Antibodies specific to a deoxyribodinucleotide sequence.

Antibodies were raised in rabbits against the bovine serum albumin conjugate of dpApT. Analysis by double diffusion in agar gel and quantitative precipitation test showed the presence of antibodies specific to the hapten in the antisera. Quantitative data on the specificity of the antibodies were obtained by studying the inhibition of the binding of 3H-dpApT to the antisera by various nonradioactive mono- and oligonucleotides, using a nitrocellulose membrane binding assay. The antibodies were found to be highly specific for the dinucleotide sequence dpApT. The antibodies were able to bind to synthetic oligonucleotides containing the sequence dpApT and to denatured calf thymus DNA.     

Monoclonal antibodies specific to plant tubulin

Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two -tubulin subunits, MBT₂ and MBT₃, of which MBT₃ is a minor component, and one -tubulin, MBT₁.Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution.The properties of monoclonal antibody (K₄E₇G₃) were examined by Western blot analysis and indirect immunofluorescence studies. K₄E₇G₃ reacts with MBT₂ and MBT₃ -tubulin subunits of mung bean tubulin, but not with MBT₁ -tubulin nor with the - and -subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K₄E₇G₃ and with other monoclonal antibodies that are known to be specific to the -subunit of yeast tubulin and - or -subunit of mammalian brain tubulin. MBT₂ and MBT₃ are shown to be similar but not identical and are quite different from MBT₁ and the -subunit of sheep brain tubulin. K₄E₇G₃ reacts with peptide fragments in MBT₂ and MBT₃ that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain -tubulin. It is concluded that K₄E₇G₃ and another monoclonal antibody, K₂D₇B₈, which has similar properties, are relatively specific for plant -tubulin.In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K₄E₇G₃ and K₂D₇B₈ do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K₄E₇G₃ to react with brain tubulin subunits on Western blot analysis.     

1组曲霉单克隆抗体的制备与鉴定

目的制备并鉴定一组抗曲霉不同抗原的单克隆抗体。方法采用烟曲霉细胞壁抗原成分、分泌抗原和灭活分生孢子,分别免疫BALB／c小鼠,制备单克隆抗体,免疫荧光法鉴定单克隆抗体与曲霉属和念珠菌属抗原的交叉反应。结果获得29株稳定分泌抗曲霉单抗的杂交瘤细胞株,其中用烟曲霉细胞壁抗原成分免疫获得11株,用分泌抗原免疫获得13株,用孢子免疫获得5株;Ig亚类鉴定,11个克隆株为IgG1亚类,3个克隆株为IgG3,15个克隆株为IgM。免疫荧光法鉴定29株单抗特异性识别烟曲霉细胞壁抗原,与其他曲霉抗原有交叉反应。结论29株单克隆抗体,对于建立侵袭性曲霉感染早期诊断方法、筛选曲霉保护性抗体以及研究抗体保护机制奠定了实验基础。     

Development of monoclonal antibodies specific to urochordate intracellular epitopes

Sixteen monoclonal antibodies (MAbs) specific to 2 urochordate genera (Botryllus schlosseri and Botrylloides) intracellular epitopes were generated in mice immunized with a mixture of fresh and paraformaldehyde-fixed cells obtained from animal's blood and cells from dissociated organs. Hybridoma clones were selected by ELISA tests and immunohistochemistry assays on paraffin-embedded animal tissues. Five MAbs were tested for reactions with different zooidal organs and cell compartments; 7 MAbs were tested, separately, on 5 different botryllid colonies (3 Botryllus and 2 Botrylloides). The results revealed high polymorphism. Whereas some of the MAbs recognized, specifically, only part of the botryllid genotypes tested, others recognized only part of the cellular compartments. These MAbs will be used as an important tool in the study of botryllid ascidian immunology and developmental biology, revealing the first wide panel of MAbs specific to urochordate intracellular antigens.     

Novel conformation-sensitive antibodies specific to three- and four-repeat tau

Two types of tau isoform, three- and four-repeat tau, are found in neurofibrillary tangles--a pathological hallmark of tauopathies. Which isoform is deposited in the affected tissues depends on the tauopathy. To study how and which tau isoforms contribute to neuronal degeneration, we have developed and characterized two novel conformation-sensitive antibodies, T3R and T4R. Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. The isoform-specific residues, (305)K in three-repeat tau or (305)S in four-repeat tau, and the PHF6 motif (VQIVYK) were identified as critical sequences. Despite the high similarity of the antigens, there was no cross-reactivity between T3R and T4R. Furthermore, T3R and T4R showed reduced binding to the thioflavin-positive beta-structural form of their target. These features may enable these antibodies to act as novel indicators that allow us to observe and evaluate conformational changes in each distinct isoform of tau.     

Human-human hybridomas secreting antibodies specific to human lung carcinoma

Summary Human Namalwa cells were screened in serum-free medium and in 6-thioguanine, then fused with human lymphocytes from lymph nodes of lung adenocarcinoma cancer patients. Extensive testing using 14 lung cancer cell lines, 11 other cancer cell lines and 4 normal fibroblast lines identified monoclonal antibodies produced by 4 hybridoma clones that reacted specifically with lung adenocarcinoma cells. These monoclonal antibodies also reacted with lung adenocarcinoma tissues and not normal tissues or erythrocytes of any blood type. These hybridoma clones grew and stably secreted the antibodies in serum-free medium as well as in serum-containing medium. Editor's Statement Identification of monoclonal antibodies that recognize human lung adenocarcinoma cells with reasonable specificity represents a potentially important development that may prove useful in diagnosis and therapy of neoplastic disease. The selection procedures and methods for propagation of the human-human hybridomas described in this paper also represent some novel approaches that may be of general application. David W. Barnes     

Enhanced immunization techniques to obtain highly specific monoclonal antibodies

Despite fast advances in genomics and proteomics, monoclonal antibodies (mAbs) are still a valuable tool for areas such as the evolution of basic research in stem cells and cancer, for immunophenotyping cell populations, diagnosing and prognosis of diseases, and for immunotherapy. To summarize different subtractive immunization approaches successfully used for the production of highly specific antibodies, we identified scientific articles in NCBI PubMed using the following search terms: subtractive immunization, monoclonal antibody, tolerization, neonatal, high-zone tolerance, masking immunization. Patent records were also consulted. From the list of results, we included all available reports, from 1985 to present, that used any enhanced immunization technique to produce either polyclonal or monoclonal antibodies. Our examination yielded direct evidence that these enhanced immunization techniques are efficient in obtaining specific antibodies to rare epitopes, with different applications, such as to identify food contaminants or tumor cells.     

Human single-chain antibodies specific to the tumor necrosis factor

Six unique phage antibodies against human tumor necrosis factor (TNF) were selected from the earlier constructed naïve combinatorial library of single-chain antibodies by affinity selection. The TNF binding of these antibodies was examined by enzyme immunoassay and Western blot analysis. The specificity of the selected antibodies was determined from their binding to interferons alpha and gamma, bovine serum albumin, ovalbumin, and ubiquitin. Two antibodies (sA1 and sB3) were converted into a soluble single-chain antibodies as individual molecules. Their affinity proved to be 2.5 and 13.7 nM, respectively.     

Sequence motifs specific for cytosine methyltransferases

Using a new alignment method, the sequences of 13 m5C methyltransferases (MTases) have been examined. Five extremely well-conserved blocks of sequence have been detected and have been used as fixed points for the alignment of the 13 sequences. Following this initial alignment, five further blocks of similarity have been identified to give a total of ten recognizable blocks of sequence homology that are all arranged in a common order. The structures of these MTases consist of a variable-length N-terminal arm followed by eight well-conserved blocks each separated by small variable-length regions. A large variable-length segment of 90 to 270 amino acids (aa) then follows. After this are two blocks, and a variable-length C-terminal segment completes the sequence. Within the final alignment, 20 aa in the protein sequences, and 86 nucleotides in the nucleotide sequences are invariant. The strongest conservation is found in proximity to a suspected functional site that contains the dipeptide proline-cysteine. Consensus patterns can be defined for the five best conserved blocks and, when used as search motifs, are able to clearly distinguish between the m5C MTases and all other identified proteins in the PIR database. This suggests they may be of use in identifying putative MTases among protein sequences of unknown function.     

Development and characterization of monoclonal antibodies to a specific domain of human estrogen receptor

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.     

Pinocytotic response of circulating erythrocytes to specific blood grouping antibodies

Human blood samples from adults and newborns of blood groups O, A, and B were treated with either anti-A blood grouping serum, ferritin-conjugated anti-A serum, free ferritin, or saline and then prepared for electron microscopy. Morphological differences were observed between the untreated erythrocytes of infants and adults. Circulating red cells of newborns were frequently vesiculated (25.5%), whereas those of adults only occasionally showed vesicles (5.5%). On the basis of morphology and incidence, the majority of these vesiculated cells seemed to be mature erythrocytes. The introduction of anti-A serum to group A erythrocytes of infants appeared to stimulate vesicle formation, but anti-A serum did not have a similar effect on group O or B cells of infants or on group A cells of adults. Vesicles which formed in response to antiserum treatment appeared to be the result of pinocytosis. In contrast to the well dispersed ferritin along the membrane of agglutinated adult cells, the ferritin particles on the infants' cells were frequently clustered at irregular intervals. These accumulations seemed to lead to invaginations of the cell membrane, resulting in ferritin-lined intracytoplasmic vesicles. The addition of free ferritin or ferritin-conjugated antibodies of the wrong specificity to red cells did not increase vesicle formation.     

Use of bacterial DHFR-II fusion proteins to elicit specific antibodies

Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented.     

Immunopurification of highly specific antibodies to calmodulin from Neurospora crassa

Abstract Highly specific antibodies against calmodulin from Neurospora crassa were produced in rabbits. These antibodies were immunopurified by chromatography on Neurospora calmodulin-Sepharose, and had a titer for purified calmodulin from Neurospora crassa and bovine testis of 80 ng/ml and 2.5 μg/ml respectively. By immunoblot, as little as 8 ng of pure Neurospora calmodulin could be detected, and the antibodies revealed calmodulin in crude homogenates from Neurospora crassa and Aspergillus nidulans while in homogenates from Allomyces arbuscula, Saccharomyces cerevisiae, Dictyostelium discoideum and bovine testis, calmodulin remained undetected.