Pool Of Monoclonal Antibodies Research Articles

Use of a pool of monoclonal antibodies in diagnosing cells from serous cavities.

The usefulness of monoclonal antibodies that recognize markers of neoplastic lesions in complementing conventional cytology was evaluated by the avidin-biotin-peroxidase complex, indirect immunoperoxidase technique. In order to enhance the sensitivity of the traditional method, a pool of seven combined monoclonal antibodies (Pool C7), which reacts specifically with cells of epithelial origin and is able to distinguish between mesothelial and malignant cells, was tested on cytologic smears of 262 serous effusions. The effusions were benign or neoplastic, mainly from breast, ovary and lung cancers. Immunocytochemical method showed an 100% specificity and 100% of predictivity whereas the sensitivity was 98%, 96% and 95% for breast ovarian and lung carcinomas, respectively. The results demonstrated that the pool when used together with conventional methods, is useful in analysis of serous effusions in diagnostic investigations.

Epizootiological aspects of type 1 and type 4 equine herpesvirus infections among horse populations.

The dissemination of equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) among various horse populations in Japan was investigated through the isolation and typing of virus strains from horses with respiratory diseases. Type specific monoclonal antibody pools were used for the typing of isolates. The 42 strains of EHV-1 and 64 strains of EHV-4 were isolated from 4593 nasal swabs and/or blood plasma samples collected from 3326 horses during a period from 1979 to 1990. All the strains of EHV-1 were isolated from racehorses only and during the winter season exclusively, when the epizootic of respiratory diseases occurred among racehorse populations at two Training Centers of the Japan Racing Association. In contrast, the strains of EHV-4 were isolated from horses irrespective of the season, facility, or horse population; foals and yearlings in breeding farms and our institute, rearing horses in rearing farms, and racehorses. Especially, 4 strains of EHV-4 were isolated from plasma samples containing buffy coat cells. We believe this is the first reported case of EHV-4 cell-associated viremia in the world. All 87 strains isolated from aborted fetuses were identified as EHV-1. The results suggest that EHV-1 is responsible for epizootic respiratory diseases in racehorses in the winter and abortion among mares at the late stage of gestation, and that EHV-4 causes respiratory diseases throughout the year among all horse populations.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus

Monoclonal antibodies to the hemagglutinin protein, fusion protein, phosphoprotein, matrix protein, and nucleoprotein of measles virus were evaluated as detector antibodies in capture enzyme immunoassays (EIAs) for the detection of specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to measles virus. A pool of monoclonal antibodies to hemagglutinin protein and nucleoprotein proved optimal and was further evaluated. Specific IgM was detected in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of clinical measles cases and vaccinees, in 26% of healthy persons, and in 36% of infants 8 months postvaccination; consequently, IgA antibodies were not a useful indicator of recent measles infection. A significant increase in IgG antibodies between paired specimens was detected in 92% of clinical cases and all vaccinees. Only 59% of infant specimens had persistent IgG antibodies as detected by capture EIA at 8 months postvaccination, whereas all specimens had antibodies as detected by hemagglutination inhibition and plaque neutralization. An alternative indirect EIA, in which antigen was directly absorbed to the solid phase, was more sensitive than the capture design, detecting IgG antibodies in all infants postvaccination. When standardized with a microneutralization assay for the detection of persistent antibodies, the indirect IgG EIA gave predictive values for positive and negative tests exceeding 90%. Our capture IgM and indirect IgG EIAs provide a practical combination of serologic tests for the determination of acute measles virus infection and past exposure to measles virus or vaccine, respectively.

Comparison of polyclonal antisera and anti-43kDa antigen monoclonal antibodies for the culture-amplified antigenic detection of Mycoplasma pneumoniae by immunoblotting

Mycoplasma pneumoniae is a common etiologic agent of community-acquired respiratory disease whose rapid laboratory diagnosis is complicated. We developed a culture-amplified immunological antigen detection assay which employs a monoclonal antibody for the recognition of a polypeptide antigen of 43kDa in an immunoblotting technique. Polyclonal antisera to the same antigen were obtained from rabbit and hamster immunization with affinity chromatography-purified antigen. The latter sera, sera from natural human infection and experimental hamster infection, and a pool of anti-43kDa monoclonal antibodies were compared to the single anti-43kDa monoclonal antibody, OC2F5. Among these, only polyclonal hamster anti-43kDa sera were able to enhance antigen detection quantitatively. This enhancement however did not result in a significant benefit to the culture-amplified system.

In Vitro Effects of Hematopoietic Growth Factors on the Proliferation, Endoreplication, and Maturation of Human Megakaryocytes

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.

In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

Detection of border disease virus in sheep efferent lymphocytes by immunocytochemical and in situ hybridisation techniques.

The prefemoral efferent lymphatics of four sheep persistently infected with a non cytopathic (NCP) isolate of border disease virus (BDV) were cannulated. Recovered lymphocytes were examined for the presence of virus by an immunocytochemical technique employing a pool of monoclonal antibodies which recognise the 120K non-structural polypeptide of NCP BDV. The results revealed that 9.5% of the lymphocytes carried virus antigen. Lymphocytes from two of the sheep were studied by in situ hybridisation using a viral antisense RNA probe complementary to the region of the BDV genome coding for the 120K polypeptide. This showed that 70-80% of the cells were infected, confirming the greater sensitivity of the in situ hybridisation technique.

Secretory Component‐Binding Properties of Normal Serum IgM

Our aim was to investigate why serum IgM is poorly transferred into secretions in normal subjects. Indeed, the low IgM level in secretions contrasts with the capacity of monoclonal IgM to bind to secretory component (SC), but it is not well established to what extent normal serum IgM can do so. The mean SC affinity was studied with a polyclonal IgM preparation from 250 normal subjects and with a representative pool of 100 different monoclonal IgM. The SC-binding percentages varied as a function of the IgM/SC molar ratio according to a common hyperbolic curve, with similar association constants: Ka = 4.19 +/- 2.61 x 10(7) M-1 (polyclonal pool) and Ka = 5.80 +/- 2.73 x 10(7) (monoclonal pool). It thus appears that the large difference in IgM concentrations between blood and secretions cannot be due to an SC-binding defect of serum IgM, but is probably explained by its low diffusion from blood to the extravascular compartment.

Synthesis of complement component C5 by human B and T lymphoblastoid cell lines

Human B and T lymphoblastoid cell lines were shown to synthesize C5. C5 synthesis was quantitated with an enzyme-linked immunosorbent assay (ELISA) that utilized a pool of C5-specific monoclonal antibodies (mAbs). Some level of C5 synthesis was detected in all eight of the B and T cell lines examined. In three of the cell lines, C5 was detected in both culture supernatants and whole cell detergent lysates, whereas in the other five cell lines, C5 was detected only in the cell lysates. Lymphoblastoid cells with both distributions of C5 were shown to synthesize a messenger RNA that was similar in size to the C5 mRNA expressed by the HepG2 hepatoma cell line. Estimates of the concentration of the C5 transcript in poly(A)+ RNA from lymphoblastoid and HepG2 cells suggested that C5 mRNA levels in the lymphoblastoid cell lines were comparable and about one-tenth of the levels in HepG2 cells. Lymphoblastoid C5, isolated by immunoaffinity chromatography from the supernatants of 35S-labeled cultures, had the same subunit composition as plasma-derived C5, but had an alpha subunit of slightly smaller relative mass.

Detection ofNeisseria Gonorrhoeaeby Dot-Enzyme Immunoassay Using Monoclonal Antibodies

A highly sensitive and specific dot-enzyme immunoassay for the detection of Neisseria gonorrhoeae was developed using a pool of monoclonal antibodies (MAbs). The MAbs were obtained following immunization of mice with lithium acetate extracted outer membrane (OM) preparations. Western immunoblotting experiments demonstrated that MAbs NG26 and NG38, both IgG2a, reacted with lipopolysaccharides (LPS) and with the major OM protein, P1, respectively, MAb NG28, an IgG3, did not react in Western immunoblotting, MAbs NG28 and NG38 failed to react with OM treated with proteolytic enzymes or with semi-purified preparation of LPS. MAb NG26 reacted with the same LPS preparation. Binding radioimmunoassay with live bacteria showed that all the MAbs adsorbed to cell surface-exposed antigenic determinants. The limit of detection of the dot-enzyme immunoassay was between 1 and 4 x 10(4) cfu per dot. Using a panel of 177 strains of N. gonorrhoeae, MAbs NG28 and NG38 recognized only P1A and P1B strains respectively. MAb NG26 reacted with P1A, P1B and non-typable strains. These MAbs did not react with other Neisseria species or other bacterial species. Using this pool, the dot-enzyme immunoassay had a sensitivity of 93.2% and a specificity of 100%.

Detection of influenza virus by centrifugal inoculation of MDCK cells and staining with monoclonal antibodies.

Two methods for detection of influenza virus in 451 clinical respiratory specimens were compared: (i) 24-well-plate centrifugation with Madin-Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza viruses A and B (Centers for Disease Control, Atlanta, Ga.) in an indirect immunofluorescence assay after incubation for 40 h, and (ii) conventional tissue cell culture with primary monkey cells and hemadsorption. For 100 of these specimens, direct examination of smears by the direct fluorescence assay with monoclonal antibodies (Boots Cell Tech/API Analytab Products, Plainview, N.Y.) was also performed. Influenza A virus was recovered from 28 specimens by tissue cell culture after incubation for an average of 4.75 days (range, 2 to 14 days). Influenza B virus was recovered from 35 specimens by tissue culture after incubation for an average of 5.4 days (range, 3 to 14 days). By the centrifugation assay, 23 specimens were positive for influenza A virus and 30 were positive for influenza B virus. All specimens positive by the centrifugation assay were also positive by conventional tissue cell culture. The sensitivities of the centrifugation assay were 82% for detection of influenza A virus and 86% for influenza B virus (84% overall); the specificity of the assay was 100%. Of the 100 specimens studied by direct examination, 15 were positive for influenza virus by both conventional culture and centrifugation assay; however, the direct-smear results for these 15 specimens were negative in 13 cases and inconclusive in 2. The centrifugation assay is a rapid and specific method for detection of influenza A and B viruses in clinical specimens, and it can serve as a valuable and cost-efficient adjunct to conventional culture methods.

Detection of Murine Typhus Infected Fleas with an Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.

Pneumocystis Antigen Appearance during Immunosuppression

ABSTRACTThe sequential appearance of Pneumocystis carinii (Pc) antigens during the progression of immunosuppression in rats was studied using the immunoblotting technique and specific immunologic probes. Putative Pc soluble antigens, with molecular weights of approximately 70 and 90 kd, were detected as early as 2 wk after initiation of immunosuppression in rats using a pool of monoclonal antibodies produced to Pc isolated from lungs using enzymatic digestion. Monoclonal antibodies produced to Pc isolated by massaging the lung tissue using a Stomacher apparatus and infecuon‐derived sera did not detect soluble antigens until at least the 6lh wk of immunosuppression. Analysis of Pc pellets obtained from Stomacher‐ and lavage‐processed lungs revealed that the lower molecular weight antigens ( 40, 45 and possibly 55–60 kd) were recognized early during the immunosuppression process.

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.

Evaluation of a monoclonal antibody pool for rapid diagnosis of respiratory viral infections

A pool of monoclonal antibodies (MAbP) was evaluated both as a method of cell culture confirmation and as a rapid diagnostic screen for viral infection in respiratory secretions. The MAbP was used in a two-step fluorescent staining procedure on cells harvested from cultures (phase 1) and on exfoliated nasopharyngeal or tracheal cells (phase 2). Antibodies in the MAbP were directed against respiratory syncytial virus, adenoviruses, parainfluenza virus types 1, 2, and 3, and influenza viruses A and B. Individual antiviral antibody stains were used to identify specific viruses from MAbP-positive specimens. In phase 1 (cell culture confirmation only), 241 respiratory specimens were tested. MAbP sensitivity and specificity were 91 and 94%, respectively. In phase 2, 376 respiratory specimens were evaluated by direct staining of exfoliated cells, and these results were compared with results of cell culture isolation. The sensitivity and specificity of the MAbP used in direct specimen testing were 69 and 97%, respectively. These results indicate that the MAbP is highly specific and fairly sensitive for detection of seven different respiratory viruses in one testing procedure. The MAbP is a rapid screening technique for respiratory secretions and is potentially a cost-effective approach to viral detection.

Homologous regions shared by adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium

A λgt11 library of Mycoplasma genitalium genomic DNA was generated, and clones were identified using a pool of monoclonal antibodies directed against different epitopes of the 140 kDa adhesin protein. Because the 140 kDa protein of M. genitalium and the 170 kDa P1 adhesin of M. pneumoniae share biological properties such as a tip-associated location, cytadherence function and immunologic crossreactivity, we performed Southern blot analysis using these cloned partial 140 kDa gene fragments and 14 subclones that span the P1 structural gene of M. pneumoniae. Homologous regions of the two genes were identified.

Pneumocystis antigen appearance during immunosuppression.

The sequential appearance of Pneumocystis carinii (Pc) antigens during the progression of immunosuppression in rats was studied using the immunoblotting technique and specific immunologic probes. Putative Pc soluble antigens, with molecular weights of approximately 70 and 90 kd, were detected as early as 2 wk after initiation of immunosuppression in rats using a pool of monoclonal antibodies produced to Pc isolated from lungs using enzymatic digestion. Monoclonal antibodies produced to Pc isolated by massaging the lung tissue using a Stomacher apparatus and infection-derived sera did not detect soluble antigens until at least the 6th wk of immunosuppression. Analysis of Pc pellets obtained from Stomacher- and lavage-processed lungs revealed that the lower molecular weight antigens (less than or equal to 40, 45 and possibly 55-60 kd) were recognized early during the immunosuppression process.

Utility of monoclonal antibody coagglutination to identify Neisseria gonorrhoeae.

The Phadebact monoclonal gonococcus coagglutination test was evaluated with 1367 (996 anogenital and 371 pharyngeal) neisserial isolates from patients who gave positive diagnostic test results for Neisseria gonorrhoeae. The overall correlation with carbohydrate utilisation was 99.7%. The Phadebact test had a specificity of 100% (286/286) and a sensitivity of 99.7% (1077/1080). The three non-reactive strains were epidemiologically linked and were of a very unusual serovar. Unlike polyclonal antibody based tests, the monoclonal antibody test provides reliable identification of gonococci from anogenital and pharyngeal sites. Because non-reactive strains are rare, however, negative anogenital isolates from heterosexual patients should be tested biochemically. The use of two reagents comprising separate pools of monoclonal antibodies against gonococcal protein IA and IB permitted the identification of an appreciable number of double infections, which would otherwise have been missed. Genital, rectal, and pharyngeal isolates from the same patient should be identified individually.