Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes non-specific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidin-biotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

Summary The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes nonspecific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidinbiotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Background: Development of functional monoclonal antibodies against intractable GPCR targets.Results: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.Conclusion: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.Significance: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.Summary: The CXC chemokine receptor-2 (CXCR2) is a member of the large ‘family A’ of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor.The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.     

Preparation of peroxidase: antiperoxidase (PAP) complexes for immunohistological labeling of monoclonal antibodies

The production of mouse peroxidase:antiperoxidase (PAP) complexes suitable for immunohistological use in conjunction with monoclonal antibodies is described. Three approaches were explored: 1) production of conventional polyclonal PAP complexes; 2) conversion of rabbit PAP to "pseudo-mouse PAP" by incubation with monoclonal mouse anti-rabbit immunoglobulin; 3) formation of PAP complexes from monoclonal mouse antiperoxidase. PAP complexes prepared by the latter technique gave the best immunohistological labeling reactions, being stable on storage and compatible with a wide range of human monoclonal antibodies. Gel filtration revealed that monoclonal PAP is of lower molecular weight than conventional PAP complexes (fulfilling theoretical predictions based on the monospecificity of monoclonal antibodies).     

A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2)

Background: Development of functional monoclonal antibodies against intractable GPCR targets.

Results: Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.

Conclusion: The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.

Significance: The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.

Summary: The CXC chemokine receptor-2 (CXCR2) is a member of the large ‘family A’ of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor.

The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specific antibodies.     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Summary Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specifie antibodies.     

Molecular biotherapy with human monoclonal antibodies

Human monoclonal antibodies are theoretically superior to murine monoclonal antibodies for many reasons. However, there are many hurdles in their preparation and clinical use, including the source of B lymphocytes, immortalization techniques, screening methods, scale-up limitations, purification steps, and the problems associated with conjugating to cytotoxic substances. Chimeric mouse/human antibodies are an alternative approach. Optimum clinical utilization will require a better understanding of target antigens and the ability to formulate cocktails.     

Immunoelectron microscopical visualization of ribonucleoproteins in the chromatoid body of mouse spermatids

The chromatoid body (CB), a cytoplasmic organelle present only in germ cell line, was studied at the electron microscopic level in mouse spermatids using cytochemical techniques and specific antibodies directed against sn-RNPs, hnRNPs, and ribosomal proteins. We found that specific staining for DNA as well as the use of monoclonal anti-DNA antibodies show a complete absence of DNA in the CB. The CB remains stained, however, after the application of the ethidium bromide-PTA technique, suggesting the presence of RNA within this organelle. snRNP as well as hnRNP proteins are demonstrated within the CB by means of specific monoclonal or polyclonal antibodies, especially during earlier spermiogenic stages. Monoclonal antibodies directed against the large ribosomal subunit proteins P1/P2 detect these antigens on the CB essentially along the internal threads of dense fibrillar material. Our findings suggest that the CB may function as a source of mRNA and/or of its partially processed precursors during the late stages of spermiogenesis, when the spermatid nucleus becomes gradually inactive.     

兔支气管败血波氏杆菌单克隆抗体的制备及鉴定

目的建立能稳定分泌抗兔支气管败血波氏杆菌（Bb）的单克隆抗体杂交瘤细胞株,为今后进一步建立该菌的免疫检测技术奠定基础。方法以Bb分离株BLJ05的灭活菌液为免疫原,腹腔免疫BALB/c小鼠,采用常规杂交瘤技术制备Bb单克隆抗体（McAb）,用间接ELISA、Western-blot等方法对McAb特性进行鉴定。结果获得两株能稳定分泌抗Bb单克隆抗体的杂交瘤细胞株,分别命名为A7D5和D6B2,其小鼠腹水抗体效价分别为1∶409600和1∶102400;且不与兔大肠杆菌、多杀性巴氏杆菌、产气荚膜梭菌等兔的常见病原菌反应,特异性强。两株单抗亲和力实验表明A7D5亲和力略高于D6B2。ELISA相加试验表明它们针对相同的抗原表位。结论成功建立了两株能稳定分泌抗兔支气管败血波氏杆菌单克隆抗体的杂交瘤细胞株,效价高、特异性强,为今后建立该菌的免疫检测技术建立奠定了基础。     

Detection of the microtubule cytoskeleton of the coccidian Toxoplasma gondii and the hemoflagellate Leishmania donovani by monoclonal antibodies specific for beta-tubulin

Seven monoclonal antibodies specific for mammalian beta-tubulin demonstrate the microtubule cytoskeleton of Toxoplasma gondii and Leishmania donovani by indirect immunofluorescence microscopy. Immunoblots of T. gondii and L. donovani proteins separated by SDS polyacrylamide gel electrophoresis confirm the specificity of the monoclonal antibodies for tubulin. Differential staining of flagellar and subpellicular microtubule populations was not seen in L. donovani with these antibodies. All seven antibodies also detected the subpellicular microtubules of T. gondii, but the polar ring and conoid of this organism was not visualized by any of them. This technique provides a rapid and specific way to assess microtubular organization in whole organisms.     

Immunofluorescent staining method for use with monoclonal antibodies

This note describes an immunofluorescent staining method for cells in the S-phase which have been allowed to take up bromodeoxyuridine into their DNA in place of thymine. The technique involves the use of fluorescinated monoclonal antibodies against bromodeoxyuridine and allows rapid and accurate estimation of cells in the S-phase, the technique does not require interpretation by skilled technicians.     

蓖麻毒素与其单克隆抗体相互作用动力学研究

表面等离子体激元共振(SPR)是一种可微量、实时、动态地监测生物分子相互作用的生物传感技术。蓖麻毒素为核糖体失活蛋白,具有很强的细胞毒性作用。通过SPR技术研究了两种抗蓖麻毒素的单克隆抗体C5、D12与蓖麻毒素相互作用的动力学,计算出两者的亲和常数分别为2.49×108mol-1·L和7.9×108mol-1·L,并对两种抗体的抗原表位进行了分析。     

抗-D抗体及其应用

Rh血型是仅次于ABO血型系统的人类红细胞抗原系统,至今已发现40多种抗原,但与临床密切相关的是D,C、c、E、e等5种抗原,其中最主要的是D抗原。相应的抗-D抗体无论是在临床输血检测,还是在Rh(D)新生儿溶血病、溶血性输血反应等的防治方面均具有非常重要的意义。传统的抗-D抗体的制备需用人的血清,来源受限。各种抗-D人源性单克隆抗体和基因工程抗体已经成为发展方向。     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with porcine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.     

Determination of monoclonal antibody specificity by mixed precipitation ingel

The enzyme-immunodiffusion technique is advanced which permits testing monoclonal antibodies included in the precipitate line formed in gel by polyclonal antibodies with the corresponding antigen. Rat or mouse monoclonal antibodies were mixed with polyclonal rabbit antiserum to the antigen at issue. The precipitate formed by immunogen and rabbit polyclonal antibodies included monoclonals.     

Human monoclonal antibodies

Summary The technology for the production of murine monoclonal antibodies has been refined enormously since its introduction in 1975. However, the technology for generating human monoclonal antibodies has only recently come into its own. In this review, three currently available approaches to the production of human monoclonal antibodies are described. These include the hybridoma technique, based on the fusion of antibody-producing human B lymphocytes with either mouse or human myeloma or lymphoblastoid cells; the EBV immortalization technique, based on the use of Epstein-Barr virus (EBV) to immortalize antigen-specific human B lymphocytes; and the EBV-hybridoma technique, based on a combination of the first two methods.The EBV-hybridoma system retains the advantageous features of the other two systems while overcoming their pitfalls and may be the current method of choice for producing human monoclonal antibodies with a defined specificity.Recipient of a W.H.O. training scholarship in Tropical Diseases.Fellow of the National Cancer Institute of Canada.     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

Summary The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with poreine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.Supported by the Deutsche Forschungsgemeinschaft, Carvas SFB 90     

Screening of monoclonal antibodies using antigens labeled with acetylcholinesterase: application to the peripheral proteins of photosystem 1

An original immunoenzymatic screening method, based on the use of antigens labeled with the stable enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is described. The high turnover of this enzyme results in a very sensitive detection of antibodies. In this method, monoclonal antibodies from the supernatants of hybridoma cultures are immobilized on a solid phase coated with anti-mouse immunoglobulins and react simultaneously with the appropriate antigen labeled with biotin molecules. In a second step, biotinylated acetylcholinesterase is in turn associated to the system via avidin interactions and subsequently detected by a colorimetric assay. The method appears more sensitive and easier to use than either the corresponding radioimmunological test using a 125I-iodinated antigen or the same type of enzymatic immunoassay performed with biotinylated horseradish peroxidase instead of biotinylated AChE. The combined use of microtiter plates, solid-phase separation, and colorimetric detection allows a high level of automation of the method which makes it very efficient to process a large number of samples. This technique has been successfully applied to the screening of monoclonal antibodies directed against peripheral proteins of the photosystem 1 (PS1) membrane complex in photosynthesis. A complete set of antibodies recognizing these PS1 components was selected. The same technique was also tested in competition immunoassays and appears to be a very precise and useful tool for quantifying PS1 polypeptides in different biological extracts, including sodium dodecyl sulfate-denatured membranes. This can be of special interest for studying the biogenesis of membrane complexes.     

Antibody engineering,a strategy for the development of monoclonal antibodies

Antibody engineering is the selection process enabling the isolation of hybridoma clones, each of which produces an antibody with predefined qualities. The state of the art of hybridoma technology is reviewed with emphasis on the results obtained by antibody engineering in our laboratories for the development of monoclonal antibodies for specific use in diagnostic tests. The perspective for in vitro monoclonal antibody production as well as the application of monoclonal antibodies for diagnostic reagents, industrial purification and therapeutic use are indicated.