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1.
Leukemia ; 29(2): 406-14, 2015 Feb.
Article in English | MEDLINE | ID: mdl-24903480

ABSTRACT

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.


Subject(s)
Antibodies, Bispecific/chemistry , Antibodies/chemistry , Burkitt Lymphoma/immunology , Immunotherapy/methods , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, CD20/chemistry , CD55 Antigens/chemistry , CD59 Antigens/chemistry , Cell Separation , Cloning, Molecular , Complement System Proteins , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , Killer Cells, Natural/cytology , Macrophages/cytology , Mice , Mice, SCID , Microscopy, Fluorescence
2.
Lett Appl Microbiol ; 59(3): 253-62, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24834970

ABSTRACT

UNLABELLED: Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. ). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. SIGNIFICANCE AND IMPACT OF THE STUDY: A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) - a pathological condition caused by Bartonella henselae infection - has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins - GroEL and 17-kDa - to develop a serological-based ELISA, showing promising results with the potential for further development as an effective tool for the differential diagnosing of B. henselae infection.


Subject(s)
Antibodies, Bacterial/blood , Bartonella henselae/immunology , Cat-Scratch Disease/diagnosis , Adolescent , Adult , Amino Acid Sequence , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Case-Control Studies , Cat-Scratch Disease/blood , Chaperonin 60 , Child , Child, Preschool , Diagnosis, Differential , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin M/blood , Lymphoma/diagnosis , Male , Molecular Sequence Data , ROC Curve , Tuberculosis, Pulmonary/blood , Tuberculosis, Pulmonary/diagnosis , Young Adult
3.
Int J Immunopathol Pharmacol ; 22(1): 73-83, 2009.
Article in English | MEDLINE | ID: mdl-19309554

ABSTRACT

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its death receptors, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricidal activity. The anti-TRAIL-R2 MB2.23 efficiently and specifically bound to membrane-associated TRAIL-R2 on different leukemic cell lines and could act as a direct agonist in vitro, initiating apoptotic signaling as well as complement-dependent cytotoxicity and antibody-dependent cell cytotoxicity, providing a rationale for further investigations of MB2.23 in anticancer therapy.


Subject(s)
Immunoglobulin Fragments/therapeutic use , Leukemia/drug therapy , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Animals , Antibody-Dependent Cell Cytotoxicity , Apoptosis/drug effects , CHO Cells , Cricetinae , Cricetulus , Humans , Leukemia/pathology , Peptide Library , Receptors, TNF-Related Apoptosis-Inducing Ligand/immunology , Recombinant Proteins/therapeutic use
4.
Protein Eng Des Sel ; 20(6): 267-71, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17545175

ABSTRACT

The fragmentation of DNA is a useful procedure for many molecular biology procedures. However, most methods used to fragment DNA are poorly controllable, and cannot be used to create small fragments. We describe a method to generate random DNA fragments of a predictable size to be cloned in expression vectors for the construction of display libraries. The DNA is allowed to form complexes with archaeal histones from Methanothermus fervidus (HMf) and the HMf/DNA core complex is naturally protected from nuclease DNaseI activity, giving rise to DNA fragments of approximately 60 bp and multiples thereof. We found that by varying the wt/wt ratio between DNA and HMf, the concentration of DNA and the incubation time with DNaseI, DNA fragments of desired size can be obtained. This approach should be applicable to the efficient fragmentation of DNA for the construction of phage display polypeptide libraries, as well as any other molecular biology procedures in which small DNA fragments of defined size are required.


Subject(s)
Archaeal Proteins/chemistry , DNA/chemistry , Histones/chemistry , Methanobacteriaceae/metabolism , Cloning, Molecular , DNA/metabolism , DNA Fragmentation , Deoxyribonuclease I/chemistry , Peptide Library , Plasmids
6.
Arch Dis Child ; 89(6): 512-5, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15155392

ABSTRACT

AIMS: To determine coeliac disease prevalence by an anti-transglutaminase antibody assay in a large paediatric population; to evaluate acceptance of the screening programme, dietary compliance, and long term health effects. METHODS: Cross-sectional survey of 3188 schoolchildren (aged 6-12) and prospective follow up of diagnosed cases. Main outcome measures were: prevalence of coeliac disease defined by intestinal biopsy or positivity to both human tissue transglutaminase and anti-endomysium antibodies in HLA DQ2-8 positive subjects; percentage of children whose families accepted screening; dietary compliance as defined by negativity for anti-transglutaminase antibodies; and presence of clinical or laboratory abnormalities at 24 month follow up. RESULTS: The families of 3188/3665 children gave their consent (87%). Thirty biopsy proven coeliacs were identified (prevalence 1:106). Three other children testing positive for both coeliac related autoantibodies and HLA DQ2-8 but refusing biopsy were considered as having coeliac disease (prevalence 1:96). Of 33 cases, 12 had coeliac related symptoms. The 30 biopsy proven coeliacs followed a gluten-free diet. Of 28 subjects completing 18-24 months follow up, 20 (71.4%) were negative for anti-transglutaminase antibodies, while eight were slightly positive; symptoms resolved in all 12 symptomatic children. CONCLUSIONS: Prevalence of coeliac disease is high in Italian schoolchildren. Two thirds of cases were asymptomatic. Acceptance of the programme was good, as was dietary compliance. Given the high prevalence and possible complications of untreated coeliac disease, the availability of a valid screening method, and evidence of willingness to comply with dietary treatment population mass screening deserves careful consideration.


Subject(s)
Antibodies/blood , Celiac Disease/diagnosis , Mass Screening/methods , Transglutaminases/immunology , Celiac Disease/epidemiology , Child , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay/methods , Female , Follow-Up Studies , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Italy/epidemiology , Male , Patient Compliance , Prospective Studies , Transglutaminases/blood
7.
Gut ; 51(2): 177-81, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12117875

ABSTRACT

BACKGROUND AND AIMS: Coeliac disease (CD) is a multifactorial disorder which has an autoimmune component characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). The aim of this study was to investigate whether binding of antibodies to the enzyme influences tTG activity. METHODS: tTG activity was assayed in the presence of immunoglobulin A (IgA) and immunoglobulin G (IgG) purified from the serum of coeliac patients, CUB 7402 (an anti-tTG mouse monoclonal antibody), and human anti-tTG monoclonal antibodies derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. For our studies we used calcium treated and untreated recombinant human tTG. Furthermore, the effects of antibodies were determined by immunohistochemical detection of tTG activity in sections of human umbilical cord. RESULTS: IgG and IgA from CD patients inhibited tTG activity in vitro in a dose dependent manner, with a different rate of inhibition among patients. The monoclonal antibody CUB 7402 and human monoclonal antibodies displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme, both in vitro and in situ. Preincubation of tTG with CaCl(2) caused loss of the inhibitory effect due to CUB 7402 but not that caused by human monoclonal antibodies. CONCLUSIONS: Purified CD IgA, IgG, as well as human anti-tTG monoclonal antibodies inhibited the enzymatic activity of human tTG both in vitro and in situ.


Subject(s)
Antibodies, Monoclonal/pharmacology , Celiac Disease/enzymology , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/immunology , Transglutaminases/immunology , Animals , Calcium Chloride/pharmacology , Celiac Disease/immunology , Cell Line , Cells, Cultured , Dogs , GTP-Binding Proteins/analysis , Humans , Immunoglobulin A/pharmacology , Immunoglobulin G/pharmacology , Immunohistochemistry/methods , Mice , Protein Glutamine gamma Glutamyltransferase 2 , Recombinant Proteins/pharmacology , Transglutaminases/analysis , Transglutaminases/antagonists & inhibitors , Transglutaminases/metabolism , Umbilical Cord/enzymology
8.
Scand J Gastroenterol ; 37(6): 679-84, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12126246

ABSTRACT

BACKGROUND: The main autoantigen recognized by the sera of patients with coeliac disease (CD) is tissue transglutaminase (tTG). A human-recombinant form of tTG was used to develop an ELISA to measure anti-tTG serum antibodies for the diagnosis of CD. Preliminary retrospective reports suggest that the human tTG-based ELISA could identify coeliac patients missed by the IgA-anti-endomysium antibody test (AEA). Whether the human recombinant tTG ELISA is sufficiently accurate to become the main diagnostic CD tool in everyday clinical practice is unknown. The objective was to determine, in a prospective study, the sensitivity and specificity of an ELISA test based on the use of human tTG compared with AEA, to analyse the discordant cases for HLA DQ2-8 and for clinical and intestinal biopsy characteristics. METHODS: 1106 patients referred to a gastrointestinal outpatient clinic for symptoms attributable to CD, 52 first-degree relatives of CD patients and 200 healthy controls were tested for both anti-human tTG and AEA antibodies. RESULTS: Out of 1158 subjects, 146 were tested positive for anti-tTG antibodies and 140 were biopsy-proven coeliacs. The AEA test identified 126/1158 coeliacs who also tested positive for anti-tTG antibodies. The 14 patients missed by the AEA test carried the typical HLA-DQ for CD; they had normal levels of total serum IgA and had milder pathology than those with both anti-tTG and AEA positivity (P < 0001). CONCLUSIONS: These results prove that human tTG-based ELISA is an excellent diagnostic tool for CD, for mass screening by both the specialist and the general clinic.


Subject(s)
Autoantibodies/analysis , Celiac Disease/diagnosis , Enzyme-Linked Immunosorbent Assay , Transglutaminases/blood , Adolescent , Adult , Aged , Ambulatory Care Facilities , Biopsy, Needle , Case-Control Studies , Celiac Disease/epidemiology , Child , Child, Preschool , Female , Gastric Mucosa/pathology , HLA-DQ Antigens/analysis , Humans , Infant , Infant, Newborn , Male , Mass Screening/methods , Middle Aged , Probability , Prospective Studies , Reference Values , Reproducibility of Results , Sensitivity and Specificity , Statistics, Nonparametric , Transglutaminases/analysis
9.
J Biotechnol ; 74(4): 303-15, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11526909

ABSTRACT

The creation of diversity in populations of polypeptides has become an important tool in the derivation of polypeptides with useful characteristics. This requires efficient methods to create diversity coupled with methods to select polypeptides with desired properties. In this review we describe the use of in vivo recombination as a powerful way to generate diversity. The novel principles for the recombination process and several applications of this process for the creation of phage antibody libraries are described. The advantage and disadvantages are discussed and possible future exploitation presented.


Subject(s)
Antibodies/genetics , Genetic Variation , Peptide Library , Recombination, Genetic , Animals , Biotechnology , Genetic Vectors , Genomics , Humans , Integrases , Protein Engineering , Viral Proteins
10.
J Immunol Methods ; 253(1-2): 233-42, 2001 Jul 01.
Article in English | MEDLINE | ID: mdl-11384684

ABSTRACT

Antibodies against most antigens can be isolated from high quality phage antibody libraries. However, not all antibodies binding a particular antigen are necessarily found when standard selections are performed. Here we investigate the effect of two different selection strategies on the isolation of antibodies against a number of different antigens, and find that these different strategies tend to select different antibodies, with little overlap between them. This indicates that the full diversity of these libraries is not tapped by a single selection strategy and that each selection strategy imposes different selective criteria in addition to that of antigen binding. To fully exploit such libraries, therefore, many different selection strategies should probably be employed for each antigen. The use of alternative strategies should be considered when selection apparently fails, or when the number of different antibodies recognizing an antigen needs to be maximised. Furthermore, the microtitre selection strategy developed is likely to prove useful in the application of phage antibody libraries to the human genome project, allowing the high throughput selection of antibodies against multiple antigens simultaneously.


Subject(s)
Antibodies/immunology , Peptide Library , Antibodies/genetics , Antibody Diversity , Antigens/immunology , Clone Cells/immunology , Humans
11.
J Immunol ; 166(6): 4170-6, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11238668

ABSTRACT

Celiac disease (CD) is an intestinal malabsorption characterized by intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and tissue transglutaminase, an autoantigen located in the endomysium. Tissue transglutaminase belongs to the family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. The role of gliadins in eliciting the immune response in CD and how transglutaminase is linked to the primary reaction are still unclear. In this work, we report the production and analysis of six phage Ab libraries from the peripheral and intestinal lymphocytes of three CD patients. We were able to isolate Abs to transglutaminase from all intestinal lymphocytes libraries but not from those obtained from peripheral lymphocytes. This is in contrast to Abs against gliadin, which could be obtained from all libraries, indicating that the humoral response against transglutaminase occurs at the local level, whereas that against gliadin occurs both peripherally and centrally. Abs from all three patients recognized the same transglutaminase epitopes with a bias toward the use of the V(H)5 Ab variable region family. The possible role of these anti-transglutaminase Abs in the onset of CD and associated autoimmune pathologies is discussed.


Subject(s)
Autoantibodies/biosynthesis , Autoantibodies/genetics , Celiac Disease/enzymology , Celiac Disease/immunology , GTP-Binding Proteins/immunology , Transglutaminases/immunology , Adult , Amino Acid Sequence , Antibody Affinity , Autoantibodies/metabolism , Autoantigens/immunology , Autoantigens/metabolism , Binding, Competitive/immunology , Clone Cells , Epitope Mapping , Fetal Blood/immunology , Fluorescent Antibody Technique, Direct , GTP-Binding Proteins/metabolism , Gene Library , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Gene Rearrangement, B-Lymphocyte, Light Chain , Genetic Vectors/immunology , Humans , Immune Sera/metabolism , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Inovirus/genetics , Inovirus/immunology , Molecular Sequence Data , Muscle Fibers, Skeletal/immunology , Mutation , Protein Glutamine gamma Glutamyltransferase 2 , Solubility , Transglutaminases/metabolism
12.
Gut ; 47(5): 628-31, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11034577

ABSTRACT

BACKGROUND: Coeliac disease is one of the commonest underdiagnosed diseases in general practice. The autoantigen recognised by the sera of patients with coeliac disease has recently been identified as tissue transglutaminase. AIMS: We evaluated a simple non-invasive immunological dot blot assay for coeliac disease, suitable for use by the general physician in the ambulatory setting. The sensitivity and specificity of this dot blot assay based on recognition of recombinant human transglutaminase were compared with those of antiendomysial antibodies and an enzyme linked immunosorbent assay. METHODS: Serum samples were analysed from 64 healthy controls, 58 first degree relatives of coeliacs, 74 diseased controls, and 70 biopsy confirmed untreated patients with coeliac disease. Dot blot assay and enzyme linked immunosorbent assay were performed using recombinant human transglutaminase as antigen. RESULTS: The dot blot assay, which can be performed in 20 minutes, was positive in all 70 untreated coeliacs (sensitivity 100%). Among the three control groups, there were three false positive tests by dot blot (specificity 98%), all belonging to the group of healthy subjects. The antiendomysial antibodies test missed five untreated coeliac patients (sensitivity 93%) and was negative in all three control groups (specificity 100%). The specificity of the immunosorbent assay was 99% for IgA and 98% for IgG, while sensitivity was 93% for IgA, 47% for IgG, and 100% for IgA and IgG combined. CONCLUSIONS: The dot blot assay is highly accurate in detecting untreated subjects with coeliac disease and can be performed in the general physician's medical office during the course of a routine examination. This innovative test is a practical, reliable alternative to both the immunofluorescent based antiendomysial test and immunosorbent assay for detection of transglutaminase antibodies for the diagnosis of coeliac disease.


Subject(s)
Celiac Disease/diagnosis , Mass Screening/methods , Reagent Kits, Diagnostic , Adolescent , Adult , Antibodies/immunology , Celiac Disease/immunology , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , False Positive Reactions , Female , Humans , Immunoblotting , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Infant , Male , Middle Aged , Reagent Kits, Diagnostic/standards , Sensitivity and Specificity , Transglutaminases/immunology
13.
Am J Gastroenterol ; 95(5): 1253-7, 2000 May.
Article in English | MEDLINE | ID: mdl-10811336

ABSTRACT

OBJECTIVE: Tissue transglutaminase is the autoantigen recognized by the sera of celiac patients. An enzyme-linked immunosorbent assay (ELISA) based on guinea-pig tissue transglutaminase was recently used to measure serum tissue transglutaminase antibodies for the diagnosis of celiac disease. We determine the sensitivity and specificity of an ELISA test based on the use of human recombinant transglutaminase, compared with the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies. METHODS: Serum samples were tested from 65 patients with intestinal biopsy proven celiac disease, from 10 patients with Crohn's disease, and from 150 healthy blood donors. RESULTS: Human transglutaminase ELISA identified 64 of 65 celiac patients, whereas the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies identified 58 of 65 and 60 of 65 subjects, respectively. The three tests showed comparable specificity. CONCLUSIONS: These results proved that the human tissue transglutaminase-based ELISA represents a cost-effective strategy for identifying both symptomatic and atypical forms of celiac disease and could mean that intestinal biopsy need no longer be the gold standard for diagnosing this clinical condition. Furthermore, early identification and treatment of patients with celiac disease in an outpatient setting could have significant implications for reducing long-term morbidity and can produce major savings in future health care costs.


Subject(s)
Autoantibodies/blood , Celiac Disease/diagnosis , Transglutaminases/immunology , Adolescent , Adult , Animals , Child , Child, Preschool , Crohn Disease/immunology , Enzyme-Linked Immunosorbent Assay , Female , Guinea Pigs , Humans , Immunoglobulin A/blood , Male , Middle Aged , Muscle Fibers, Skeletal/immunology , Recombinant Proteins/immunology , Sensitivity and Specificity
14.
Hum Antibodies ; 9(4): 199-205, 2000.
Article in English | MEDLINE | ID: mdl-11341173

ABSTRACT

Celiac disease (CD) is an autoimmune enteropathy characterized by intestinal malabsorption and immunological responses to dietary gliadins and an auto antigen located in the endomysium. The latter has recently been identified as the enzyme tissue transglutaminase (tTG). The linkage between gliadins, tTG and the autoimmune response has still to be clarified. In this work we report the production and analysis of a phage antibody library from the peripheral blood lymphocytes (PLB) of a CD patient. The library contained polyreactive and monoreactive antibodies to alpha-gliadin, to the dietary antigen beta-lactoglobulin, but not to tTG. The majority of the VH regions of the anti-alpha-gliadin antibodies belonged to the VH 4 family. The possibility of exploiting phage display antibodies as tools to study the molecular events associated with CD is discussed.


Subject(s)
Antibodies/blood , Autoimmune Diseases/immunology , Celiac Disease/immunology , Gliadin/immunology , Lymphocytes/immunology , Transglutaminases/immunology , Adult , Animals , Antibodies/genetics , Enzyme-Linked Immunosorbent Assay/methods , Female , Gliadin/chemistry , Gliadin/metabolism , Humans , Mice , Mice, Inbred BALB C , Peptide Library , Transglutaminases/metabolism
15.
FEBS Lett ; 411(1): 27-31, 1997 Jul 07.
Article in English | MEDLINE | ID: mdl-9247136

ABSTRACT

The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties. The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities. Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display. We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell. This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Bacteriophage M13 , Endotoxins/genetics , Insecticides , Bacillus thuringiensis Toxins , Cloning, Molecular , Gene Expression , Genetic Vectors , Hemolysin Proteins , Peptide Library , Recombinant Fusion Proteins/genetics
16.
Gene ; 185(1): 27-33, 1997 Jan 31.
Article in English | MEDLINE | ID: mdl-9034309

ABSTRACT

fd and IKe are two similar filamentous phage which infect their hosts by means of pili found on the host membrane: fd infects bacteria bearing F pili, whereas IKe infects bacteria bearing N or I pili. Infection is mediated by the gene 3 protein (g3p), which of the nine proteins found in both phage is the most diverse. Previous attempts to incorporate g3p from one phage into the other by complementation have been unsuccessful [Bross et al. (1988) J. Gen. Microbiol. 134, 461-471]. Here we have grafted different parts of IKe g3p to the end of fd g3p and so augmented the host range of fd phage. We show that phage bearing such chimeric g3p are able to infect bacteria bearing both N and F pili providing they contain at least the receptor domain of IKe g3p, the infection of N bearing bacteria occurring at a level 70,000 times greater than background. This level of infection can be increased tenfold by including the glycine-rich domain as well. Addition of the penetration domain does not improve the level of infection above that of the receptor domain alone, indicating that the fd penetration domain is functional in the infection of bacteria bearing either N or F pili. Similarly derived fd phagemid also show increased infection of bacteria bearing N pili, albeit at much lower levels, suggesting that efficient infection requires more than one functional g3p on the surface of the phage.


Subject(s)
Binding Sites/genetics , Chimera/genetics , DNA-Binding Proteins/genetics , Inovirus/genetics , Protein Binding/genetics , Viral Fusion Proteins/genetics , Bacteria/chemistry , Bacteria/virology , Blotting, Western , Capsid Proteins , Cloning, Molecular , Fimbriae, Bacterial/chemistry , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/physiology
17.
J Immunoassay ; 13(2): 181-96, 1992.
Article in English | MEDLINE | ID: mdl-1385481

ABSTRACT

Y7, a murine monoclonal IgG1 kappa antibody against a human monoclonal IgM lambda DJ molecule, was affinity purified on an IgM lambda immunoaffinity column. As detected by enzyme-linked immunosorbent assay (ELISA) the isolated Y7 monoclonal antibody was shown to be not cross-reactive with human IgG, human secretory IgA, mu chain, lambda + kappa chains and another human monoclonal IgM lambda BR. Binding to the polyclonal human IgM standard in the same assay was about 30 percent. The epitope specificity of affinity purified and biotinylated Y7 MoAb was localized only in the nonreduced pepsin Fab fragments of IgM lambda DJ immunogen. As the immunogen was determined to be a specific antibody to phosphorylcholine, the specificity of Y7 MoAb was further ascertained in its capacity to induce 95% inhibition of immunogen binding for phosphorylcholine.


Subject(s)
Antibodies, Anti-Idiotypic/chemistry , Antibodies, Monoclonal/chemistry , Animals , Antibody Specificity , Enzyme-Linked Immunosorbent Assay , Epitopes , Humans , Immunochemistry , Immunoglobulin G/chemistry , Immunoglobulin M , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin lambda-Chains , Mice , Phosphorylcholine/immunology , Waldenstrom Macroglobulinemia/immunology
18.
Eur J Biochem ; 198(1): 211-6, 1991 May 23.
Article in English | MEDLINE | ID: mdl-2040281

ABSTRACT

The class I of the high mobility group (HMG) proteins is formed by phosphoproteins which are associated with AT-rich DNA sequences in the nucleus. Three HMGI proteins have previously been described in proliferating rodent cells (HMG Y, HMG I and HMGI-C). All three proteins exhibit microheterogeneity. The microheterogeneity of mouse HMG Y has been investigated in detail and shown to be due to phosphorylation of the protein which is sensitive to alkaline-phosphatase treatment. HMG I is similarly modified. Human cells have up to now only been found to contain HMG Y and HMG I. A search for the third protein, HMGI-C, in human cells was carried out and the protein was found in a hepatoma cell line, but not in normal or transformed T-cells. This HMGI-C protein was found to be modified by phosphorylation, part of which was found to be phosphatase insensitive. An unexpected additional finding in this study was that human cells contain two HMG17 proteins which differ in their N-terminal primary sequences.


Subject(s)
High Mobility Group Proteins/chemistry , Alkaline Phosphatase/chemistry , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Rodentia , Tumor Cells, Cultured
19.
J Pediatr Gastroenterol Nutr ; 11(3): 337-41, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2246715

ABSTRACT

A dot immunobinding assay to detect anti-alpha-gliadin-specific antibodies in the sera or whole blood of enteropathic patients is described here. The method is based on the adsorption of alpha-gliadin as a spot onto nitrocellulose sheets. After incubation with the patient sample, the detection of specific antibodies is performed with alkaline phosphatase-conjugated goat anti-human (IgA or IgG) antibodies. Twenty-one celiac serum samples together with 18 enteropathic or disease controls and 44 healthy controls were analyzed. The classical ELISA test and the dot test gave comparable results. The dot test gave reliable result even when whole blood was tested. The method proved to be simple and sensitive.


Subject(s)
Celiac Disease/immunology , Gliadin/immunology , Celiac Disease/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Immunosorbent Techniques
20.
Appl Environ Microbiol ; 55(2): 446-53, 1989 Feb.
Article in English | MEDLINE | ID: mdl-2541663

ABSTRACT

The entire coding region of the Bacillus thurigiensis HD73 crystal protein gene was subcloned from plasmid pJWK20 into the integration vector pUG2-15. This plasmid expresses chloramphenicol resistance when integrated into the Bacillus subtilis chromosome in the outH locus near the recE region. The correct molecular organization of the integrated plasmid was verified by hybridization to Southern blots of chromosomal DNA digests. Production of the toxic crystal protein was monitored at different time points during the life cycle of B. subtilis. Toxicity assays against Anagasta (Ephestia) larvae, direct electron microscopy crystal detection, and immunoblotting assays proved that the expression of the gene in B. subtilis is time regulated and restricted mainly to the sporulation stage. RNase protection experiments defined the transcription initiation start point and the transcription timing. All tests were made in a strain containing one to three copies of the integrated plasmid and in a strain subjected to an amplification regimen.


Subject(s)
Bacillus subtilis/genetics , Bacillus thuringiensis/genetics , Bacterial Proteins , Bacterial Toxins , Cloning, Molecular , Endotoxins/genetics , Genes, Bacterial , Genes , Animals , Bacillus thuringiensis Toxins , Crystallization , Endotoxins/isolation & purification , Endotoxins/pharmacology , Hemolysin Proteins , Insecta/drug effects , Insecticides/pharmacology , Larva , Plasmids , Recombinant Proteins/isolation & purification , Restriction Mapping , Transcription, Genetic
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