Antigenic characterisation of monoclonal antibodies againstSarcocystis muris by Western blotting and immuno-electron microscopy

The antigenic specificities of 13 monoclonal antibodies (mAbs) raised againstSarcocystis muris cystozoites were examined by Western blotting and immuno-electron microscopy against homologousS. muris and heterologousS. gigantea, S. tenella, S. arieticanis, S. capracanis, S. miescheriana andToxoplasma gondii antigens. Four mAbs reacted in Western blots againstS. muris antigens: SM-4 and-17 recognized single antigenic bands (31000 and 34000 MW, respectively) and SM-2 and-3 reacted against multiple bands (ranging from 12500–30000 and 13000–50000 MW, respectively). Similar antigens were also recognized by polyclonal immune sera from chronically infected mice. None of the mAbs cross-reacted with heterologousSarcocystis spp. orT. gondii. Ultrastructural studies performed with colloidal-gold conjugates demonstrated that three mAbs reacted with specific antigenic elements inS. muris cystozoites: SM-3 and-4 labelled pellicular determinants and SM-19 labelled micronemes. None of the mAbs crossreacted with heterologousSarcocystis spp., whereas polyclonal immune sera from chronically infected sheep, goats and pigs cross-reacted with a variety of antigens in allSarcocystis spp. except the primary cyst-wall determinants.Abbreviations cz Cystozoite - fi tibril - gs ground substance - mi micronemes - pe pellicle - pw primary cyst wall - rh rhoptries     

ELISA tests and monoclonal antibodies for EBV

Monoclonal antibodies have now been prepared against a number of the major polypeptides associated with the different EBV-induced antigenic complexes. These antibodies have made it possible to purify and characterize most of these polypeptides and to identify the viral genes encoding for these proteins. In addition, new ELISA tests have been developed which measure antibodies in different immunoglobulin classes against the purified polypeptides. These assays have been useful in the diagnosis of EBV-associated diseases in clinical laboratories. The development of these antibodies and their application to the development of the new ELISA tests will be reviewed in this paper.     

Comparison of immunoperoxidase staining with indirect immunofluorescence, ELISA, and Western blotting assays for detecting anti-HTLV-I antibodies in systemic lupus erythematosus.

Serum antibodies against human T cell leukaemia virus type I (HTLV-I) were investigated in 12 patients by four methods: indirect immunoperoxidase staining, indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA), and strip radioimmunoassay based on the Western blotting assay. Seven patients had systemic lupus erythematosus (SLE) and five various autoimmune diseases with one or more circulating autoantibodies. Serum samples from three patients were found to be HTLV-I-positive by the ELISA assay and sera from five patients showed a non-specific reaction by indirect immunofluorescence. These sera were negative when tested by indirect immunoperoxidase staining and Western blotting assay. All four methods gave positive results when tested with samples from 19 HTLV-I carriers and 16 patients with adult T cell leukaemia. Indirect immunoperoxidase staining and Western blotting assay are probably useful and more specific assays for the detection of anti-HTLV-I antibodies in samples from patients with autoimmune diseases.     

Competitive ELISA employing monoclonal antibodies specific for dothistromin

Dothistromin (DOTH) is a fungal toxin occurring in Pinus radiata needles contaminated with Dothistroma pini. Monoclonal antibodies (MAbs) of high affinity were prepared against DOTH and incorporated into competitive ELISAs. MAbs were secreted by hybri‐doma prepared from mice immunized with DOTH conjugated, through the aromatic ring of the anthraquinone, to bovine serum albumin. DOTH could be quantitated at 2–60 ng ml^‐1 (0.1–2 ng/assay) and at 8–300 ng ml^‐1 (0.4–15 ng/assay) using peroxidase‐labelled MAb 10C12A5 and MAb 6A2D4 respectively in indirect competitive ELISA. The corresponding limits of detection of DOTH were < 300 pg and 1 ng ml^‐1 respectively. The furan ring was an important structure in the epitope recognized by both MAbs.     

Evaluation of seven oestrogen receptor beta antibodies for immunohistochemistry,western blotting,and flow cytometry in human breast tissue

Two oestrogen receptors, ER alpha and ER beta, exist. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. The aim of this study was to determine the utility of seven ER beta antibodies (14C8, 8D5, PAI313, PPG5/10, N19, 9.88, and D7N) raised against different domains of ER beta in three commonly used laboratory applications, namely immunohistochemistry, western blot, and flow cytometry, using human breast material. For immunohistochemical analysis of frozen material, PAI313 and D7N gave stronger and more specific signals than 14C8, 8D5, and PPG5/10. In paraffin sections, 14C8, closely followed by PPG5/10, gave by far the most superior nuclear immunoreactivity, compared with the other antibodies tested. In general, flow cytometry results mirrored the immunohistochemistry data for paraffin sections, with antibodies ranked 14C8 > 8D5> or = PAI-313 > PPG5/10 >D7N. For western blotting, 8D5 and D7N yielded the strongest and most consistent bands, with weaker bands seen with the others. It is concluded that ER beta protein can be detected using specific antibodies. However, there is considerable variation between the specificity and application of these antibodies, highlighting the fact that careful optimization is required when selecting an antibody for use in a particular laboratory technique.     

A sensitive blotting system for detection of alpha-fetoprotein variants with monoclonal and polyclonal antibodies

Human alpha-fetoprotein (AFP) variants from cord sera were separated by isoelectric focusing in agarose gels under native conditions, transferred to nitrocellulose paper and detected with polyclonal and monoclonal antibodies (Moabs). Rabbit anti-AFP recognized up to 9 individual electrophoretic variants in the range of pH 4.5 to pH 5.2. The reactivity of 8 Moabs ranged from weak to strong and showed variability in the pattern of AFP bands recognized. Moabs were separated into 3 groups according to the number of bands detected: group 1 detected 6 to 7 bands; group 2 recognized only one band; and group 3 recognized 4 bands. The sensitivity of the system with polyclonal antibodies was 0.15 ng of AFP in complete cord serum and varied between 300 and 0.2 ng with Moabs.     

ELISA for detection of Mycoplasma pneumoniae antigens using monoclonal antibodies

Seven monoclonal antibodies directed against major antigens of Mycoplasma pneumoniae were selected for the development of an antigen detection assay. Three of these were directed to the 170,000-dalton adhesin of M. pneumoniae. The test was an antigen-capture enzyme immunoassay using the different monoclonal antibodies for capture of antigen and a polyclonal rabbit antiserum as detection reagent. With three of the monoclonal antibodies a detection limit of approximately 2 ng M. pneumoniae protein was obtained, as determined by titration of M. pneumoniae organisms in buffer. The detection limit of the assays was only slightly less when the other four monoclonal antibodies were used. In artificially infected nasopharyngeal aspirates the detection limit was approximately 10 times lower. The fact that no significant differences in the detection limit of the assays were recorded using monoclonal antibodies directed against different antigens indicates that these antigens were available for reaction with antibodies irrespective of their location in intact M. pneumoniae cells. In the assay there were no significant cross-reactions with a number of bacterial species potentially colonizing the respiratory tract, except for a protein A-positive strain of Staphylococcus aureus. Our test is equally sensitive to another recently described ELISA using polyclonal antibodies. In comparison with other recommended methods such as immunoblot and culture-amplified antigen detection assays, the ELISA is more rapid and less laborious.     

New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry

Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost-effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL-ER-6F11 and NCL-PGR respectively, which are effective in heat-treated formalin-fixed, paraffin-embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL-ER-6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer. © 1997 John Wiley & Sons, Ltd.     

Development of monoclonal antibodies and ELISA specific for the mouse vascular endocan

Human vascular endocan is a proteoglycan exhibiting tumorigenic activity through both its glycan and protein cores. Endocan mRNA is identified as being one of the most significant molecular signatures defining a poor prognosis in lung, breast, kidney, prostate, and thyroid malignancies. The survival inversely correlates with endocan expression in tumor tissue from hepatocarcinoma, and in serum from lung cancer. In mouse, endocan mRNA is also increased in tumor vessels. However, mouse endocan has not yet been fully characterized. Here, we produced a panel of rat monoclonal antibodies directed against mouse endocan, leading to the development of a specific mouse/rat endocan ELISA. Mouse endocan serum level was measured at a median of 0.96 ng/mL and 1.08 ng/mL in 129Sv mice and C57bl6, respectively. These results also provide new tools to characterize and explore the role of endocan in mouse and rat models of human diseases. These results present mouse vascular endocan as a circulating molecule similar to human endocan.     

Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.     

Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies

Hybridomas producing anti-creatine kinase (CK) and anti-lactate dehydrogenase (LDH) antibodies were screened by enzyme linked immunosorbant assay (ELISA) with antigen coated onto plastic wells. Out of seven antibodies positive for each isoenzyme of CK, four antibodies failed to bind to the radiolabeled antigen in solution phase radioimmunoassay (RIA) or native antigen in competitive ELISA. Moreover, out of nine antibodies shown reactive with LDH-1 and seven antibodies binding to LDH-5 by ELISA, not a single antibody bound to either radiolabeled or native antigen in solution. Our results along with other recent studies strongly suggest that antigen conformation on solid phase may frequently be different from in solution and that screening by ELISA in which antigen is attached to solid phase is often inappropriate for determining the presence of useful monoclonal antibodies.     

An ELISA method for quantification of Tamm-Horsfall protein using monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MoAbs) to Tamm-Horsfall protein (THP) were established. The isotype and reaction pattern of the MoAbs with THP from rat, rabbit, guinea pig and man were employed for the selection of clones. At least four epitopes were recognised on human THP. One of these epitopes differed from the others in its dependence on the state of aggregation of the THP. An ELISA procedure was developed for quantification of THP in urine requiring no other sample treatment than dilution in the assay buffer. In this ELISA, THP showed an increased immunoreactivity after freezing.     

Competitive ELISA employing monoclonal antibodies specific for coronafacoyl amino acid conjugates

Coronatine (COR) is composed of two structural components, coronafacic acid (CFA) and the amino acid coronamic acid (CMA), linked by an amide bond. Monoclonal antibodies (MAbs) were prepared against COR and incorporated into competitive ELISAs. MAbs were secreted by hybridoma cells which had been prepared from mice immunized with COR and conjugated, through the free carboxyl group on CMA, to ovalbumin, the available amino groups of which had been increased by derivatization with propyl diamine via free carboxyl groups. COR and coronafacoyl valine could be quantified at 6.800 ng ml^‐1(0.34–40 ng/assay) using peroxidase‐labelled MAb 8H3G2 in an indirect competitive ELISA. The corresponding limit of detection was 1 ng ml～’. Reduction and subsequent acetylation of the ketone oxygen of the CFA moiety of COR reduced the affinity of MAb 8H3G2 some 250 times, whereas methylation of the free carboxyl group on COR slightly enhanced the affinity four‐fold. These and other results suggest that CFA and the amide bond are important structural features of the epitope recognized by MAb 8H3G2.     

Western blot screening for monoclonal antibodies against human separase

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.     

Rapid, simple and sensitive antigen capture ELISA for the quantitation of myoglobin using monoclonal antibodies

A rapid ELISA method has been developed to quantitate myoglobin in serum. An antigen capture enzyme-linked immunoassay with IgG1 mouse monoclonal antibodies produced by cell clones MGB20-4A1.1 and MGB20-3C1.2 were used for myoglobin detection. These monoclonal antibodies are specific for different epitopes of the myoglobin molecule. Monoclonal antibodies from the hybridoma clone MGB20-4A1.1 were adsorbed to microtiter plate wells. The plates were washed with PBS containing 0.05% Tween 20 and then 20 microliter of standard serum or serum of patients and 200 microliter of peroxidase labeled monoclonal antibodies MGB20-3C1.2 were added to each well. Plates were incubated for 90 min at 37 degrees C and enzyme activity was determined using o-phenylenediamine as a substrate. The ELISA assay described is a rapid and sensitive procedure to assess the quantity of myoglobin within the range 2-1000 ng/ml serum. 120 samples can be tested in 3 h.     

Grass pollen allergens: antigenic relationships detected using monoclonal antibodies and dot blotting immunoassay

Monoclonal antibodies against major rye-grass pollen allergens have been used to detect cross-reactive determinants in other grass pollen extracts. Antibody binding was detected by the dot blotting immunoassay in which alkaline phosphatase-conjugated anti-mouse IgG was used as secondary antibody. Taxonomically ordered variations were found between pollen of 22 grass species representing all major natural groups. One of the antibodies, that bound to the 28 to 30Kd allergen, showed high species specificity; immunoblotting showed binding to similar polypeptides in Festuca elatior but to none of the other grasses tested. This simple assay has applications in standardizing grass pollen extracts.     

A new model ELISA,based on two monoclonal antibodies,for quantification of fatty acid synthase

A new model ELISA, based on two monoclonal antibodies, was developed for the quantification of fatty acid synthase (FAS). In this sandwich assay, a monoclonal antibody M6 was used as a capture on Nunc MaxiSorp ELISA/EIA Modules and another monoclonal antibody M3, labeled with biotin, was used as a detection antibody. More than 10 molecules of biotin were labeled on the anti-FAS monoclonal antibody using modified biotinylation conditions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/mL. Recoveries were 98.54-121.95%, averaging 106.05%. The average FAS concentration obtained from the total 55 healthy volunteers blood was 4.07 +/- 1.81 ng/mL, 4.25 +/- 2.14 ng/mL in women (n = 37) and 3.70 +/- 0.74 ng/mL in men (n = 18). When compared with the previously developed polyclonal-monoclonal ELISA, a different pattern of FAS levels was observed in the supernatant of two cultured breast cancer cell lines in a time course study and there was no linear correlation between the two assays using 215 human blood samples. Thus, this new model FAS-ELISA could be used as an independent assay in measuring clinical samples. In summary, this monoclonal-monoclonal FAS-ELISA is sensitive, accurate, and precise in quantification of fatty acid synthase and has potential as a complementary tool in testing clinical samples.     

适用免疫组化的突触素与嗜铬素A 单克隆抗体的制备与临床初步评价

目的:制备突触素与嗜铬素A 免疫组化单克隆抗体,期望可初步应用于临床。方法:设计Syn 与CgA 融合基因,并构建至表达载体上表达获得融合蛋白。经过抗原免疫、细胞融合后筛选获得目标抗体。通过临床样本对比验证,研究Syn 和CgA 两者的特异性,评价相关系数。结果:通过筛选,共获得2 株分别针对Syn 与CgA 的抗体3D9 和4A12。通过对比抗体试剂共同验证19 种蜡块组织,统计结果分析,结果符合性较好,相关系数分别达到r =0.989 2 与r =0.993 9。结论:该方法成功制备获得了可初步适用于免疫组化的突触素与嗜铬素A 抗体,该方法可为免疫学抗体研究提供一定的借鉴意义。     

An improved ELISA for the detection of antibodies to ovine and caprine lentiviruses, employing monoclonal antibodies in a one-step assay

An improved ELISA for the detection of antibodies to ovine and caprine lentiviruses has been developed. The assay employs two monoclonal antibodies (Mabs) directed against the major core protein (p28) of the virus in a modified double antibody sandwich blocking procedure. The modification was necessary to circumvent the consequences of using low affinity Mabs and to lower the chance of false-negative results. A novel one-step concept was developed in which washing between steps was largely omitted. The new assay was designated complex trapping blocking (CTB)-ELISA. A range of sera from sheep and goats was tested comparatively in CTB-ELISA, in an indirect ELISA and in an agar gel precipitation test. The CTB-ELISA proved sensitive and highly specific and in addition reliable and easy to perform.     

A highly specific two-site ELISA for pneumococcal C-polysaccharide using monoclonal and affinity-purified polyclonal antibodies

A two-site ELISA for the detection of pneumococcal C-polysaccharide (PnC) has been developed. A monoclonal antibody directed against the phosphorylcholine residue of the PnC was used as catcher and an affinity-purified polyclonal anti-PnC rabbit antiserum for detection. Polyclonal antibodies against the PnC as well as capsular antigens were obtained by immunizing rabbits with type 1 pneumococci. Antibodies against the phosphorylcholine determinant of PnC could be removed by affinity purification. Remaining antibodies reacted in an ELISA with type 1 capsular polysaccharide as well as with PnC. Only in the fraction with the highest antibody activity against PnC, phosphorylcholine exhibited a slight inhibitory action. It is concluded that the purified antibody preparation reacted with an antigenic determinant shared by the two polysaccharides, in all probability a determinant associated with 2-acetamido-4-amino-2,4,6-trideoxygalactose which is the only monosaccharide component in common between PnC and the type 1 capsular polysaccharide. By the use of this affinity-purified antibody preparation, reactions with alpha-streptococci, occurring with non-purified serum, were abolished. The sensitivity and specificity of the test was determined using capsulated and non-capsulated pneumococci and alpha-streptococci known to cross-react with unpurified serum against the pneumococcal C-polysaccharide.