Monoclonal Reagents Research Articles

Failure of a diagnostic monoclonal immunofluorescent reagent to detect Legionella pneumophila in environmental samples

Three commercial diagnostic fluorescein-labeled antibodies, one monoclonal and two polyclonal, were compared to evaluate their abilities to detect Legionella pneumophila in environmental samples. The monoclonal conjugate failed to detect L. pneumophila in the 12 environmental samples studied by direct immunofluorescence. In contrast, the two polyclonal conjugates detected L. pneumophila in all 12 samples by both direct and indirect immunofluorescence. However, isolates recovered by culture from the 12 samples demonstrated equal immunofluorescence with all three conjugates. The reason for the failure of the monoclonal antibody to detect L. pneumophila in the environmental samples remains unknown. Laboratories considering the use of the monoclonal conjugate to screen environmental samples for L. pneumophila should be aware of this finding.

Culture confirmation and typing of herpes simplex virus isolates from clinical specimens with new monoclonal antibody typing reagents

FITC-labelled monoclonal antibody typing reagents for herpes simplex virus (HSV) (PathoDx, Diagnostic Products Corp., Los Angeles, California) were compared to another commercially available FITC-labelled monoclonal antibody typing reagent for culture confirmation and typing of HSV isolates from clinical specimens. A total of 344 clinical specimens were evaluated for culture confirmation and typing by the two systems. There were 296 HSV-positive cultures identified by cytopathic effect (CPE). The PathoDx typing reagents identified 143 isolates as HSV-1, and 151 isolates as HSV-2, with two isolates staining with both the type 1 and type 2 reagent. The PathoDx typing reagents had a 100% concordance with the other set of typing reagents.

Monoclonal antibodies to carcino‐embryonic antigen

Several approaches were made to produce new monoclonal antibodies (MoAb) to human colonic tumours using different immunogens, such as colon cell lines, and tissue and serum from patients with colon cancer. Six MoAb were produced but all were found to be reactive with carcino-embryonic antigen (CEA). Through tissue reactivity and competitive inhibition studies it was found that four epitopes could be detected: CEA-1 detected by MoAb I-1 and O-1; CEA-2 detected by MoAb 17C4; CEA-3 detected by MoAb LK-4 and XPX-13 and CEA-4 detected by MoAb JGT-13. CEA-2 was also found on granulocytes and probably detects normal cross-reacting antigen (NCA). In formalin-fixed tissues the CEA-specific epitopes were absent from normal adult tissues, but this was an artefact of the fixation procedure and fresh, non-fixed colonic tissues were clearly reactive. All six MoAb reacted with greater than 70% of colonic tumours when tested by the immunoperoxidase technique and gave different patterns of reactivity reflecting the differential expression of the CEA 1-4 epitopes. Despite the apparent specificity of these MoAb for colon cancer, serum testing using MoAb gave similar results to CEA polyclonal antibodies, that is the MoAb gave no obvious advantage. Thus efforts to produce new reagents to colon cancer yielded only anti-CEA monoclonal reagents. Clearly, other new approaches are required to produce better diagnostic reagents for this common disease.

HOSAICISM OF BLOOD GROUP SPECIFICITY OF BRUSH BORDER OF HUMAN ENTEROCYTES

The pattern of differentiation of the enterocyte along the villus is considered to be dependent on the age of the cell and uniform in cells occupying comparable location on the villus. Using a polyclonal antibody against A blood group specific components of enterocyte brush border and three monoclonal antibodies we have demonstrated the expression of the A and B antigens in the brush border and Golgi apparatus of enterocytes of individuals of the expected blood groups, by light and electron microscopy, with the immunogold technique. The polyclonal anti-A serum was specific for the brush border whereas the monoclonal reagents also bound the endothelial cells and erytrocytes in the sections. Four of 16 blood group A individuals showed a mosaic pattern of expression of the A antigen, both with the polyclonal serum and the monoclonal anti-A reagent. Some enterocytes showed a normal staining and some other enterocytes at similar position on the villus showed no staining at all. An anti-Le a antibody showed that all the 4 individuals with the mosaic pattern were non secretor. Abnormalities of blood group antigens may reflect abnormal expression of glucosyl-transferase genes or disturbances of their acceptors. Enterocytes may therefore display a non homogeneous pattern of differentiation in morphologically similar cells occupying comparable location on the villus.

1 Monoclonal antibodies as blood grouping reagents

The large volume requirements for high quality ABO and Rh(D) typing reagents can now be supplied by selected monoclonal antibodies. Superior anti-A and anti-B monoclonal reagents can be prepared, from blends of at least two antibodies, to optimize the intensity of agglutination for slide tests and the potency for the detection of the weaker sub-groups, including Ax and Bw, by tube techniques. New quality control steps have been described for some highly sensitive anti-A/anti-B antibodies to avoid the detection of traces of A on B cells or traces of B on A1 cells, which results from the non-specific activity of A and B transferases. Excellent anti-A,B reagents may also be made by blends of at least two antibodies to optimize both A and B reactions, but the need for their continued use is now debatable. The development of high titre IgM monoclonal anti-D reagents offers simple rapid saline Rh(D) typing of both patients and donors, but they cannot reliably detect weak D (Du) and some D variants, e.g. the epitopes on D category VI cells. However, this can be achieved by blending an IgM anti-D with IgG (polyclonal) anti-D which can detect these types after conversion of negative saline tests to an antiglobulin phase. In addition, high grade Du, D categories and variants can be reliably detected (for typing donors) by selected monoclonal IgM and IgG anti-Ds by use of suitably enhanced tests without the use of an antiglobulin test.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal Antibody in the Identification of Haemophilus Somnus

Electrophoretic comparisons of outer membrane proteins of Haemophilus somnus isolates revealed 2 major protein bands (46 and 14 kilodaltons [kD]) common to all isolates tested. A monoclonal antibody raised against H. somnus reacted to the 46-kD band. Coagglutination tests were performed using a monoclonal antibody coagglutination assay. The monoclonal reagent was produced by incubating Cowan strain Staphylococcus aureus suspension, used as a source of crude protein A, with mouse ascitic fluid monoclonal antibody or goat anti-H. somnus hyperimmune serum. Bacteria to be tested were suspended at a concentration of 4.5 x 10(9) cells/ml. The coagglutination test was performed by the addition of 50 microliters of the monoclonal reagent to 50 microliters of the bacterial suspension on a glass plate and manual rotation for 2-3 minutes. The coagglutination assay using Cowan strain Staphylococcus aureus protein A, coupled with the monoclonal antibody, agglutinated 10 different H. somnus isolates. The antibody reagent did not coagglutinate with Actinobacillus suis, A. equuli, Pasteurella haemolytica, P. multocida, or P. pneumotropica under similar test conditions.

Identification of Monoclonal Antibodies Capable of Differentiating Antigenic Varieties of Eastern Equine Encephalitis Viruses

We have isolated and characterized 3 monoclonal antibody (Mab) reagents useful in the serological identification of varieties of eastern equine encephalitis (EEE) viruses. These antibodies were specific for the E1 glycoprotein of their homologous viruses. One Mab, 1B5C-3, reacted specifically with all North American (NA) EEE viruses isolated over a 50 year period. This antigenic stability of NA isolates was genetically confirmed by oligonucleotide fingerprinting. Evolutionary stability is a unique feature among alphaviruses. The Mab, 1C1J-4 reacted specifically with 1 South American isolate of EEE virus. A third Mab, 1B1C-4, was EEE virus complex reactive. While none of these antibodies had virus neutralizing activity, the identified reactivities could be demonstrated in the more rapid serological tests of enzyme-linked immunosorbent assay and indirect immunofluorescence.

Two Ig framework I epitopic specificities recognized by a rabbit antiserum and a monoclonal antibody to mouse VH chains.

The reactivity of 23 mouse monoclonal Ig with a rabbit polyclonal antiserum to VH of anti-alpha(1----6)dextran 19.22.1 and with a monoclonal anti-VH of anti-DNP MOPC315, when correlated with amino acid sequence, identified several residues in the first and third framework regions as being of potential importance in forming the epitope. Inhibition studies using synthetic peptides corresponding to residues 1-15 of the monoclonal Ig used to produce the poly- and monoclonal reagents provide evidence that the epitopes are predominantly, if not exclusively, specific for the N-terminal strand of the domain. Examination of known x-ray structures of mouse VH suggests that the primary difference between the two epitopes in the N-terminal strands is determined by the peptide chain structure due to Pro at position 9. Pro 9 appears essential for the epitope reactive with anti-VH MOPC315.

Monoclonal antibodies to Chlamydia psittaci Guinea pig inclusion conjunctivitis (GPIC) strain

Monoclonal antibodies to a strain of Chlamydia psittaci isolated from guinea pig inclusion conjunctivitis (GPIC) were developed. Only five of the 15 hybridomas isolated produced antibodies specific for the GPIC strain, while seven others produced antibodies which cross reacted with other strains and another species. Strain-specific and species-specific monoclonal antibodies were isotyped as IgG 2a and IgG 3 respectively. It appears that the GPIC strain has at least two epitopes, one of which is specific for the strain and the other common to the species. These monoclonal reagents may be used to immunotype GPIC agents, better than available methods and may be of potential use in the development of vaccines against chlamydial infections.

Gonococcal infection within Scotland: Antigenic heterogeneity and antibiotic susceptibility of infecting strains

Two panels of monoclonal antibody reagents were used to serotype all strains of Neisseria gonorrhoeae isolated from four separate geographical areas serving two million of the five million Scottish population. Serotype 1B isolates accounted for 60% of the 869 strains examined and were more prevalent than 1A isolates in each geographical area. A total of 11 1A serovars and 47 1B serovars were recognised. Only two of the 11 1A serovars (Aedgkih/Arost and Aedih/Arst) were found in every centre but these accounted for over 90% of the 1A isolates. Although there was a total of 47 different 1B serovars over 80% of the isolates were accounted for by the ten most commonly encountered serovars. There were, however, marked geographical differences within both major and minor serovars. There was a highly significant difference (P less than 0.001) between protein 1A and 1B serovars with respect to their susceptibility to penicillin. Within each protein 1 type there were also differences in antibiotic susceptibility. Penicillinase-producing N. gonorrhoeae (PPNG) were found in all centres and accounted for 24 (2.8%) of the 869 isolates. The majority of the PPNG (71%) were serotype 1A and with one exception were serovar Aedih/Arst. PPNG strains accounted for 37% (16) of the 43 Aedih/Arst isolates. Epidemiological, diagnostic and therapeutic implications arising from the distinct geographical differences in the pool of circulating gonococci are discussed.

Percutaneous umbilical blood sampling and umbilical vein transfusions

Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.

Group A variants defined with a monoclonal anti‐A reagent

Unusual serologic findings mimicking the B(A) phenomenon were noted in two group B blood donors using blended murine monoclonal anti-A reagent. With additional studies, including a binding experiment using affinity chromatography, the presence of aberrant group A activities was confirmed. This observation suggests that persons with atypical B(A) phenotype may warrant further investigation to delineate the ABO blood group.

Serological characterization of polycyclic aromatic hydrocarbon diolepoxide -DNA adducts using monoclonal antibodies

Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide-DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH-DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH-DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus demonstrating their specificity for PAH-DNA adducts and not the PAHs alone. Monoclonal antibodies produced against BADE-DNA also bound to chrysene diolepoxide-DNA but not to BPDE-DNA or to two other PAH-DNA adducts. These data demonstrate that significant serological cross-reactivity exists between different PAH-DNA adducts and support our contention that human antibodies to PAH-DNA can often bind to more than a single type of PAH-DNA adduct. However, our ability to produce monoclonal antibodies with restricted specificity suggest that the production of highly monoclonal antibody reagents for use in assays designed to measure PAH-DNA adducts in biological samples is feasible.

Monoclonal antibodies directed against human Rh antigens in tests with the red cells of nonhuman primates

Monoclonal antibodies against Rh related antigens on human red cells often crossreact with the red cells of the highest subhuman primate species. Depending on specificity of antibody, the species tested, and technique used, these reactions can be either species-specific or type specific. In tests with chimpanzee red cells, some of the latter type reactions have specificities related to the R antigen of the R-C-E-F blood group system of chimpanzee; specificities of some others seem to be unrelated to any known chimpanzee blood groups. Monoclonal anti-D reagents that give uniformly positive reactions with human D-positive (common and rare types) red cells, display wide individual differences in tests with chimpanzee blood. This indicates that there are minute structural variations of antibody molecules from one monoclonal anti-D antibodies apparently have no bearing on recognition of the D combining site on the human red cells, but come into play when in contact with chimpanzee rbcs. Some of the monoclonal antibodies directed against Rh and LW molecules are distinguished by unusually strong reactions with the red cells of the Old World monkeys (macaques and baboons), which is in contrast with negative or weak reactions of the same antibodies with the red cells of anthropoid apes and human bloods. One may recall, that polyclonal anti-Rh sera do not react with the blood of rhesus monkeys, the phenomenon that was the source of controversy surrounding the discovery of the rhesus factor of the human blood.

Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens from marrow transplant patients: evaluation of a direct fluorescein-conjugated monoclonal antibody reagent

An FITC-conjugated monoclonal antibody reagent containing three CMV-specific monoclonal antibodies was evaluated for the rapid detection of CMV in bronchoalveolar lavage (BAL) cytospin preparations by direct IF (DFA). Eighty-six BAL samples from 72 marrow transplant patients were inoculated into both centrifugation and standard cell culture. CMV was detected in 49/86 (57%) BAL samples. DFA detected 37/46 (80%) samples which were positive in centrifugation culture. While DFA staining lacked the sensitivity (overall sensitivity 38/49, 78%) to replace either standard or centrifugation culture, the total laboratory time needed to complete the DFA was only 1.5 h and its concurrent use with centrifugation culture can provide rapid specific diagnosis of CMV pneumonia.

Flow cytometric assays for monitoring production of recombinant HIV-1 gp160 in insect cells infected with a baculovirus expression vector

A baculovirus expression system for the HIV-1 envelope glycoprotein gp160 has been used as a model for development of flow cytometric assays for monitoring production of cell-associated recombinant antigen. Using monoclonal antibodies to the transmembrane (gp41) or envelope (gp120) portion of gp160, gp!20, but not gp41, could be reproducibly detected on the surface of insect cells 48 h after infection with the recombinant baculovirus. In contrast, fixation and permeabilisation of infected cells prior to staining, to allow access of monoclonal reagents to the intracellular compartment, markedly improved the sensitivity of detection, with reactivity to both monoclonal antibodies observed at 24 h post-infection. Specificity of the intracellular immunofluorescence was verified by demonstrating that the appropriate native or recombinant HIV-1 protein blocked reactivity of monoclonal antibody with infected cells. In addition, it was observed that production of gp160 following baculovirus infection was associated with a marked increase in the 90° light scatter of insect cells, as determined by flow cytometry, and that this correlated with the kinetics of cell-associated gp160 production as determined by immunofluorescence. These procedures should be of great utility for routine monitoring of recombinant proteins produced in insect cells in response to infection with recombinant baculovirus.

Identification of a Subset of Group B Donors Reactive with Monoclonal Anti-A Reagent

Two red blood cell (RBC) units labeled group B were returned to the source blood center after they had been retyped as AB by a transfusion service. The discrepancy could be reproduced, but only with the use of the transfusion service's reagent, Ortho Diagnostic's anti-A Bioclone (a licensed, blended, murine monoclonal anti-A reagent). RBCs from 35 of 3,458 random group B donors (1%) reacted with the monoclonal anti-A after immediate centrifugation. Reactivity was associated with high serum levels of B-gene-specified transferase and was caused by the MH04 component, a potent anti-A capable of detecting some examples of Ax RBCs. It is probable that the potency of MH04 permitted detection of low levels of A determinants synthesized by the donors' unusually strong B-gene-specified transferase. Transfer of N-acetylgalactosamine by B-gene-specified transferases, reported in vitro, has not been detected previously in vivo. Use of highly sensitive monoclonal reagents may result in clinically ambiguous blood grouping results.

Phenotypic and functional differences in NK and LAK cells in the peripheral blood of sooty mangabeys and rhesus macaques

Greater than 75% of the sooty mangabey monkeys at the Yerkes Regional Primate Research Center are naturally infected with SIV without any apparent clinical symptomology. On the other hand, experimental infection of rhesus macaques with SIV results in a clinical syndrome similar to human AIDS. These differences with regard to SIV infection prompted us to examine the natural immunosurveillance system of peripheral blood mononuclear cells (PBMC) from SIV-infected and uninfected monkeys of these two species. Phenotypic and functional studies of precursor and effector NK and LAK cells in the PBMC from these two species were carried out using monoclonal reagents, flow microfluorometry (FMF), and the standard in vitro 51Cr release assay against prototype K562 (NK sensitive) and RAJI (NK resistant, LAK susceptible) target cell lines. Data indicate that both NK and LAK cell activities in the PBMC of sooty mangabeys were significantly (P less than 0.01) greater than those in rhesus macaques. The predominant NK effector cells and LAK cell precursors were shown to be Leu 19-CD8+ in the PBMC of sooty mangabeys and Leu19+ CD8- in the PBMC of rhesus macaques as determined by panning depletion techniques and FMF analysis. On the other hand, the predominant LAK effector cells were found to be dual marked Leu 19+ CD8+ in rhesus macaques and Leu 19- CD8+ in sooty mangabeys. These qualitative and quantitative differences were not due to SIV infection of these two species since PBMC from both SIV-seropositive and virus-positive and SIV-sero-negative and virus-negative monkeys gave similar results. Moreover, of importance is the finding that the functional NK and LAK precursor cells are CD8+ and CD8- in sooty mangabeys and rhesus macaques, respectively. These data may have implications for the natural SIV/SMM virus-positive asymptomatic state of sooty mangabeys and may provide useful tools for tracing the ontogeny and lineage derivation of NK and LAK cells.

Isotype analysis of the anti–CENP‐B anticentromere autoantibody: Evidence for restricted clonality

Utilizing the centromere B fusion protein (CENP-B) and specific, matched monoclonal antiisotype reagents in an enzyme-linked immunosorbent assay, we found that anti-CENP-B autoantibodies were skewed to the IgG1 isotype. The overall kappa:lambda light chain ratio was 2:1, although several individual sera showed a strong predominance of one of the light chains. Isoelectric focusing of light chain-skewed sera showed polyclonal patterns. Our findings are consistent with the anti-CENP-B autoantibody response being a chronic, antigen-driven response.

Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and O polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica O:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A- and M-antigen structures, an interpretation supported by competitive binding studies with O polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous alpha 1,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by alpha 1,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element alpha-D-Rha4NFo(1----3)alpha-D-Rha4NFo bracketed by alpha 1,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated alpha 1,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.