Biomedical subjects

R L Raison

Publications and source records attributed to R L Raison.

At least 37 records · Page 2Linked to original sources

PubMed1992

Hagfish humoral defense protein exhibits structural and functional homology with mammalian complement components.

A genomic clone and cDNA fragment encoding a portion of a humoral recognition molecule from the hagfish were isolated and sequenced. The serum protein has previously been described as having structural features that are immunoglobulin-like. Amino acid sequence obtained from the 77-kDa H1 heavy chain facilitated the isolation of a genomic clone containing at least two coding regions. Through use of primers derived from the genomic sequences, a 231-base-pair cDNA fragment was obtained by PCR from liver RNA. Comparison of the deduced 120-amino acid sequence from the N terminus of H1 with known protein sequences revealed substantial sequence similarity with the beta chain of the murine fourth complement component C4 and with the related third and fifth complement molecules C5 and C3 and the major histocompatibility complex-encoded sex-limited protein. Observation of structural and functional similarities associated with the sequence similarity indicate that these molecules share an evolutionary relationship: the polypeptide chain structure of hagfish complement-like protein (CLP) resembles that of C4; CLP contains a hidden thioester group on the 70-kDa chain; CLP binds to streptococcal cells and enhances the phagocytosis of yeast by hagfish leukocytes. These data suggest that CLP forms part of a non-clonally-derived complement-related humoral defense system in the hagfish.

Amino Acid Sequence↗

PubMed1991

Low affinity binding of mouse immunoglobulin to human CD5+ B cells.

Polyreactive immunoglobulin (Ig) secreted by CD5-bearing B cells has the capacity to bind a broad range of self and foreign antigens. Flow cytometric analysis was used to detect low-affinity binding of mouse Ig molecules to the surface of CD5-bearing B cells from patients with chronic lymphocytic leukaemia (CLL). Mouse Ig isotypes G, A, and M, and IgG subclasses G1, G2a, and G3 bound to the B cell surface via a mechanism not involving the antigen-binding site of the mouse Ig molecule. Fab and F(ab')2 fragments of mouse Ig associated with the CLL cells in a similar manner to intact Ig, indicating that Fc receptor interactions were not involved. Dissociation of the mouse Ig from the B cell surface by three washing steps distinguished this lower-affinity binding from high-affinity binding which occurs through the antigen-binding site of a mouse monoclonal antibody (MoAb) when it recognizes a specific cell surface epitope. Blocking studies suggest that the low-affinity binding occurred via surface IgM and surface IgD (sIgM/sIgD) on the CD5-bearing B cells. The results are consistent with the expression of polyreactive Ig on the surface of CD5-bearing B cells in CLL.

Animals↗

PubMed1991

Monoclonal antibody against sheep kappa light chain.

The production of a monoclonal antibody specific for sheep kappa light chain protein is described. The monoclonal antibody was designated McM11 and its specificity was verified using western blots of sheep IgG and slides of efferent lymph cells. The specificity of McM11 was confirmed by specific recognition of fusion proteins expressed by recombinant phage containing sheep kappa cDNAs. N terminal sequence of the light chain recognized by McM11 showed homology to kappa type light chains. McM11, together with McM6, a lambda specific monoclonal antibody, was shown by two color FACS analysis of sheep blood lymphocytes to recognize all sheep light chains.

Amino Acid Sequence↗

PubMed1990

Distinct Ig H chains in a primitive vertebrate, Eptatretus stouti.

Serum Ig from the Pacific hagfish, Eptatretus stouti, was isolated by affinity chromatography using a specific mAb (H.45). Analysis of the approximately 210-kDa molecule by SDS-PAGE under reducing conditions revealed two H chains of approximately 77 kDa (H1) and approximately 70 kDa (H2) and L chains of approximately 30 kDa. H1 and H2 were shown to differ with respect to their peptide maps, amino-terminal amino acid sequences, and reactivity to the mAb H.45, suggesting that they represent discrete H chain isotypes. Two-dimensional nonreducing/reducing SDS-PAGE demonstrated that H and L chains were covalently linked predominantly as H-H-L and H-L configurations. Noncovalently bound L chains were also found. H-H-L complexes were shown to contain H1-H2 heterodimers of H chains in addition to H1-H1 homodimers.

Amino Acid Sequence↗

PubMed1990

Single-step purification of immunoglobulin M on C1q-Sepharose.

A rapid and simple affinity chromatography method for purifying IgM from myeloma serum and ascites fluid is described. Complement protein C1q is coupled to Sepharose with an efficiency of 35%, giving 1.7 mg of C1q bound/ml of gel. This C1q-Sepharose selectively binds IgM from crude samples at 5 degrees C, with a capacity of 0.4 mg of IgM/ml of gel. The bound IgM may be eluted simply and isocratically by bringing the gel to room temperature for 2 h, or by washing with buffer containing 0.5 M KI. The eluted IgM is highly pure by SDS-PAGE and double immunodiffusion analysis, although IgG may be a potential contaminant. The C1q-Sepharose is stable for at least 18 months.

Chromatography, Affinity↗

PubMed1988

A new microsphere-based immunofluorescence assay for antibodies to membrane-associated antigens.

The screening of panels of hybridoma supernatants for specific secreted monoclonal antibodies is often achieved by cellular immunofluorescent staining and flow cytometric analysis. In some circumstances such assays are difficult because the required antigen-bearing cell population is not suitable for use in flow cytometry, has limited cell cycle expression or poor in vitro growth. A method is presented here that provides a solution to these difficulties. A system was developed, using polyacrylamide microspheres coupled with cell membranes, which permitted the production of an easily stored, standardised antigen source which could be used in subsequent flow cytometric assays. Studies comparing the binding of antibodies to whole cells and cell membrane-coupled microspheres indicate a strong qualitative and quantitative correlation in the expression of surface antigens. It is shown here that membrane antigens can be stored coupled to microspheres for months and still retain good reactivity with the appropriate antibodies. This same technique could be used in studies of antigens other than those of the mammalian cell membrane - for example, membrane antigens of sub-cellular organelles such as the mitochondrion and endoplasmic reticulum, plant or bacterial membrane antigens, or antigens not associated with membranes such as viral proteins.

Antibodies, Monoclonal↗

PubMed1988

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells.

A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a cross-reactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.

Animals↗

PubMed1988

Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins.

A new protein crosslinking agent, 2,3-dibromopropionyl-N-hydroxysuccinimide ester, has been synthesized and characterized. The potential use of this compound as a temperature-controllable heterobifunctional crosslinking agent has been investigated using model systems and its reactivity compared with that of chlorambucil-N-hydroxysuccinimide ester. The coupling of 14C-labeled phenylethylamine to lysozyme has been used to illustrate the feasibility of the use of this crosslinking agent for the synthesis of immunotoxins.

Amino Acids↗

PubMed1988

Radioimmunolocalization and selective delivery of radiation in a rat model system: comparison of intact and fragmented antibody.

Monoclonal antibody (MoAb) fragments are known to have advantages over intact immunoglobulins for radioimmunoscintigraphy. It is less clear whether they are as effective in the delivery of radioimmunotherapy. The imaging and dosimetric properties of an intact MoAb, K-1-21, reactive against human kappa light chains (LC) were compared with that of its F(ab')2 and Fab fragments using a normal rat model system. Two days after injection of 131I-K-1-21 into rats bearing antigen-sepharose implants, gamma camera images showed specific localization of the MoAb to the target (kappa LC) but not to the control (lambda LC) implant. Better images were obtained with K-1-21 F(ab')2 than with Fab or intact antibody. Mean kappa implant: blood ratios were 8.6 +/- 3.9 for Fab, 7.9 +/- 1.8 for F(ab')2 and 2.0 +/- 0.3 for intact K-1-21. The improvement associated with the use of 131I-K-1-21 fragments was, however, achieved at the expense of lower absolute values of activity at the target site. Thus the absorbed dose delivered to the implant by the intact K-1-21 was double that delivered with F(ab')2 and six times that delivered with Fab. As intact K-1-21 also delivered a greater radiation dose to normal tissues, F(ab')2 fragments may have the greatest overall advantages for therapy with radionuclide MoAb conjugates.

Animals↗

PubMed1987

Quantitation of free kappa light chains in serum and urine using a monoclonal antibody based inhibition enzyme-linked immunoassay.

The murine monoclonal antibody K-1-21 with specificity for free but not heavy chain-associated kappa chains was used to develop an inhibition enzyme immunoassay for quantitation of free kappa chains in serum and urine. The assay utilizes a biotin conjugate of the monoclonal antibody and has an effective working concentration range of 0.5-50 micrograms/ml free kappa chain. Normal serum and urine yielded mean values of 1.2 and 4.9 micrograms/ml respectively using this assay system. Serum levels in patients with kappa chain myelomatosis ranged from 2 to 1700 micrograms/ml while serum from patients with lambda chain malignancies exhibited normal free kappa chain levels. The importance of the ability to readily quantitate light chains in serum was demonstrated by the finding in one patient of an absence of free kappa chains in the urine despite elevated levels of serum kappa chains. A significant drop in serum-free kappa chain levels was observed in this patient following a course of chemotherapy. The assay should prove valuable in the diagnosis, prognosis and monitoring of B cell neoplasias characterized by free kappa light chain secretion.

Antibodies, Monoclonal↗

PubMed1987

Localization of an idiotope on the L chain dimer and intact IgG1 immunoglobulin from the patient Mcg.

A monoclonal anti-idiotype (M3.9) raised against the covalently linked Mcg lambda chain dimer binds with a similar affinity to the Mcg IgG immunoglobulin and covalent heterodimers of Mcg with other human L chains. Despite having identical amino acid sequences, the two light chains in the Mcg dimer adopt different conformations with monomer 1 acting as a heavy chain analog and monomer 2 behaving like a light chain component of an Fab. As the lambda chain in the Mcg IgG and at least one hybrid L chain dimer (Mcg X Weir) assumes a conformation similar to that of monomer 2 and the binding of anti-idiotype requires only the presence of a single Mcg lambda chain, we conclude that the idiotope is restricted to the monomer 2 type of the Mcg lambda chain conformational isomer. Cooperative binding of two molecules of rhodamine 123 in the main cavity of the Mcg dimer block the binding of the anti-idiotype whereas the binding of one molecule of bis(DNP)lysine has no significant effect on the idiotype-anti-idiotype system. Previous crystallographic analyses indicated that bound rhodamine 123 protrudes outside the rim while bis(DNP)lysine is completely immersed in the cavity. At high concns bis(DNP)lysine penetrates through the floor of the main cavity and forms a virtually irreversible complex with the dimer. Production of this complex is accompanied by conformational changes, which are presumed to be correlated with observed inhibition of binding with the anti-idiotype M3.9. Expression of the idiotope probably involves more than one linear sequence since reduction and alkylation of the intra- and inter-chain disulphide bonds in 8 M urea leads to a complete loss of binding of the anti-idiotype. The inhibition data suggest involvement of residues on or near the rim of the main cavity. Distribution of potential contact residues for rhodamine 123 is asymmetric only in the case of aspartic acid 97, which is located on the cavity rim in only one conformational isomer (monomer 2). The homologous residue in monomer 1 is directed away from the cavity and is unlikely to participate in the epitope recognized by M3.9. Attempts to define the epitope in more detail by simulation with multiple peptides have been initiated in collaboration with the laboratory of H. M. Geysen.

Amyloidosis↗

PubMed1987

Cellular requirements for mixed leucocyte reactivity in the cyclostome, Eptatretus stoutii.

Unfractionated peripheral blood leucocytes from allogeneic hagfish proliferated vigorously in one-way mixed leucocyte reaction (MLR) at 20-22 degrees. Maximum responsiveness was observed after 5 days of co-culture of responder and irradiated stimulator cells. Separation of leucocytes on the basis of laser scatter parameters yielded a small leucocyte population which responded but did not stimulate in MLR and a large leucocyte population capable of stimulating but not responding. Adherent cell depletion of the total leucocyte population did not affect the ability to respond in MLR but did ablate the stimulatory capacity of hagfish leucocytes. Together with previous phenotypic studies, these results confirm the presence of lymphocyte and macrophage/accessory cell populations in hagfish peripheral leucocytes.

Animals↗

PubMed1986

Evolutionary development of lymphocyte heterogeneity: leucocyte subpopulations in the Pacific hagfish.

Flow cytometric analysis of forward angle versus 90 degree scatter patterns of hagfish peripheral blood revealed two distinct leucocyte populations with size characteristics analogous to mammalian monocytes/granulocytes (hagfish large leucocytes) and small lymphocytes (hagfish small leucocytes). A cell population enhanced for the small leucocytes was obtained by density gradient centrifugation. Over 70% of the small leucocyte population consistently stained with a rabbit antiserum directed against polypeptide determinants on hagfish immunoglobulin, while staining of the large cell population was greatly reduced (less than 10%). A panel of monoclonal antibodies raised against a crude hagfish leucocyte preparation distinguished the two cell populations and revealed the existence of subpopulations of both small and large leucocytes.

Animals↗

PubMed1985

A monoclonal antibody with selectivity for human kappa myeloma and lymphoma cells which has potential as a therapeutic agent.

K-1-21 is a monoclonal antibody which binds to human free kappa light chains and recognises a determinant, KMA selectively expressed on kappa myeloma and lymphoma cells. KMA is absent on plasma cells and resting B cells from normal adults but can be detected on some foetal B cells and a small proportion of activated B cells. Expression of KMA is greatest on cycling cells. K-1-21 is an IgG1 antibody that elicits ADCC but will not cap the KMA determinant unless a second ligand is present. K-1-21 has potential for practical application.

Antibodies, Anti-Idiotypic↗

PubMed1985

Conformation dependence of a monoclonal antibody defined epitope on free human kappa chains.

Chemically and enzymatically modified kappa chains were tested by inhibition radioimmunoassay for their ability to block the binding of antibody K-1-21 with native kappa chains. Complete reduction and carboxymethylation of intrachain disulphide bonds destroyed the free kappa-chain epitope, a result confirmed by Western blotting of unreduced and reduced kappa monomers and dimers. Purified V kappa fragments failed to block the homologous interaction while inhibition was obtained with a pepsin digest yielding predominantly the C kappa region. Dimeric kappa chains were less effective than monomers in the inhibition assay, although HPLC analysis of immune complexes demonstrated the binding of two antibody molecules per molecule of dimer. Thus, the epitope on free kappa chains recognized by K-1-21 is dependent upon conformational integrity of the C kappa domain, the decreased binding activity of dimeric chains possibly being due to minor conformational changes induced by C-domain interactions.

Animals↗

PubMed1985

Purification of two allergens from Dermatophagoides pteronyssinus using monoclonal antibodies.

Fractionation of soluble extracts from the house dust mite Dermatophagoides pteronyssinus (DP) by SDS-PAGE and crossed immunoelectrophoresis (CIE) revealed at least fifty distinct protein components. Western blotting and crossed radioimmunoelectrophoresis (CRIE) indicated that fewer than one quarter of these components were allergens as determined by their ability to bind IgE from allergic individuals. Following immunization with a crude extract, two monoclonal antibodies were raised against distinct components which exhibited IgE binding capacities in Western blot and CRIE. Affinity chromatography using these monoclonal antibodies yielded components which elicited positive skin test reactions in patients allergic to DP.

Allergens↗

PubMed1985

Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies.

Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenic bands. These two bands, of Mr 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast, the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

Animals↗

PubMed1985

Co-expression of an epitope on human free kappa-light chains and on a cytoplasmic component in activated T cells.

K-1-21 is a murine monoclonal antibody that reacts with human kappa-light chains in free form but not when they are associated with immunoglobulin heavy chains. K-1-21 was unexpectedly shown to bind to a determinant, STA (Sezary T cell antigen), detected by immunofluorescence in the cytoplasm but not on the surface of Sezary T cells isolated from peripheral blood (4/4 cases) and in Sezary T cells from lymph node and bone marrow (one patient). STA was detected in F2/F7, CCRF-CEM, Molt-4, and CCRF-HSB (four human T ALL cell lines), in JURKAT (a human T cell leukemia line), and in MLA144 (a Gibbon T cell lymphoma line). It also occurred in Leu-3a+ antigen-specific T cell clones (6/6 tested). Moreover, although STA was absent from freshly isolated normal T cells, its expression could be evoked in E+ cells from peripheral blood by in vitro culture with phytohemagglutinin. Thus, STA appears to be a cytoplasmic marker for activated T cells. Cytoplasmic inhibition immunofluorescence studies indicated that K-1-21 binding to STA in Sezary cells or T cell lines was inhibited by preincubation of the K-1-21 antibody with purified kappa-Bence Jones protein. STA from radiolabeled MLA144 cell lysates was immunoprecipitated by K-1-21 and was identified on polyacrylamide gel electrophoresis under reducing conditions as a protein of m.w. 57,000. Additional experiments are underway to define the molecular basis of the interesting cross-reactivity between a determinant in T cells and the K-1-21 reactive epitope on free kappa-light chains.

Antigen-Antibody Reactions↗