| 1. |
- Olovsson, Matts, 1958-, et al.
(författare)
-
Immunizing with mouse trophoblast to obtain mouse-human cross-reacting antibodies against normal and neoplastic human trophoblast.
- 1999
-
Ingår i: Hybridoma. - 0272-457X .- 2168-7897. ; 18:6
-
Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies raised against living, early invasive mouse trophoblast cells were screened on paraffin sections from first- and third-trimester placentas and from hydatidiform moles and choriocarcinoma. Several mouse-human cross-reacting antibodies were recognized, which implies that mouse trophoblast cells can be used as immunogen for producing antibodies against human trophoblast. Among the new antibodies obtained, some were selected for further study. That panel includes a trophoblast specific antibody with capacity to differ between invasive and noninvasive molar tissues, and two antibodies, which detect antigen epitopes in the normal, but not in the neoplastic trophoblast.
|
|
| 2. |
- Olovsson, Matts, 1958-, et al.
(författare)
-
Methods to produce stage-specific monoclonal antibodies against mouse trophoblast cells by intrasplenic and in vitro immunizations.
- 1993
-
Ingår i: Hybridoma. - 0272-457X .- 2168-7897. ; 12:3
-
Tidskriftsartikel (refereegranskat)abstract
- Monoclonal antibodies recognizing epitopes expressed by invasive mouse trophoblast cells were obtained by immunizing male mice of the NMRI strain with mouse egg-cylinders obtained by culturing blastocysts on agarose for 4 days. Immunization was achieved by intrasplenic and in vitro methods. The procedures described outline a rational way of producing and selecting antibodies which are stage-specific for the mouse trophoblast at implantation and early placentation. Preimplantation stage-specificity was tested and adhesive blastocysts only were detected by 4 antibodies. Thus, these antibodies become markers for that developmental stage which characterizes an adhesive blastocyst ready to implant. Postimplantation stage-specificity was demonstrated by 6 antibodies, which labelled trophoblast cells of day 7 implantations only. Thus, there is a specific expression of some epitopes by the invasive trophoblast. Cell surface epitopes were detected on adhesive blastocysts by 4 antibodies and on the invasive trophoblast of the egg-cylinder by 7 antibodies. Shedded antigens from egg-cylinders in vitro were detected by 5 antibodies. Four of these antibodies detected substances released only from early implantation stages. A functional effect was demonstrated for an IgM antibody which impaired hatching and outgrowth of blastocysts in vitro.
|
|
| 3. |
- Sheikholvaezin, Ali, et al.
(författare)
-
Construction and purification of a covalently linked divalent tandem single-chain Fv antibody against placental alkaline phosphatase.
- 2006
-
Ingår i: Hybridoma. - : Mary Ann Liebert Inc. - 0272-457X .- 2168-7897 .- 1554-0014 .- 1557-8348. ; 25:5, s. 255-263
-
Tidskriftsartikel (refereegranskat)abstract
- Multivalency is a recognized means to increase the functional affinity of single-chain antibody fragments (scFvs) for optimizing tumor uptake at radioimmunotargeting. A unique divalent tandem single-chain Fv antibody [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) H7, has now been generated. The antibody differs from other dimeric single-chain constructs in that a linker sequence (L) is introduced between the repeats of VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed as a His-tagged TrxA fusion protein in the Escherichia coli strain Origami B. Following cleavage with AcTev protease, the antibody was obtained in soluble and active form in E. coli and could be purified by Ni-NTA and cation-exchange chromatography. Purity and immunochemical properties were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blot, and Biacore analyses. The [sc(Fv)2] displayed proper stability and could be purified to homogeneity. This antibody also maintained immunoreactivity at 42 degrees C with only slight decrease at 52 degrees C. The high affinity displayed by the original antibody H7, 6.7 x 10(9) M(-1), was only slightly decreased to 1.2 x 10(9) M(-1) as determined by Biacore. The generation of such a divalent single-chain Fv with a molecular weight around 60 kd would be of value for clinical applications such as radioimmunolocalization and radioimmunotherapy.
|
|
| 4. |
- Sheikholvaezin, Ali, et al.
(författare)
-
Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase.
- 2006
-
Ingår i: Hybridoma. - : Mary Ann Liebert Inc. - 0272-457X .- 2168-7897 .- 1554-0014 .- 1557-8348. ; 25:4, s. 181-192
-
Tidskriftsartikel (refereegranskat)abstract
- Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.
|
|
| 5. |
|
|
| 6. |
- Mobini, Reza, 1965, et al.
(författare)
-
A monoclonal antibody directed against an autoimmune epitope on the human beta1-adrenergic receptor recognized in idiopathic dilated cardiomyopathy.
- 2000
-
Ingår i: Hybridoma. - : Mary Ann Liebert Inc. - 0272-457X. ; 19:2, s. 135-42
-
Tidskriftsartikel (refereegranskat)abstract
- A monoclonal antibody (MAb M16) was obtained by immunizing Balb/C mice with free peptide H26R, corresponding to the second extracellular loop of the human beta1-adrenergic receptor (beta1AR), against which functional autoantibodies have been detected in patients with idiopathic dilated cardiomyopathy. The MAb was found to be of IgG2b type and directed against a conformational epitope, encompassing the sequence recognized by the human autoantibodies. BIAcore measurements yielded an equilibrium constant of 6.5 X 10(7) M1 with an association rate constant (kon) of 6.5 X 10(4) M(-1) sec(-1) and a dissociation rate constant (koff) of 1.0 X 10(-3) sec(-1). It immunoprecipitated only poorly the solubilized beta1AR of Sf9 cell membranes. Functionally, the MAb was capable of not only reducing the number of the maximal binding sites to the beta1-adrenergic receptor of transfected Sf9 cell membranes, but also of displaying a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These properties, which the MAb shares with the human autoantibodies, makes it an interesting tool for passive transfer studies in mice.
|
|
| 7. |
|
|
| 8. |
- Rousseau, K, et al.
(författare)
-
New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7
- 2003
-
Ingår i: Hybridoma. - : Mary Ann Liebert Inc. - 0272-457X. ; 22:5, s. 293-299
-
Tidskriftsartikel (refereegranskat)abstract
- The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions. The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health. MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree. MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller. MUC7 has anti-fungal activity, and both mucins interact with bacteria. The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures. Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins. Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins. All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis. These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.
|
|
| 9. |
- Sjogren-Jansson, Eva, et al.
(författare)
-
Production of human monoclonal antibodies in dialysis tubing
- 1991
-
Ingår i: Hybridoma. - : Mary Ann Liebert Inc. - 0272-457X. ; 10:3, s. 411-419
-
Tidskriftsartikel (refereegranskat)abstract
- Human x mouse hybridoma cells were grown in dialysis tubing (DT) to obtain large quantities of human monoclonal antibodies (MAb). Hybridoma cells were grown inside the DT, which was placed in a tissue culture flask containing medium. The medium inside the DT was supplemented with different additives which may be selected depending on the intended use of the MAb. About 10-50 times higher concentrations of immunoglobulin (Ig) were obtained after cultivation in DT compared to conventional tissue culture (CTC) for 2 days. The purity of the MAb was high which facilitated further antibody purification. Production of human MAb in DT proved to be excellent for evaluation studies in laboratory scale. It does not require expensive equipment and several hybridomas can be grown simultaneously.
|
|
| 10. |
|
|
