Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.     

A monoclonal anti-idiotype specific for human polyclonal IgM rheumatoid factor.

One of the hallmarks of rheumatoid arthritis (RA) is the production of high titers of rheumatoid factor (RF) antibody directed against the Fc portion of IgG. Anti-Id that recognize the majority of monoclonal RF from patients with B cell dyscrasias are reactive with only 1 to 2% of these polyclonal RF from RA patients. We describe a new monoclonal anti-Id, 4C9, that recognizes a L chain determinant on polyclonal IgM RF from patients with RA but does not recognize a panel of monoclonal RF from patients with B cell malignancies. 4C9 reactivity is found in the serum of 34/43 RF-positive RA patients and in 12/12 RF-positive synovial fluids, but in only 1/14 RF-negative sera from RA patients and 1/22 sera containing monoclonal IgM RF. 4C9 reactivity is highly enriched in purified IgM RF from nine RA patients and represents a variable percentage of total IgM RF up to a maximum of 23%. Furthermore, 4C9 reactivity is enriched in the synovial fluid of three of five RA patients compared with serum, suggesting that 4C9-reactive IgM RF are synthesized within the joint. IgG RF from RA synovial fluids are not 4C9 reactive, indicating either that different genes are used to encode IgM and IgG RF in RA patients, or that IgG RF have somatically mutated away from idiotypic reactivity.     

Production of human monoclonal and polyclonal antibodies in TransChromo animals

We have developed TransChromo (TC) technology, which enables the introduction of megabase-sized segments of DNA into cells. We have used this approach to derive mice that carry megabases of human DNA by the use of a human chromosome fragment (HCF) as a vector. TC technology has been applied to the construction of the TC Mouse,trade mark which incorporates entire human immunoglobulin (hIg) loci. TC Mouse expresses a fully diverse repertoire of hIgs, including all the subclasses of IgGs (IgG1-G4). Immunization of the TC Mouse with various human antigens produced antibody responses comprised of human antibodies. Furthermore, it was possible to obtain hybridoma clones expressing fully human antibodies specific for the target human antigen. However, because of the instability of the Igkappa locus-bearing HCF2, the efficiency of hybridoma production was less than one-tenth of that observed in normal mice. An instant solution to this problem was to cross-breed the Kirin TC Mouse carrying the HCF14, which was stable in mouse cells, with the Medarex YAC-transgenic mouse carrying about 50% of the hIgVkappa gene segments as a region that is stably integrated into the mouse genome. The resulting mouse, dubbed the KM Mouse, performed as well as normal mice with regard to immune responsiveness and efficiency of hybridoma production. Another application of TC technology is the production of polyclonal antibodies in large animals such as chickens and cows. To test the efficacy of human polyclonal antibodies derived from TC animals, feasibility studies were performed using antisera and purified gamma-globulin from TC mice immunized with Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), or Japanese encephalitis virus (JEV). The TC mouse-derived antisera and gamma-globulin showed a much higher titer and efficacy in terms of the neutralizing activity of the pathogens in vitro and in vivo than either human serum or gamma-globulin prepared from human blood.     

应用单克隆抗体测定人弓形虫IgM抗体的研究

为了检测人血清弓形虫ＩｇＭ抗体,采用抗人ＩｇＭ单克隆抗体和特异性抗弓形虫单克隆抗体建立捕获ＥＬＩＳＡ法,并与ＰＣＲ方法进行了比较。结果检测１０６５份献血员血清,检出阳性３例,用ＰＣＲ方法检测呈阳性结果;检测２３例类风湿病人血清及２份弓形虫ＩｇＧ抗体阳性血清均为阴性反应。说明该方法不受类风湿因子（ＲＦ）和特异性ＩｇＧ抗体的干扰,同时也表明捕获ＥＬＩＳＡ检测人血清中弓形虫ＩｇＭ抗体特异性,敏感性良好。     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli , and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli ; of 13 non- E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non- E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non- E. coli.     

Production and specificity of monoclonal antibodies and polyclonal antibodies to Escherichia coli

Monoclonal antibodies were produced to whole cells of heat-treated Escherichia coli. Balb/c mice were immunized with a pool of five strains of heat-treated E. coli, and the resulting hybridomas were screened by indirect immunoassay. E. coli strains other than those used for immunization were used for screening to detect hybridomas producing antibody that reacted with a large number of E. coli strains. Of 864 hybridomas, 32 reacted strongly with either two or all three of the strains used for screening; 15 were successfully cloned. Antibody from hybridoma 6H2 reacted with 35 of 68 (51%) E. coli; of 13 non-E. coli tested, only Enterobacter agglomerans was weakly positive. Hybridoma 9B12 antibody reacted with all six E. coli tested. Hybridoma 9B12, however, stopped producing antibody. Five hybridomas produced antibody which reacted with a majority of the bacteria tested whereas antibodies from two other hybridomas reacted with several E. coli and non-E. coli. Polyclonal antibodies produced to two strains of E. coli varied in the numbers of E. coli with which they reacted; both antisera cross-reacted with several non-E. coli.     

Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins

Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape.     

Comparison of the specificity of polyclonal antibodies to the tick-borne encephalitis virus in immunoenzyme analysis and immunoblotting

The present study deals with the optimization of the conditions of immunoblotting (IB) and the enzyme-linked immunosorbent assay (ELISA) with the use of mouse polyclonal immunoglobulins to tick-borne encephalitis virus. In contrast to ELISA, the results of IB depended, to a great extent, on the composition and pH of blocking buffer mixtures. In some cases IB permitted the detection of a heretofore unknown virus-specific polypeptide with a molecular weight of 58-60 KD. The results of the study lead to the conclusion on the impossibility of the direct transfer of data concerning the specificity of individual preparations of immunoglobulins in ELISA or IB due to differences in information.     

Use of cooperative monoclonal antibodies in immunoenzyme analysis for determining human tumor necrosis factor

A cooperative sandwich enzyme immunoassay (EIA) based on the newly produced pair of cooperative monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was developed and characterized. It was found that, when used simultaneously, cooperative mAbs was capable to bind TNF from its preformed complexes with soluble TNF receptors (sTNF-R), thus providing the effective TNF detection in ex vivo samples by the respective one-step cooperative EIA. While demonstrating typical analytical characteristics regarding variability, dynamic range and specificity, a cooperative EIA offers an advantageous combination of high sensitivity (< 2 pg/ml) and short-time TNF capture protocol (1 hour). Application of cooperative EIA for TNF detection in clinical samples has demonstrated an increased serum TNF levels in patients with the mixed connective disease and infectious endocarditis that positively correlated with severity of systemic inflammatory reactions. Production and EIA application of cooperative mAbs would be promising in development of standardized and clinically applicable immunoassays for cytokines.     

An immunohistochemical study performed with monoclonal and polyclonal antibodies to mouse epidermal growth factor

We developed two monoclonal antibodies, E5 and F5, which react with mouse epidermal growth factor (mEGF) and urogastrone. These antibodies and a rabbit antiserum to mEGF were used for immunohistochemical analysis of staining reactions in rodent and human tissues. Our results do not confirm the published reports of EGF-like immunoreactivity in Brunner's glands, human submandibular glands, human kidney tissue, or rodent brain sections.     

Generation and characterization of polyclonal and monoclonal antibodies to human NTPDase2 including a blocking antibody

Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) is an ectonucleotidase that modulates P2 receptor activation by hydrolyzing ATP to ADP. In rodents, NTPDase2 is expressed by several specialized cell types such as vascular adventitial cells, neuroglial cells, hepatic portal fibroblasts, gustatory type I cells, and cells within the connective tissues of reproductive and gastrointestinal organs. Much less is known regarding the expression and function of NTPDase2 in humans. Here, we developed specific research tools to study human NTPDase2. We generated mouse monoclonal antibodies and rabbit polyclonal antibodies specific to human NTPDase2 and validated their specificity by western blot, immunocytochemistry, immunohistochemistry, and flow cytometry. In addition, one monoclonal antibody named hN2-D5 _s specifically inhibits human NTPDase2 enzymatic activity but not mouse nor rat NTPDase2. Using these antibodies, NTPDase2 immunoreactivity was detected on glial cells of the human enteric nervous system suggesting a function of the enzyme in intestinal motility. In conclusion, the new antibodies described in our work are novel tools that will enhance future studies of NTPDase2 expression and function in humans.     

Preparation and characterization of monoclonal antibodies to human apolipoprotein E]

Monoclonal antibodies to human apolipoprotein E (apoE) were prepared and characterized. Antibodies of 3D12F11 clone were shown to be specific to apoE and belong to IgG1 subclass. Dissociation constant of antigen-antibody complex in solution was determined as 3.5 +/- 0.5 nM. The antibodies interacted neither heparin- nor lipid-binding sites of human apoE molecule. The obtained antibodies may be useful for metabolic and structural investigations of apoE as well as for clinical studies.     

The diagnostic value of different test systems for the screening determination of antibodies to the human immunodeficiency virus]

Evaluation of the quality of enzyme immunoassay (EIA) systems by their sensitivity and specificity is inadequate, mainly due to the impossibility of detecting early or latent HIV infection in humans as it manifests by seroconversion only to a few HIV proteins. The additional evaluation criterion (confirmation rate) has been introduced, and an original method for the integral evaluation of the quality of assay systems intended for the diagnosis of HIV infection by EIA techniques has been proposed.     

An immunoenzyme test system for the detection of antibodies to Campylobacter]

An enzyme immunoassay system for the detection of antibodies to bacteria of the genus Campylobacter in human blood serum has been developed. The system is based on the use of ethanol-treated C. jejuni and C. coli whole cells as antigen. The study of sera obtained from healthy donors in this assay has made it possible to establish the value of the tentative diagnostic titer: 320.     

Comparative immunochemical characterization of polyclonal and monoclonal antibodies to aflatoxin B1

Four hybrid clones (MM-(AB₁)-1, MM-(AB₁)-2, MM-(AB₁)-3, and MM-(AB₁)-4) were obtained by hybridoma technology involving the immunization of BALB/c mice with a BSA conjugate of aflatoxin B₁ carboxymethyloxime derivative. Antibodies produced by these clones varied in their ability to recognize the aflatoxin B₁ analogues. The sensitivity of enzyme immunoassay based on all monoclonal antibodies was higher compared to analysis based on polyclonal rabbit antibodies (0.1 and 0.4 ng/ml, respectively).     

Adsorption equilibrium in immuno-affinity chromatography with polyclonal and monoclonal antibodies

The effects of pH, ionic strength, anion species, and antibody concentration on the adsorption equilibrium between immobilized antibodies and antigens were studied by use of anti-BSA, anti-HSA, anti-BlgG, and monoclonal anti-HSA coupled to Sepharose 4B. The polyclonal antibodies possessed average binding affinities of the order of 10(8)M(-1), and the heterogeneity was accounted for by assuming a normal distribution of the free energy of antibody-antigen combination. The monoclonal antibody, on the other hand, showed a homogeneous affinity of the Langmuir type. Bound antigens could be eluted by lowering pH or adding a chaotropic anion, and their purity was very high. The antibody ligand was sufficiently stable for repeated use.     

Use of monoclonal antibodies in immunoenzyme analysis for demonstrating antigenic monovalency

To prove the monovalence of the antigen a method has been developed consisting of ELISA with the use of monoclonal antibodies in combination with the antibody neutralization test. Yersinia pestis capsular antigen was disintegrated by heating at 100 degrees C for a short time and subsequently passed through a column packed with Sephadex G-50. The portions of the eluate, showing high activity in the antibody neutralization test and low activity in ELISA (the double antibody sandwich scheme), contained mainly the monovalent antigen. This antigen was replaced by the polyvalent antigen from the antibody complex, but if such complex had been previously fixed by treatment with glutaraldehyde, no replacement of the monovalent antigen by the polyvalent one occurred.     

The comparative evaluation of spectrophotometric and chemiluminescent detection in an immunoenzyme test of antibodies to the antigens of the bovine leukosis virus]

Comparative evaluation of the sensitivity limit in the detection of antibodies to bovine leukemia virus in the enzyme immunoassay with the use of chemiluminescent and spectrophotometric detection techniques was carried out. In this assay 3-amino-1,4-phthalazinedion was used as chemiluminescent substrate and ortho-phenylenediamine, as chromogenic substrate. The chemiluminescent signal was registered by means of a special luminometer designed at the Institute of Biochemistry (Lithuanian Acad. Sci.). The use of the chemiluminescent substrate permitted the detection of proteins in amounts 2-3 times lower than those detected by the spectrophotometric technique.     

Preparation and characterisation of monoclonal and polyclonal antibodies to maize membrane auxin-binding protein

Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP auxin-binding protein - DEAE diethylaminoethyl - Ig immunoglobulin - kDa kilodalton - NAA naphthalene-1-acetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

A novel platform to produce human monoclonal antibodies

A new technology has been developed that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps, and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.