mAb Clone Research Articles

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts

The angiogenic inducers cysteine-rich angiogenic protein 61 (Cyr61) and connective tissue growth factor (CTGF) are structurally related, extracellular matrix-associated heparin-binding proteins. Both can stimulate chemotaxis and promote proliferation in endothelial cells and fibroblasts in culture and induce neovascularization in vivo. Encoded by inducible immediate early genes, Cyr61 and CTGF are synthesized upon growth factor stimulation in cultured fibroblasts and during cutaneous wound healing in dermal fibroblasts. Recently, we have shown that adhesion of primary human fibroblasts to immobilized Cyr61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans (HSPGs) (Chen, N., Chen, C.-C., and Lau, L.F. (2000) J. Biol. Chem. 275, 24953-24961), providing the first demonstration of an absolute requirement for HSPGs in integrin-mediated cell attachment. We show in this study that CTGF also mediates fibroblast adhesion through the same mechanism and demonstrate that fibroblasts adhesion to immobilized Cyr61 or CTGF induces distinct adhesive signaling responses consistent with their biological activities. Compared with fibroblast adhesion to fibronectin, laminin, or type I collagen, cell adhesion to Cyr61 or CTGF induces 1) more extensive and prolonged formation of filopodia and lamellipodia, concomitant with formation of integrin alpha(6)beta(1)-containing focal complexes localized at leading edges of pseudopods; 2) activation of intracellular signaling molecules including focal adhesion kinase, paxillin, and Rac with similar rapid kinetics; 3) sustained activation of p42/p44 MAPKs lasting for at least 9 h; and 4) prolonged gene expression changes including up-regulation of MMP-1 (collagenase-1) and MMP-3 (stromelysin-1) mRNAs and proteins sustained for at least 24 h. Together, these results establish Cyr61 and CTGF as bona fide adhesive substrates with specific signaling capabilities, provide a molecular basis for their activities in fibroblasts through integrin alpha(6)beta(1) and HSPG-mediated signaling during attachment and indicate that these proteins may function in matrix remodeling through the activation of metalloproteinases during angiogenesis and wound healing.

Determination of Myloid Antigen Expression on Childhood Acute Lymphoblastic Leukaemia Cells: Discrepancies Using Different Monoclonal Antibody Clones

Prospective clinical studies including large numbers of patients have led to the conclusion that co-expression of myeloid antigens in childhood acute lymphoblastic leukaemia (My+ ALL) does not have prognostic significance. However, reports of the frequency of My+ ALL in children vary widely across laboratories using different mAb clones and staining and analysing procedures. Taking two commonly accepted thresholds of positivity for myeloid antigens (20 and 30%), we analysed the immunoreactivity of the most widely employed mAb clones against CD13 (SJ1D1, L138 and My7) and CD33 (My9, P67.6 and D3HL60) and compared the proportions of My+ ALL detected by these clones in childhood ALL. The correlation between myeloid antigen expression and the presence of the t(12;21) translocation was analysed concomitantly in the same samples.The percentage of ALL cases positive for myeloid markers varied significantly depending on the mAb clone and the positive threshold. Among patients with B-ALL, the proportion of CD13+ ALL was significantly lower using SJ1D1 than using L138 or My7, while the proportion of CD33+ ALL was significantly higher for My9 than for P67.6 or D3HL60. Analysis of the co-expression of CD13 and CD33 on B-ALL cells using combinations of mAb clones showed that this frequency was either underestimated by the SJ1D1/D3HL60 or overestimated by the L138/P67.6 and My7/My9 combinations. A correlation between CD13/CD33 positivity and the t(12;21) translocation was uniformly observed in B-ALL patients for a positive threshold of 30%, whereas SJ1D1/D3HL60 detected no correlation between t(12;21) and CD13/CD33 ppositivity when the threshold was lowered to 20%. These data show that the mAb clones commonly used to detect the CD13 and CD33 surface antigens have variable immunoreactivity against childhood ALL cells, which may partly explain the conflicting reports concerning the prognostic significance of myeloid antigen expression in paediatric ALL and its association with different translocations. The present findings may also be of clinical importance for therapeutic choices.

Determination of Myeloid Antigen Expression on Childhood Acute Lymphoblastic Leukaemia Cells: Discrepancies Using Different Monoclonal Antibody Clones

Prospective clinical studies including large numbers of patients have led to the conclusion that co-expression of myeloid antigens in childhood acute lymphoblastic leukaemia (My+ALL) does not have prognostic significance. However, reports of the frequency of My+ ALL in children vary widely across laboratories using different mAb clones and staining and analysing procedures. Taking two commonly accepted thresholds of positivity for myeloid antigens (20 and 30%), we analysed the immunoreactivity of the most widely employed mAb clones against CD13 (SJ1D1, L138 and My7) and CD33 (My9, P67.6 and D3HL60) and compared the proportions of My+ ALL detected by these clones in childhood ALL. The correlation between myeloid antigen expression and the presence of the t(12;21) translocation was analysed concomitantly in the same samples.The percentage of ALL cases positive for myeloid markers varied significantly depending on the mAb clone and the positive threshold. Among patients with B-ALL, the proportion of CD13+ ALL was significantly lower using SJ1D1 than using L138 or My7, while the proportion of CD33+ ALL was significantly higher for My9 than for P67.6 or D3HL60. Analysis of the co-expression of CD13 and CD33 on B-ALL cells using combinations of mAb clones showed that this frequency was either underestimated by the SJ1D1/D3HL60 or overestimated by the L138/P67.6 and My7/My9 combinations. A correlation between CD13/CD33 positivity and the t(12;21) translocation was uniformly observed in B-ALL patients for a positive threshold of 30%, whereas SJ1D1/D3HL60 detected no correlation between t(12;21) and CD13/CD33 positivity when the threshold was lowered to 20%. These data show that the mAb clones commonly used to detect the CD13 and CD33 surface antigens have variable immunoreactivity against childhood ALL cells, which may partly explain the conflicting reports concerning the prognostic significance of myeloid antigen expression in paediatric ALL and its association with different translocations. The present findings may also be of clinical importance for therapeutic choices.

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a: Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation–permeabilization kits (Cytofix/Cytoperm™, Fix and Perm™, Intraprep™, Intrastain™, Permeacyte™ and Permeafix™) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood ( n=4), normal bone marrow ( n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL ( n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm™ kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm™ kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm™, Intraprep™, Intrastain™ or Permeafix™, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.

Use of peptide ligands to analyze the fine specificity of antibodies against asialo GM1

We recently described clone 10, a monoclonal Fab fragment that binds to asialo GM1 (GA1), and three mutated Abs derived from it that also bind GA1 and have a three to four times increase in avidity. We selected a phage display linear heptapeptide library with these four Abs, and an IgM mAb, 156, which binds to GM1 and GD1b, but not to GA1. Peptides with the same motif, KL/VWQXXX, were selected by clones 10 and the two heavy chain mutants 227 and 109. In contrast, the light chain mutant L3 58 selected an entirely different peptide motif, TFGLQSL. Moreover, a different motif, K/S WTNL/M PP, was selected by mAb 156. Although mAbs clone 10 and its mutants 109, 227 and L3 58 all bind only to GA1, differences in their fine specificity were revealed by binding to peptide ligands.

Redox regulation of β2-integrin CD11b / CD18 activation

Although the central role of β2-integrin CD11b / CD18 in neutrophil functions is well recognized, signaling pathway that regulate integrin activation remain to be elucidated. We analyzed the contribution of oxido-reduction mechanisms in this signaling. Exogenously added H2O2 induced CD11b / CD18-dependent neutrophil adhesion and expression of an integrin activation neoepitope recognized by monoclonal antibody (mAb) clone 24. H2O2-triggered β2-integrin activation was inhibited by tyrosine kinase inhibitors and by complexing sulfhydryl groups with phenylarsine oxide (PAO). CD11b / CD18-dependent adhesion and mAb 24 antigen expression triggered by physiological agonists such as TNF-α were inhibited by diphenylene iodonium (DPI, an inhibitor of flavoprotein oxidoreductase), by free radical scavengers, by tyrosine kinase inhibitors and by PAO. No inhibition was observed when adhesion was induced by the integrin-activating KIM 185 mAb. Taken together, these results emphasize the importance of an oxidative S-thiolation step(s) in the tyrosine kinase-dependent signaling pathway leading to β2-integrin activation. H2O2 would directly mediate this oxidative reaction and bypass the initial agonist / receptor pathway to promote integrin-dependent adhesion. The putative oxidase(s) involved in this process is not NADPH oxidase, since adhesion of neutrophils from patients with chronic granulomatous disease was normal and inhibited by scavengers and DPI. These data shed a new light on the regulation of integrin activation required for cell migration into inflamed organs.

Redox regulation of beta2-integrin CD11b/CD18 activation.

Although the central role of beta2-integrin CD11b / CD18 in neutrophil functions is well recognized, signaling pathway that regulate integrin activation remain to be elucidated. We analyzed the contribution of oxido-reduction mechanisms in this signaling. Exogenously added H(2)O(2) induced CD11b/CD18-dependent neutrophil adhesion and expression of an integrin activation neoepitope recognized by monoclonal antibody (mAb) clone 24. H(2)O(2)-triggered beta2-integrin activation was inhibited by tyrosine kinase inhibitors and by complexing sulfhydryl groups with phenylarsine oxide (PAO). CD11b/CD18-dependent adhesion and mAb 24 antigen expression triggered by physiological agonists such as TNF-alpha were inhibited by diphenylene iodonium (DPI, an inhibitor of flavoprotein oxidoreductase), by free radical scavengers, by tyrosine kinase inhibitors and by PAO. No inhibition was observed when adhesion was induced by the integrin-activating KIM 185 mAb. Taken together, these results emphasize the importance of an oxidative S-thiolation step(s) in the tyrosine kinase-dependent signaling pathway leading to beta2-integrin activation. H(2)O(2) would directly mediate this oxidative reaction and bypass the initial agonist/receptor pathway to promote integrin-dependent adhesion. The putative oxidase(s) involved in this process is not NADPH oxidase, since adhesion of neutrophils from patients with chronic granulomatous disease was normal and inhibited by scavengers and DPI. These data shed a new light on the regulation of integrin activation required for cell migration into inflamed organs.

Production and characterisation of a monoclonal antibody specific for apoptotic bodies derived from several tumour cell lines

We have recently shown that apoptotic bodies (apobodies) derived from rat colon carcinoma cell lines (PROb) after sodium butyrate (NaB) treatment were able to cure rats with induced peritoneal carcinomatosis ( Boisteau et al., 1997). A specific immune response was assumed to be involved since the serum of cured rats contained antibodies specific for apobodies. In the present study, a mAb (clone 6E8) produced by immunisation of rats with apobodies strongly recognized apobodies but had little reactivity with parental tumour cell lines, as demonstrated by enzyme-linked immunosorbent assay (ELISA), immunostaining and flow cytometry. Immunoelectron microscopy showed that 6E8 mAb mainly stained the hyaloplasm or cytosol of apobodies. A protein was detected at 67 kDa by immunoprecipitation of apobodies with mAb, followed by immunoblotting, using serum of rats immunised with apobodies. The 6E8 mAb recognized apobodies derived from several rat or human colon cancer cell lines and a rat glioma cell line, regardless of the apoptosis stimulus used (NaB, staurosporine or UV). Our results clearly show that 6E8 mAb defines an epitope specifically generated during apoptosis, which suggests that the protein recognized may be involved in the molecular cascade of apoptotic cell death.

Natural polyreactive immunoglobulin A antibodies produced in mouse Peyer's patches.

To understand the biological function of natural immunoglobulin A (IgA) antibodies in Peyer's patches (PP), we generated IgA monoclonal antibody (mAb) clones from the PP of normal, unimmunized, specific pathogen-free BALB/c mice and examined their reactivities by enzyme-linked immunosorbent assay (ELISA). Many of these antibodies reacted with more than one antigen examined, suggesting that they were polyreactive Abs. Two mAbs agglutinated several different strains of commensal bacteria isolated from mice. To examine the genetic features of these polyreactive mAbs, the VH genes of seven different IgA mAbs were sequenced. The VH genes from the VGAM, J558 and 7183 families were compared with sequence from the mAbs with distinct VDJ rearrangements. One of the mAbs that agglutinated bacteria was encoded by a germline VH gene, but the VH region of the other polyreactive mAbs contained between seven and 11 mutated sites. No indication of antigenic selection was observed in the pattern of these mutated sites. Our results show that polyreactive IgA Abs are present in PP as a part of the normal B-cell repertoire. These polyreactive Abs may establish a natural immune homeostasis, and function as a polyreactive sensor to detect pathogenic invasion and to control immune response in the gut.

Development of a monoclonal antibody to 6β-hydroxycortisol and its application in an enzyme-linked immunosorbent assay (ELISA) for 6β-hydroxycortisol in urine

A novel monoclonal antibody to 6β-hydroxycortisol (6β-OHC) was generated and incorporated into an antigen-coated indirect enzyme-linked immunosorbent assay (ELISA) using 6β-OHC-protein conjugate as the steroid-coating antigen. The monoclonal antibody is specific to 6β-OHC and 6β-OHC-3-carboxymethyloxime. Cross-reactivity with other structurally related steroids such as cortisol, cortisone, and 6β-hydroxycortisone was less than 10%. Two different clones (clone 5C1 and 19F) of the monoclonal anti-6β-OHC antibody have been developed, each with slightly different sensitivity and specificity. The sensitivity of the MAb clones was not significantly improved when compared to the rabbit polyclonal antibodies in this study, but still within the accepted detection limit for 6β-OHC in both human and laboratory animals. The assay had a detection limit of 200 ng/ml, an intraassay variation of 6.4% and an interassay variation of 7.3%. The application of the anti-6β-OHC-MAb-based-ELISA was tested by measuring the urinary output of 6β-OHC in human before and after enzyme induction by rifampicin treatment. The mean 24-h urine output of 6β-OHC in human subjects was 485 ± 100 μg and 1478 ± 281 μg before and after rifampicin administration, respectively. In conclusion, the monoclonal anti-6β-OHC antibody developed in this study has the required specificity and sensitivity as an alternative method for measuring urinary 6β-OHC in the detection of enzyme induction or enzyme inhibition of CYP3A in humans and laboratory animals.

Characterization of C3-binding proteins on mouse neutrophils and platelets.

In the mouse, MCR1 and MCR2 on B lymphocytes are encoded by alternatively spliced Cr2 gene transcripts. Immune adherence receptors that bind C3 are present on mouse platelets and unstimulated neutrophils, yet they are not MCR1 or MCR2. To examine C3b- and C3d-binding proteins on mouse platelets and neutrophils, we performed C3b and C3d affinity chromatography as well as immunoprecipitation studies using previously described Ab to MCR1/MCR2 (mAb clones 8C12, 7G6, and 7E9 and polyclonal Ab BRN-1). Mouse neutrophils contained a 190-kDa membrane protein that specifically bound to C3b-Sepharose. Preabsorption of neutrophil proteins with anti-MCR1/MCR2 Ab did not affect the recovery of the 190-kDa C3b-binding protein by subsequent C3b affinity chromatography. Thus, this protein is immunologically distinct from the previously described MCR1 and MCR2 proteins. By virtue of its size and C3b-binding capacity, the 190-kDa protein was named C3bR-190. C3bR-190 was also apparent on platelets, but in reduced amounts. BRN-1 anti-MCR1/MCR2 Ab immunoprecipitated proteins of 125 and 150 kDa from surface-radiolabeled mouse platelets, which specifically bound to C3d-Sepharose. However, these proteins were not identified by mAb to MCR2, thus distinguishing them from previously described MCR2. These proteins were named C3dR-125 and C3dR-150. Therefore, we have identified a 190-kDa C3b-binding protein on mouse neutrophils and 125- and 150-kDa C3d-binding proteins on mouse platelets. These appear to be distinct from the heretofore identified mouse B lymphocyte MCR1 and MCR2. The identity of these C3b- and C3d-binding proteins on mouse neutrophils and platelets awaits further study.

Close Association of the First and Fourth Extracellular Domains of the Duffy Antigen/Receptor for Chemokines by a Disulfide Bond Is Required for Ligand Binding

It has been demonstrated that the promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fya/b, and Fy3 epitopes, localized in the first and fourth extracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displacement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fya, and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libraries with an anti-Fy6 monoclonal antibody led to the identification of the motif Phe22-Glu23, the mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulated on activation normal T cell expressed)) to DARC transfectants. These results characterized the core of the Fy6 epitope and provided definitive proof of the tight relationship between Fy6 and the chemokine receptor site. Analysis of red cells treated by sulfhydryl group-modifying reagents suggested that the chemokine receptor function of DARC required the integrity of disulfide bond(s) but not that of free sulfhydryl group(s). Accordingly, mutation of cysteines 51 and 276 abolished chemokine binding to DARC transfectants. Altogether, our results suggested that the chemokine binding pocket of DARC included sequences located in the first and fourth extracellular domains which are brought into close vicinity by a disulfide bridge.

Inhibition of a porphyromonas gingivalis colonizing factor between actinomyces viscosus ATCC 19246 by monoclonal antibodies against recombinant 40-kDa outer-membrane protein

1. 1. Porphyromonas gingivalis, an important pathogen in human periodontal disease, aggregates with Actinomyces viscosus ATCC 19246. 2. 2. Monoclonal antibodies (mAbs) against purified recombinant 40-kDa outer-membrane protein (r40-kDa OMP) of P. gingivalis 381 inhibited its coaggregation with A. viscosus ATCC 19246 in a dose-dependent manner. 3. 3. Five mAb clones against r40-kDa OMP were selected. The isotype of the five was IgG1. 4. 4. Pg-ompA2 inhibited the coaggregation of several strains of P. gingivalis with A. viscosus ATCC 19246 cells.

Reversal of immunosuppression inducible through ultraviolet-exposed skin by in vivo anti-CD11b treatment.

In both human in vitro models and murine in vivo adoptive transfer studies, UV-induced class II MHC+ CD11b+ leukocytes that infiltrate the epidermis appear to mediate UV-induced immunosuppression. In the present study, their role is further probed using an anti-CD11b mAb (clone 5C6), which is effective in vivo in blocking CD11b+ monocyte/macrophage diapedesis into inflammatory lesions. A single exposure, low dose UV protocol (72 mJ/cm2) that resulted in tolerance only when dinitroflurobenzene was applied 48 h later through the UV-irradiated skin, but not through a distant non-UV-irradiated site, was used. In vivo anti-CD11b treatment in non-UV-irradiated mice did not block contact sensitivity responses. However, the ability to induce a primary contact sensitivity response was completely restored in UV-irradiated mice receiving anti-CD11b. This restoration was associated with partial restoration of papillary dermal class II MHC+ NLDC-145- cells. In vivo anti-CD11b treatment also blocked tolerance induction, which was associated with a 50% reduction in the infiltration of class II MHC+ CD11b+ Gr-1+ monocyte/macrophages into UV-irradiated skin. In addition, anti-CD11b treatment partially protected against epidermal UV injury, in that the epidermal structure was better preserved and the keratinocytes were less severely damaged. CD11b+ leukocytes may thus affect UV-irradiated skin through at least two mechanisms: 1) a class II MHC+ CD11b+ Gr-1+ monocyte/macrophage population inducing a state of tolerance to Ag(s) acquired in UV-irradiated skin, and 2) CD11b+ leukocytes capable of inflicting additional injury to both keratinocytes and constitutive APC damaged by UV photons.

Identification and characterization of monoclonal antibodies that partially block human natural antibody binding to pig endothelial cell xenoantigens

Abstract: The shortage of human donors for clinical transplantation has led to a serious consideration of the use of non‐human species as organ donors. The major barrier to the clinical use of xenografts from species such as the pig in human transplantation has been the aggressive nature of the immune‐mediated rejection of the graft. We have recently identified the molecular weights of several endothelial cell surface proteins that may be targets of human antibody‐mediated responses to pig aortic endothelial cells (PAEC). In this series of experiments, we produced a panel of rat monoclonal antibodies (Mabs) to PAEC in an effort to identify Mabs that detect pig xenoantigens. Mabs were selected based on flow cytometric binding to PAEC, pig platelets, and various pig cell lines, including a pig kidney cell line (LLC‐PK1) reported to react with human natural antibodies (HNA). Eleven of the eighty‐three antibodies produced were cytotoxic for PAEC. Six of the cytotoxic clones recognized a 44 kDa protein and two of the clones recognized a 115 kDa protein expressed on the surface of PAEC. Since PAEC target antigens recognized by human natural antibodies include both 115 and 44 kDa antigens, these Mab clones were selected for further study. Several distinct patterns of tissue reactivity were demonstrated within this group of antibodies by immunohistochemical analysis; however all monoclonal antibodies were highly reactive with endothelial cells in all tissues examined. Two monoclonal antibodies recognizing antigens that are highly expressed on pig endothelial cells (92–98%) and pig platelets (74–92%), but moderately expressed on pig splenocytes (33–38%), were capable of reproducibly blocking 48–53% of human IgM binding to pig endothelial cells when analyzed with flow cytometry. This data suggests that these Mabs may recognize epitopes of potential significance in the human‐to‐pig xenograft reaction.

Anti-PAP MAbs clone 1.3B5, 2.5H2, 5.1F5, 6.1C8, and 7.1E5

Anti-PAP MAbs clone 1.3B5, 2.5H2, 5.1F5, 6.1C8, and 7.1E5

Natural autoantibody against apolipoprotein A-I. Detection and characterization of the monoclonal antibody established from normal unimmunized BALB/c mice.

During the course of studying the immunogenicity of soybean lipids, we observed frequent production of the mAbs that bind to apolipoprotein A-I (apoA-I) when spleen cells from unimmunized normal BALB/c mice were employed for fusion. Of the 986 colonies from six fusions, 38 (3.9% of the total) were directed against apoA-I and 13 mAbs (IgM) were established for further analysis. The following lines of evidence indicate that this family of mAb may form a novel family of natural autoantibodies against apoA-I: 1) The mAbs were shown to bind effectively to high density lipoprotein from various species, including BALB/c mouse, and immunoblotting analyses revealed that the mAbs bound specifically to the 28-kDa protein of high density lipoprotein. 2) The 28-kDa protein was purified to homogeneity and identified as apoA-I by amino acid sequence analyses and by its cross-reactivity with a xenogenic anti-apoA-I mAb (clone A/11). 3) Differing from the xenogenic anti-apoA-I mAb, the present mAb did not bind to native apoA-I, whereas an effective binding was observed only when the apoA-I had formed a complex with neutral lipids containing polyunsaturated fatty acids such as trilinoleoylglycerol and 5-cholesten-3 beta-ol 3-linoleate. 4) Sera from unimmunized BALB/c mice had readily detectable Abs against apoA-I and the majority of the serum autoantibodies were of the IgA and IgM isotype. 5) The anti-apoA-I mAbs displayed a functional heterogeneity in their reactivity with polyanionic substances and some of the mAbs established showed an extensive cross-reaction with polyanionic substances such as ssDNA and cardiolipin.

Expression of stratified squamous epithelia-type cytokeratin by canine mammary epithelial cells during tumorigenesis: type I (acidic) 57 kilodalton cytokeratin could be a molecular marker for malignant transformation of mammary epithelial cells.

Two monoclonal antibodies (MAbs) were established in 20 clones of MAbs generated against cytokeratin fraction extracted from canine squamous cell carcinoma to investigate the expression of intermediate filament proteins during tumorigenesis. These MAbs were confirmed to react with purified cytokeratin by ELISA. One monoclonal antibody, MAb32 reacted all layers of epidermis except the cornified layer and mammary myoepithelial cells but not any epithelial cells. Another antibody named MAb24 exclusively reacted the basal monolayer of epidermis, the stratum germinativum. Any positive reactions with MAb24 were not detected in normal mammary gland. From the analysis by two-dimensional gel electrophoresis followed by immunoblotting, it was revealed that MAb24-recognizing cytokeratin subunit gave a molecular weight of 57 kilodalton and a isoelectric-pH value of pI5.1, indicating type I (acidic) cytokeratin. In intraductal papillomas developed in canine mammary glands, most tumor cells were positively stained with MAb32 in addition of myoepithelial cells while no positive reaction with MAb24 was seen. In ductal carcinomas, MAb24-positive cytokeratin was begun to express by tumor cells with positive reaction of MAb32 where these cells showed infiltrative growth into the stroma. We therefore proposed that 57 kilodalton-type I cytokeratin was a molecular marker for malignant transformation in canine mammary tumor and these antibodies could be useful tools to investigate the change of cytokeratin expression during tumorigenesis.

The Effect of High Mr Glutenin Subunit Composition on the Results from Tests Used to Predict Durum Wheat Quality

Abstract The relationships between protein content, sodium dodecyl sulphate sedimentation volume (SV), cooked gluten viscoelasticity (CGV), mixograph mixing development time (MDT) and pasta disc viscoelasticity (PDV) were investigated, and the effects of high M r glutenin subunit composition upon these quality test parameters determined. Durum wheat wholemeals or semolinas from 143 F2-derived F4 families from crosses between the cultivars Vic and Berillo were tested for protein content, SV, CGV, MDT, PDV and high M r glutenin subunits. To identify specific wheat endosperm proteins SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) using MAb clones P24B (specific for γ-gliadin 45) and clone 304/13 (specific for high M r glutenin subunit-B1) were used. Vic and Berillo both gave high CGV and PDV values. Vic also had high SV and MDT values, whilst those for Berillo were relatively low. Lines having high M r glutenin subunits 6 + 8 gave significantly higher SV values than those having high M r glutenin subunits 20. The effects of high M r glutenin subunit composition on CGV was inconsistent between years. The results indicate that, while both SV and CGV predict gluten strength, they are independent quality characteristics. Furthermore, MAb clone 304/13 could be used to identify breeders' lines containing high M r glutenin subunits 6 + 8 or 20.