and the binding and cytotoxic ability of the purified products was examined by cell-ELISA and non-radioactive cell proliferation assay seperately. The expression vector B3dsscFv-SEA-pET was constructed successfully and the expression product exists mainly in the inclusion body, amounting to 33% of the total protein. The refolding product remains the binding ability of the single-chain antibody and has cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37℃. This genetically engineered B3dsscFv-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and promises to be an effective reagent for tumor targeted immunotherapy.     

Regiospecific orientation of single-chain antibody and atomic force microscope (AFM) images

An antibody containing a genetically engineered lipid group at the N-terminus and a hexahistidinyl tag at the C-terminus (Lpp-scFv-His6) was immobilized in an oriented manner on the surface of liposomes. Liposomes, consisting of antibody and phosphatidylcholine, have been prepared and imaged by AFM. For AFM visualization, the resulting liposomes were bound on the surface of mica by two different mechanisms. The histidine tags present in the antibody molecules of the immunoliposome were anchored to the NiCl₂ treated mica surface. Alternatively, the immunoliposomes were immunochemically bound on antigen-coated mica surface. Both approaches yielded liposomes which were clearly imaged without damage by AFM in ambient condition.     

Survival of human monoclonal anti-Rho (D) antibodies in the rhesus monkey.

In vivo half-life of a 125I-labeled human anti-D monoclonal antibody (mAb) and that of 131I-labeled Rho-GAM was assessed in a rhesus monkey injected simultaneously with both reagents. The half-life of the mAb was 7.9 days, compared to 17 days of Rho-GAM. Survival of the second dose of mAb, given 34 days after the first injection, was identical to that of the first dose, thus showing that the human mAb did not elicit an immune response. The in vitro produced human mAbs appear to be an alternative, unlimited source of anti-D antibodies for possible use in prevention of feto-maternal Rh immunization.     

Generation of a rabbit V(H) domain antibody polyspecific to c-Met and adenoviral knob protein

Several types of bispecific antibodies with affinity to both adenoviral coat proteins and a targeted antigen have been developed with the aim of providing the specific delivery of adenoviral gene therapy vehicle. From a phage display library of combinatorial dAb2s (each with an anti-adenoviral knob protein V(H) fragment linked with an anti-c-Met V(H)), we serendipitously enriched and isolated a clone, JS5, that has polyspecificity such that it binds both the adenoviral knob protein and c-Met, despite having only one V(H) domain. Our indirect observations suggest that the polyspecificity of JS5 is developed through accumulation of antibody specificity. The method of sequential immunization of a rabbit, first with the adenoviral knob protein and then with target antigens, may provide a method by which monoclonal antibodies with stand-alone polyspecificity may be developed. Such targeted polyspecific antibodies could readily be used for re-directing adenoviral vectors to target cells.     

The structure of an antitumor C(H)2-domain-deleted humanized antibody

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.     

The effect of the catalytic topoisomerase II inhibitor dexrazoxane (ICRF-187) on CC9C10 hybridoma viability and productivity

The effect of dexrazoxane on monoclonal antibody (Mab) production by CC9C10 hybridoma cells was investigated. Dexrazoxane is a catalytic inhibitor of DNA topoisomerase II. DNA topoisomerase II has a critical role in DNA metabolism and its inhibition by dexrazoxane can prevent completion of cytokinesis. Incubation of hybridomas with dexrazoxane was found to increase specific monoclonal antibody production by up to four-fold. However, due to the growth inhibitory effects of dexrazoxane the total Mab yield decreased by 40%. Under high density culture conditions(defined here as 10⁶ cells ml^-1) specific monoclonal antibody production increased by up to 37%, which was, however, accompanied by up to a 48% decrease in Mab yield. Hybridomasthat were incubated with dexrazoxane significantly increased in size due to the inhibition of cytokinesis. Dexrazoxane was also observed to induce a delayed apoptosis in the hybridomas. The caspase inhibitor Z-VAD-fmk slightly decreased the apoptotic effects of dexrazoxane. Preincubation with the caspase inhibitorZ-Asp-CH2-DCB had no effect on dexrazoxane-treated hybridomas, but it did have antiapoptotic effects on the untreated hybridomas which normally undergo a significant basal level of apoptosis. In conclusion, dexrazoxane-induced growth inhibition (which results in higher specific antibody production) and apoptosis inhibition (which results in prolonged viability) has the potential to significantly enhance the productivity of hybridoma cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)

The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion‐exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652–662. © 2010 Wiley Periodicals, Inc.     

Effect of acetylation (O-factor 5) on the polyclonal antibody response to Salmonella typhimurium O-antigen

Antibodies directed against lipopolysaccharide (LPS) O-antigen are often critical in the immune response to Gram-negative pathogens. Mice were orally immunized with isogenic strains of Salmonella typhimurium that differ only in a minor modification of O-antigen, namely acetylation, mediated by the oafA locus. To specifically examine the effect of acetylation on the antibody response to O-antigen, antibody titers were determined against both acetylated and unacetylated LPS by ELISA. In mice immunized with an oafA+ strain, the median titer against acetylated LPS was 32-fold higher than the titer against unacetylated LPS. Mice immunized with the oafA- strain had an 8-fold higher titer against unacetylated LPS. Thus, acetylation of O-antigen alters recognition by the vast majority of individual antibodies. This differential antibody recognition of O-antigen had a statistically significant correlation with protection against subsequent challenge with virulent S. typhimurium.     

抗人肝癌单抗片段抗体HAb18F(ab')_2的冻干工艺研究

选择适合于HAb18F(ab')2片段抗体的冻干保护剂及其冻干工艺,是确保该生物制品的质量关键之一,对不同种类和不同浓度的保护剂进行了研究,结果表明10%蔗糖对HAb18F(ab')2片段抗体冻干样品的剂型、外观、残水量(<3%)、复溶时间(<10s)、纯度(>95%)及稳定性没有影响。研究优化与保护剂相结合的适用于HAb18F(ab')2片段抗体的最佳冻干工艺,为抗体片段扩大生产提供依据。     

MIP-ligand binding assays (pseudo-immunoassays)

Molecular imprint sorbent assays (MIAs) have been applied to an increasing number of analytes of medical and environmental interest: the sensitivities and selectivities of these assays are comparable to immunoassays employing biological antibodies. In a number of cases complete analytical procedures starting from raw samples (blood, plasma and urine) have been demonstrated. There have been significant advances in applying MIPs in new formats and in the use of non-radioisotope labels. Progress in the field is reviewed, with particular emphasis on the technical aspects and new innovations. It is demonstrated that many of the perceived drawbacks of molecular imprinted polymers (MIPs) do not hinder their application in competitive binding assays: Many MIAs have been applied in aqueous systems and a heterogenous distribution of binding sites is not problematic, provided the recognition sites which bind the probe most strongly are selective.     

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.     

单核增生性李氏杆菌溶血素的原核表达及其单克隆抗体的制备

为了深入研究单核增生性李氏杆菌(LM)致病机理,从其基因组中克隆李氏杆菌溶血素基因hly,并将其与原核表达载体连接在大肠杆菌BL21中表达携带His标签的李氏杆菌溶血素(LLO)融合蛋白,经镍柱纯化得到重组LLO蛋白作为免疫原并免疫小鼠。取免疫小鼠的脾细胞与骨髓瘤细胞(Sp2/0)进行融合,经过3次亚克隆后获得3株稳定分泌针对LLO蛋白单抗的杂交瘤细胞株,分别命名为Anti-LLO1、Anti-LLO2、Anti-LLO3;经ELISA测定其细胞培养上清效价分别为1:3.6×104、1:6.4×104、1:1.6×104,腹水效价分别为1:2×107、1:2×107、1:1×107;亲和力解离常数(Kd)分别为6.18×10-11、7.50×10-11、6.27×10-11;3株单抗的IgG亚类均为IgG1。经Westernblotting鉴定证明,该3株抗体均能特异地识别李氏杆菌LLO蛋白,该单抗的制备为深入研究LM的致病机理奠定了基础。     

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody,LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkin's B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548–564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitt's lymphoma cell line metabolically labelled with [³H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines. Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of¹³¹I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.This work was supported in part by USPHS grant CA39841 from the NIH     

Production of Polyclonal Antibodies against the Tegument of Sparganum (Plerocercoid of Spirometra mansoni) and Its Immunolocalization

In a previous study, the author developed a method for separation of the tegument of spargana (plerocercoids of Spirometra mansoni) from the parenchyme using urea. The present study, as a next step, was performed to evaluate which molecules are present in the outer tegument. Two major proteins, 180 and 200 kDa, are present in the tegument and we could make polyclonal antibodies against these molecules. Their immunolocalization was processed and the outermost layer of the spargana showed strong positive staining. Conclusively, we could confirm that the 180 and 200 kDa molecules might be tightly bound membrane proteins in the tegument of spargana.     

Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts

A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.