Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria.

Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria.     

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5C1 demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5C1 and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5C1 and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.     

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5Cl demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5Cl and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.     

Induction of monoclonal antibody with predefined ELNKWA epitope specificity by epitope vaccine

Since the hybridoma technique to produce monoclonal antibodies (MAbs) was discovered, thousands of MAbs with predefined protein specificity have been produced, and a natural or recombinant protein as antigen is necessary for inducing MAbs in the conventional hybridoma technique. To induce epitope-specific MAbs, we suggest an epitope vaccine as a new technique to induce MAbs with predefined epitope specificity. ELDKWA was identified as an important neutralizing epitope on HIV-1 gp41. The MAb 2F5, recognizing ELDKWA epitope, has shown broad neutralizing activity to many HIV strains, including primary isolates, but the mutant in ELNKWA epitope results in escape 2F5-based neutralization. To produce MAbs recognizing this mutated epitope for consideration of passive immunotherapy against the mutant bearing the ELNKWA epitope, MAbs with predefined ELNKWA epitope specificity were induced by synthetic epitope-peptide instead of a natural or recombinant gp41 bearing this epitope. Three MAbs were identified to recognize ELNKWA epitope on the synthetic epitope-peptide, and interestingly could bind the recombinant gp41 with ELDKWA epitope in an ELISA assay and immunoblotting analysis.     

Telomerase activation in colorectal carcinogenesis.

Telomerase activity has been detected in germ cells as well as in the developing embryo. Activity is no longer detectable in most somatic cells of the neonate, although low levels of activity persist in regenerative tissues. Telomerase has been found to be reactivated or up-regulated in the majority of cancers. The colorectal adenoma-carcinoma sequence is one of the best-characterized models of multistep tumourigenesis and is thus suitable for determining at which stage telomerase is activated. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in 96 cases of colorectal tissues, including 50 carcinomas, 31 adenomas, and 15 normal colonic tissues. For each case, histological diagnosis and telomerase activity were determined on consecutive frozen sections. In order to reduce the chance of a false-negative TRAP assay due to RNA degradation, the integrity of rRNA in the tissues was verified in each case. Twenty-five carcinomas, 30 adenomas, and all of the 15 normal colorectal mucosal samples showed no or only partial rRNA degradation and only in these cases was the TRAP assay interpreted. None of the normal tissues exhibited telomerase activity. In contrast, all of the 25 cancers and 47 per cent (14/30) of the adenomas were positive. In adenomas, telomerase activation was highly significantly related to the grade of dysplasia (p< 0.0001). All adenomas which contained high-grade dysplasia revealed telomerase activity, whereas telomerase activity was detectable in only 20 per cent (4/20) of cases with exclusively low-grade dysplasia. These results indicate that telomerase activation, which may be an obligatory step in colorectal carcinogenesis, occurs in the progression from low-grade to high-grade dysplasia in adenomas. Furthermore, in the adenoma-carcinoma sequence, telomerase activation seems to occur later than K- ras mutation but earlier than p53 mutation.     

Monoclonal IgM antibodies from cytomegalovirus-infected mice recognize the GlcNAc-containing receptor determinant of murine CMV as well as neutralizing anti-CMV IgG antibodies.

This study examines monoclonal antibodies derived from mice at different time points after infection with attenuated murine cytomegalovirus (MCMV). The antibodies obtained from mice at 3 weeks p.i. were of IgG type (designated V-antibodies) and several could neutralize the virus. Those obtained at 5 weeks p.i. were of the IgM class (designated R-antibodies), bound to uninfected (MEF, mouse embryo fibroblast) cells, and thereby blocked MCMV plaque formation. In ELISA, the IgM monoclonals (R-antibodies) bound to GalB1-3GlcNAc and GalB1-4GlcNAc, the receptor determinants for MCMV. Similarly, these GlcNAc-containing residues blocked the binding of purified IgM monoclonal antibodies (MAbs) to MEF. The R- and V-series of antibodies showed mutual binding activities; for example, IgM MAb R-2D8 bound specifically to four (V-8C4, V-1C7, V-8C7, V-9C5) of six neutralizing IgG MAbs in ELISA. The same neutralizing IgG MAbs bound to antireceptor IgM antibodies in an immunoblot assay. This suggests that the IgM monoclonals directed against the known cell surface receptor determinant are anti-idiotypic antibodies against neutralizing antiviral IgG antibodies. The neutralizing antiviral IgG MAbs bound to 60- and 66-kDa MCMV polypeptides on Western blots, suggesting that these viral polypeptides may be important in MCMV binding to this receptor. The R-series might represent anti-idiotype antibodies capable of down-regulating antiviral V-antibodies and may also represent a mechanism for the induction of IgM autoantibodies directed at cell surface glycolipids present in autoimmune CMV-associated neuropathies.     

Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.     

Real-time quantitative analysis of telomerase activity in breast tumor specimens using a highly specific and sensitive fluorescent-based assay.

Telomerase activity has been associated with almost 90% of malignant human cancers from a variety of tissue sources, making it one of the most prominent molecular cancer markers known to date. As such, telomerase has become a very attractive diagnostic and therapeutic target. The advent of the telomeric repeat amplification protocol (TRAP) has allowed for the semiquantitative detection of telomerase from limiting sample amounts. Both the standard TRAP assay and a real-time assay using Amplifluor technology with primers designed specifically for telomerase activity amplification were used to quantitatively assess telomerase activity in primary tumors and tumor-derived cell lines. We have adapted the recently developed TRAPeze XL telomerase detection kit (Intergen, Gaithersburg, MD) for use with real-time polymerase chain reaction for more accurate quantification and high-throughput capabilities. In doing so, the reliability, assay time, and accuracy of quantitation have all been dramatically improved. A comparison of the quantitative analysis for the standard TRAP assay versus the real-time assay using 19 breast tumors revealed telomerase quantitation and standardization using the real-time assay was superior to the standard assay. Our data suggest that this assay will be useful for clinical and research studies involving detection of telomerase activity as it relates to cancer diagnosis.     

Telomerase expression in intracranial tumours: prognostic potential for malignant gliomas and meningiomas.

AIM: To evaluate the diagnostic value of telomerase expression in intracranial tumours. METHODS: 98 surgical specimens from different neoplasms were analysed by the telomeric repeat amplification protocol (TRAP) and the presence of telomerase compared with the histological diagnosis and the proliferation index. RESULTS: A high degree of positivity for telomerase activity was found in glioblastomas and atypical/anaplastic meningiomas. Telomerase activity was poorly detected in anaplastic astrocytomas. CONCLUSIONS: The TRAP assay seems to be a valuable index for identifying meningeal tumours with aggressive behaviour.     

Production of monoclonal antibodies against recombinant human interferon-gamma: screening of hybridomas without purified antigen

A panel of mouse monoclonal antibodies (MAbs) against the recombinant human gamma interferon (HuIFN-gamma) has been produced for the study of the structure-function relationships of this important lymphokine. Enzyme linked immunosorbent assay (ELISA) is the current method of choice to screen hybridomas for specific MAb production. The purity of the antigen used for screening dictates the specificity of the ELISA. As often is the case in many systems, adequately purified biologically active HuIFN-gamma was not readily available for this purpose. A sandwich ELISA which allowed the use of unpurified HuIFN-gamma for hybridoma screening was developed. A rabbit antiserum against the denatured HuIFN-gamma purified by SDS-PAGE was prepared and the nonspecific binding activity was removed by adsorption to control cell proteins immobilized on Sepharose. The adsorbed immunoglobulin fraction was bound to the ELISA plate: (i) to trap HuIFN-gamma specifically from the whole cell lysate, thus providing specificity for MAb detection, and (ii) to avoid direct adsorption of the HuIFN-gamma to the ELISA plate because others have found that this prevented detection of neutralizing MAb. The sandwich ELISA detected both neutralizing and non-neutralizing MAbs with relatively low false positive reactions. This approach to the development of an ELISA method to screen hybridomas without purified antigen should be applicable to the production of MAbs to other proteins.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Telomerase and telomere length in normal and malignant human endometrium as prognostic markers

Telomerase activation and telomere length maintainence are thought to be essential for cellular immortality and oncogenesis. Normal human endometrium expresses significant telomerase activity in a menstrual phase dependent manner. In this report, we have evaluated telomerase activity and telomere length in post menopausal endometrial hyperplasias and endometrial cancers to study their usefulness as prognostic markers. Telomerase activity was measured by the TRAP assay (Boehringer Mannheim, Germany) and telomere restriction fragment (trf) by the telomere length assay kit (BD Pharmingen). Proliferation markers PCNA and Bcl2 were studied by immunohistochemistry. Senescence associated Ogal activity was studied simultaneously and correlated with the above markers. Strong telomerase activity was observed in the proliferative phase of the normal endometrium, endometrial cancers and post-menopausal endometrial hyperplasias compared to normal, secretory and resting phases of the endometrium. PCNA and Bcl2 showed high positivity in telomerase positive cases. Telomerase activity was inversely proportional to Ogal activity. Mean trf lengths became shortened as the normal tissues underwent neoplastic changes. Our study suggests that high telomerase activity and short telomere lengths could be useful prognostic markers in human endometrium.     

Detection of malignant effusions: comparison of a telomerase assay and cytologic examination

Telomerase is inactive in most somatic cells, but has been found to be reactivated in a majority of cancers. Our principal goal was to test whether the presence of telomerase activity concurred with positive cytology, and was thus of potential use in detecting cancer cells in effusions. The telomeric repeat amplification protocol (TRAP) assay and cytological examination were performed in a blinded fashion on 91 unselected effusions, for which laboratory processing was done according to standard procedures. In our series, 30% (27/91) of samples were found to be malignant by cytology. Of these, 19 (70%) were also positive in the TRAP assay. Of the 8 telomerase-negative cytology-positive samples, RNA integrity was generally poor, indicating suboptimal sample conservation for molecular analysis. Negative cytology in the presence of telomerase activity was observed in 17 effusions. Of these, 11 were from patients with advanced cancer, and thus a diagnosis of malignant effusion should be suspected. The TRAP assay for telomerase activity holds promise in the analysis of effusions, but its routine use as an adjunct to cytology awaits further confirmation of its positive predictive value.     

Monoclonal antibody characterization of Jamestown Canyon (California serogroup) virus topotypes isolated in Canada.

Jamestown Canyon (JC) virus of the California (CAL) serogroup has been isolated in 12 American states and 6 Canadian provinces. A study was undertaken to produce monoclonal antibodies (MAbs) to JC virus and to use these MAbs to assay for possible heterogeneity among naturally occurring JC topotypes in Canada. MAbs were produced to the prototype strain of JC virus using BALB/c mice. Twenty-seven secreting MAbs were obtained and three of these MAbs were propagated and studied. All three MAbs, M1 (IgG1), M2 (IgG2b), and M3 (IgG2a), were reactive by immunofluorescent antibody assay against JC-infected vero cells and by ELISA against JC antigen. MAb M2 reacted with all members of the Melao complex, MAb M1 reacted only with Keystone virus, while MAb M3 exhibited no reactivity with other CAL serogroup viruses. Only MAb M3 possessed neutralization and hemagglutination inhibition activities against JC virus. The MAbs were also tested by ELISA and for neutralizing activity against 13 JC topotypes isolated in 5 provinces from Newfoundland to Saskatchewan. ELISA confirmed closer identity of the Canadian topotypes to JC as opposed to the closely related South River virus. The MAbs verified all Canadian topotypes to be JC virus but revealed different patterns of reactivity between these topotypes and prototype JC virus.     

Monoclonal Antibodies to Conformational Epitopes of the Surface Glycoprotein of Caprine Arthritis-Encephalitis Virus: Potential Application to Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detecting Antibodies in Goat Sera

Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79–63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.     

In situ telomerase activity in pleural effusions: a promising marker for malignancy

Telomerase activity in 16 pleural effusions was studied using an in situ telomerase repeat amplification protocol (TRAP) assay on cytospin preparations. Six of nine cytologically malignant specimens contained telomerase-positive cells (67%), and in two further specimens, suspicious positive cells were seen. Two of four atypical specimens contained telomerase-positive cells, whereas two benign cases were telomerase-negative. No mesothelial cells showed telomerase reactivity. Thus, telomerase activity was specific for malignancy and it was always found only in malignant cells. The results suggest that telomerase activity measured with this in situ method can be a valuable complement in the assessment of malignancy in pleural effusions.     

Molecular Specificities of CD4+ T Cell-Reactive IgM in Human Immunodeficiency Virus (HIV-1) Infection

The molecular targets and biological properties of lymphocytotoxic sera in 11 human immunodeficiency virus (HIV)⁺ subjects in CDC stages II to IVC were investigated. A purified soluble CEM membrane used as the CD4⁺ T cell clonotypic model in immunoblotting techniques revealed a homogeneous pattern of IgM-mediated reactivity to a 43.5-kDa membrane component in 10 sera. Conversely, CEM membrane-reactive IgG from these sera were weakly reactive to various antigens with no prevalent specificity. ELISA assay against purified 25.5-, 43.5-, and 60.8-kDa CEM membrane antigens revealed a significant affinity of IgM from sera 3, 8, 9, and 10 for the 43.5-kDa receptor, thus confirming the high specificity of these cytotoxic antibodies for their substrate. Additional experiments included the idiotypic saturation of purified IgM molecules with increasing amounts of the 43.5-kDa antigen. Functional inhibition of CEM-reactive IgM by their antigen was dose-dependent. The maximum inhibition of the ELISA reactivity was detected at 1/7 antigen/antibody concentration. By contrast, a significant decline of cytotoxic properties of sera 8, 9, and 10 to CEM lymphoblasts was noted only when equivalent concentrations of the 43.5-kDa receptor and purified IgM were incubated. In addition, the 43.5-kDa antigen appeared to be the preferential target of cytotoxic IgM in HIV-1 infection, since purified cytotoxic IgM from two SLE patients were not inhibited by this molecule. Our data provide specific molecular support for earlier evidence of exacerbation by T cell-reactive antibodies of the lymphopenia associated with HIV-1 infection.