Speaking personally: monoclonal antibodies as diagnostic reagents

It is 10 years since Kohler & Milstein published the first paper describing the laboratory production of a monoclonal antibody. In those 10 yr monoclonal antibodies have caused a revolution in Immunology and in some branches of Pathology. After a surprisingly slow initial reaction, the scientific community has embraced the new technique enthusiastically, indeed overenthusiastically. Inevitably, excessive enthusiasm was followed by disappointment. There can be no doubt that monoclonal antibodies provide opportunities for major improvements in diagnostic specificity and resolving power. It is equally clear that monoclonal antibodies do not solve all analytical problems, and have a few problems of their very own. This brief review presents one man's assessment of the place of monoclonal antibodies as diagnostic reagents and discusses the properties of the antibodies and the parameters of the assay which most influence the successful use of diagnostic tests based on monoclonal antibodies.     

Direct detection of Neisseria gonorrhoeae with monoclonal antibodies characterized by serotyping reagents.

A panel of monoclonal antibodies (MAbs) directed to outer membrane protein I were generated with the ultimate aim of detecting Neisseria gonorrhoeae in patient samples by a direct immunofluorescence (IF) test. In an initial evaluation of the sensitivity of these reagents, a cocktail of six IF MAbs recognized 491 (91%) of 540 gonococci isolates from several centers in Sydney, Australia. IF MAbs designated 185 and 228 recognized serovars of WI serogroup and IF MAbs 208, 210, and 312 recognized serovars of WII/III serogroup. IF MAb 198 recognized serovars within both serogroups. Three additional IF MAbs, designated 322, 323, and 330, were then generated by using strains which failed to react with the original MAb cocktail and which belonged to particular serovars. The new cocktail of nine IF MAbs recognized 96% of the gonococcal isolates, which incidentally contained representatives of serovars shown to have a worldwide distribution in previous studies. Although subtle differences were apparent in the reaction patterns found with coagglutination (serotyping) and IF, there nonetheless seems to be merit in the approach of continually evaluating the sensitivity of diagnostic reagents such as MAbs. This is especially true with an organism such as N. gonorrhoeae, which has the capacity to regularly alter the antigenic structure of its outer membrane proteins.     

IgA类单克隆抗体血型定型试剂研制和应用

目的 研制IgA类单克隆抗体血型定型试剂.方法 根据新生物制品注册和《中国生物制品规程》等有关要求,筛选合适细胞株,建立生产工艺,对中试产品进行检定和临床试验.结果 研究、选择了2株细胞株,建立了经济、实用的生产工艺,中试和正式生产的产品所有指标均达到或超过国家标准,通过国家法定检定,临床应用1亿人份以上,没出现血型错判或误判.结论 研制成功国际唯一的IgA类单克隆抗体血型定型试剂.     

IgA类单克隆抗体血型定型试剂研制和应用

目的研制IgA类单克隆抗体血型定型试剂。方法根据新生物制品注册和《中国生物制品规程》等有关要求,筛选合适细胞株,建立生产工艺,对中试产品进行检定和临床试验。结果研究、选择了2株细胞株,建立了经济、实用的生产工艺,中试和正式生产的产品所有指标均达到或超过国家标准,通过国家法定检定,临床应用1亿人份以上,没出现血型错判或误判。结论研制成功国际唯一的IgA类单克隆抗体血型定型试剂。     

Production of Vi monoclonal antibodies and their application as diagnostic reagents

Serum antibodies to Vi antigen were detected in mice immunized with the purified antigen but not with Vi-bearing Salmonella typhi whole cells. Fusion of the spleen cells from one of the Vi antibody-producing mice with NSI myeloma cells produced four stable hybridomas that secreted antibodies to Vi. Monoclonal antibodies from these four clones were all of the immunoglobulin G class and, as determined by competition, appeared to have the same epitope specificity. Despite their immunoglobulin G nature, mouse ascitic fluids induced by one of the hybridomas strongly agglutinated the Vi-positive strains of S. typhi, S. dublin, and Citrobacter strain 5396/38. Thus, 10 clinical isolates of S. typhi but not 98 strains of other bacteria were reactive in slide agglutination tests with the monoclonal antibodies.     

Hyaluronectin: detection with monoclonal antibodies in human tumors

Two monoclonal IgG1 antibodies (MAbs) were raised against human brain hyaluronectin (HN) and used to characterize tumor HN. They were screened using an enzyme immunological technique (ELISA) combined with the HN property of specific binding to hyaluronic acid. They were shown to detect two different epitopes (HN1 and HN2) in human normal brain as well as in most tumors. Both HN1 and HN2 epitopes were found associated with mesenchymal benign or neoplastic proliferations (e.g. connective areas of fibroadenomas, extracellular matrix of fibrosarcomas) and with reactive connective tissue (e.g. stroma reaction of carcinomas, ground substance of gliomas). The results corresponded with those previously obtained with polyclonal rabbit antibodies and confirmed that HN is a constant marker of desmoplasia. Thus anti-HN MAbs recognize an antigen that is associated with tumor development and will be suitable for targeting.     

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.     

Bovine rotavirus type detection by neutralizing monoclonal antibodies

Summary A series of monoclonal antibodies were developed against bovine rotavirus Q₁₇. Among the five high affinity antibodies characterized, two (RQ 31 and RQ 4) were able to neutralize type G 6 viruses and may be specific for PB 1 type virus. Seventy seven feces from diarrheic calves were tested by double sandwich ELISA using four monoclonal and one polyclonal anti-rotavirus antibodies. The combination of mono- and polyclonal antibodies thus appears to be a more efficient strategy for detection and typing of bovine rotaviruses.     

Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-beta-adrenergic ligand antibody

A new method has been developed to raise monoclonal anti-idiotypic antibodies. Monoclonal anti-idiotypic antibodies were obtained by fusion of NS-1 myeloma cells with splenocytes of mice immunised by intravenous injections of fixed hybridoma cells bearing a monoclonal antibody specific for beta-adrenergic ligands. New screening tests were developed to analyse the resulting hybridoma supernatants for different anti-idiotypic properties. Among 23 hybridoma supernatants recognising the idiotype, 6 were found to inhibit hapten binding and 3 of these recognised beta-adrenergic receptors.     

Double immunostaining of lymph node sections by monoclonal antibodies using phycoerythrin labeling and haptenated reagents

Double immunostaining of tissue sections by monoclonal antibodies has been performed using, in immunofluorescence, combinations of reagents conjugated with different fluorochromes. These were monoclonal antibodies directly labeled with phycoerythrin (a new red fluorochrome) and haptenated monoclonal antibodies detected by fluorescein-conjugated second or third layers. All the reagents used were commercially available and the double immunostaining was easy to perform and allowed the design of several combinations useful to characterize T-cell subsets in reactive lymph nodes. The results indicate that the simultaneous detection of pairs of antigens by monoclonal antibodies can be applied successfully to tissue sections in order to investigate cellular heterogeneity in normal and pathologic conditions.     

Characterization of murine monoclonal antibodies against serogroup B salmonellae and application as serotyping reagents.

Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other O serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory.     

A and B blood group-specific monoclonal antibodies. Production and evaluation as ABO-blood typing reagents

The Monoclonal Antibody (MoAb) technology has been successfully applied to develop reagents for human blood group classification. There is no production of this kind of reagents in Venezuela, and the local demand (blood banks and clinical laboratories) is mainly supplied with imported material. Considering this we decided to apply MoAb techniques to generate murine hybridomas secreting anti-A or anti-B specific MoAb. MoAb obtained were characterized and produced in enough quantity to perform validation studies as blood typing reagents. Out of 22 hybridomas that were initially selected, 11 were anti-A secretors and 11 were anti-B secretors. Four MoAb were further characterized: Au18Kt3F, MG3 (both IgM anti-A), SS4.5 (IgG1 anti-B) and BB2-3 (IgM anti-B). Conditions were also established for growing the hybridomas Au18Kt3F and BB2-3 in the bioreactors "miniPerm" and "Tecnomouse", allowing for scale-up production of these MoAb. Avidity and specificity were estimated for each one, and the results were comparable to those obtained from commercially available reagents, making feasible its use as blood typing reagents.     

Prenatal fragile X detection using cytoplasmic and nuclear-specific monoclonal antibodies

We have been carrying out studies aimed at improving prenatal detection of the fragile X chromosome/mutation. Our current protocol requires a turnaround time (TAT) of several days. In an attempt to reduce the TAT, we have turned to the use of monoclonal antibodies (mAbs). Monoclonal antibody 1A1 (provided by Dr. Mandel of INSERM) immunostaining was performed according to a modified three-step immunocytochemical procedure. We found that cytoplasmic staining intensities, using mAb 1A1/avidin biotinylated complex/diaminobenzidine, varied from light to heavy within each sample, with controls exhibiting a majority of heavily stained cells in both chorionic villus (CV) sample and amniotic fluid cultured cells. Using mAb 1A1 and a new nuclear-specific antibody, mAb 3F11, we found that CV cultured cells harboring the FMR1 full mutation could be distinguished from controls as early as 10 weeks of gestation in both male and female specimens. Western blot analysis showed that the antibodies have similar staining patterns but that mAb 3F11 has fewer background/nonspecific bands. Our results demonstrate that it is feasible to detect fragile X full mutations within one day after obtaining cells from CV specimens taken as early as 10 weeks of gestation.     

Flow cytometric analysis of lymphocyte phenotypes in AIDS using monoclonal antibodies and simultaneous dual immunofluorescence

Simultaneous dual immunofluorescence and flow cytometry was used to study sixteen lymphocyte phenotypes in 209 men including: healthy homosexuals, lymphadenopathy patients (LAN), and AIDS patients. Significant differences between the distribution of lymphocytes in healthy homosexuals and healthy heterosexuals were decreased percentages of helper/inducer T cells (Leu 3), increased cytotoxic/suppressor T cells (Leu 2), and consequently a decreased Leu 3/Leu 2 ratio. The increased Leu 2 cells were identified as functionally cytotoxic subset Leu 2+ 15- phenotype rather than suppressor cells which are Leu 15+. Leu 2 and Leu 3 bearing cells exhibited an excess of membrane-bound immunoglobulins which were easily elutable at 37 degrees C. An increased percentage of an HLA-DR framework determinant bearing T cells were also detected. Within the NK cell family, Leu 7 cells were moderately increased and the functionally unidentified Leu 2+ 7+ population was strikingly elevated. LAN or AIDS patients were compared to healthy homosexual controls. Lower percentages of Leu 3 cells and higher percentages of Leu 2 cells were evident in LAN patients. These subsets were similar in LAN and AIDS patients. The increase in Leu 2+ cells was due to the Leu 2+ 15- cytotoxic subset. Fewer T cells had immunoglobulin in LAN and AIDS. A definite increase in Leu 2+ DR+ cells but not Leu 3+ DR+ cells occurred in AIDS compared to LAN or healthy controls. NK cell changes already present in healthy homosexuals persisted in LAN and AIDS patients. No differences in the distribution of B cells was detected in any intergroup comparisons. Changes in monocytes or pan-T cells were relatively insensitive measures of immunologic alterations among any of the groups. These results indicate many of the changes in lymphocyte subsets seen in AIDS and LAN subjects are already present in a carefully screened population of healthy homosexuals in San Francisco. Many of the changes in Leu 2 and NK family of cells suggest a possible adaptive response to viral or neoplastic challenge. Whether these interesting phenotypic alterations relate to functional changes in response to such challenge of the identified subsets waits further investigation.     

Preparation of monoclonal antibodies for detection and identification of Francisella tularensis

Monoclonal antibodies (MAbs) against Francisella tularensis were obtained. Three MAbs specifically reacted with F. tularensis, while four MAbs reacted with other members of the genus Francisella as well. Fluorescent isothiocyanate-conjugated MAbs unequivocally stained bacterial cells in specimens from experimentally infected mice. Two MAbs agglutinated F. tularensis antigen in the agglutination tests. These MAbs should improve methods for detection and identification of F. tularensis.     

Development and application of monoclonal antibodies for detection and analysis of aquareoviruses

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.     

A sensitive radioimmunoassay for the detection of monoclonal anti-idiotope antibodies

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems: the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies.     

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.