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1. Xenotran
Xenotran - adsorbent for removal anti-aGal xenoantibodies. Efficacy, biocompatibility, regeneration, sterilization and stability.
An approach to solve the problem of lacking donor organs is the transplantation of animal organs into humans. For a number of reactions (availability, size of the organs, physiology and ethical considerations), the pig is seen today as the most promising candidate for such xenotransplantations. However, the pig-to-human combination is discordant, which means that pig organs are hyperacutely rejected in humans by the binding of preformed, mostly carbohydrate-specific antibodies and the action of complement. It has been shown that preformed, naturally occurring anti-Gala (Gala1-3Gal, the linear B-disaccharide) antibodies are the most important for xenorejection of pig organs. Removal of anti-Bdi in the recipient plasma using the corresponding affinity adsorbents therefore seems to be a promising approach to overcome the phase of hyperacute rejection.
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Xenotran consists of synthetic oligosaccharide covalently coupled
to Sepharose FF via hydrophilic flexible polyacrylamide spacer.
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Immunoadsorption of human or baboon serum. Column is packed with adsorbent
and connected to peristaltic pump. The column is rinsed with PBS, the adsorption procedure
is carried out at room temperature.
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Efficacy. Reduction of anti-aGal antibodies
is determined by isotype-specific ELISA as described in [1, 2] using
B-disaccharide-polyacrylamide conjugate as coating antigen. The results
are shown in fig. 1-3.
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Regeneration, alkali treatment, sterilization and storage. After adsorption, the
column is regenerated by rinsing with PBS, acid elution of bound antibodies
with 0.1M glycin/HCl, pH 2.7, rinsing again with PBS, and then washing
with distilled water. The adsorbent is kept at 4oC in 0.05%
water solution of NaN3 or in 20% ethanol. Regeneration and alkali treatment do not affect sufficiently
efficacy of the adsorbent (Fig. 4). Sterilization is possible with: ethanol, alkili, formaline, or heating.
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Biocompatibility and specificity. No changes in total hemolytic
complement of the classic pathway, CH 50, and activated partial thromboplastin
time (aPTT) is observed (the parameters were determined before and after adsorption procedure). No reduction
of total immunoglobulins was observed (ELISA) in plasma after adsorption.
2. Allotran A, Allotran B
Allotran A and Allotran B consist of trisaccharides GalNAca1-3(Fuca1-2)Gal and Gala1-3(Fuca1-2)Gal respectively, coupled to Sepharose FF via polyacrylamide. Efficacy, regeneration, biocompapibility and other properties of the Allotrans are the same as described for Xenotran.
Depletion of AB-antibodies from intravenous immunoglobulin (IvIg) preparations
IvIgs are concentrates of immunoglobulins, especially of IgG, which are derived from the pooled plasma of a large number of donors. The use of IvIg has a steadily growing number of indications, such as antibody replacement therapy or as an immunomodulatoring autoimmune disorders. Antibody replacement appears to be of value in the prophylaxis of infections in patients suffering from low-grade B-cell malignancies, in bone marrow graft recipients, in AIDS (for the prevention of CMV-related pathology), and it has dramatically improved the survival of patients with interstitial pneumonia; IvIg appears to moderate the severity of GVHD in adult transplant recipients; it is also used successfully to treat autoimmune hemolytic anemias, which are refractory to standard forms of treatment. The use of IvIg (especially IgM-containing preparations) decreases the mortality due to sepsis in premature infants.
It is well known, that several products prepared from pooled human plasma contain elevated levels of anti-A and anti-B antibodies. Such preparations, for example IvIg, have been reported to cause moderate to severe hemolytic reactions. Some authors therefore recommend that anti-A and anti-B titers of pooled human plasma products to be used intravenously should be lower than 64 to prevent hemolysis of the red blood cells of blood group A, B, and AB recipients, respectively. One way for precaution is to exclude of O-type donors from the pooled material, but this would mean that about 1/2 of all possible donors excluded from such pools. Another possibility is the elimination of anti-A and anti-B using affinity chromatography on immunosorbents containing synthetic blood group A- and/or B-oligosaccharides.
Application for ABO-incompatible transplantations
ABO blood-group incompatibility between donor and recipient constitutes an obstacle for solid organ and bone marrow transplantation, unless the risk of a hemolytic reaction can be controlled. Hemagglutinins of the recipient's plasma are capable of reacting with A and B antigens present on red blood as well as on endothelial cells of the donor organ. However, it was shown that successful transplantations across ABO-barrier can be performed by using repeated pre- and post-operative plasmapheresis treatment. In the case of bone marrow transplantations the ABO-incompatibility results in a delayed hematopoiesis, which requires sophisticated, long-term supportive care for the patients. An alternative method to plasmapheresis for the removal of isoagglutinins is the immunoadsorption treatment by chemically synthesized affinity sorbents, containing oligosaccharides A and B as active haptens. By making ABO-incompatible kidney transplantation possible, and thus more than doubling the number of potential donors, the therapeutic immunoadsorption using A/B affinity adsorbents offer hope for an independent life to people whose only previous alternative was long-term dialysis.
We have designed biocompatible adsorbents Allotrans especially for ex vivo antibody removal from human plasma. Two cases of clinical application were documented. First one dealing with incompartible heart, the second one with incompartible lung transplantation. Biosorbents offer good parameters with respect to non-activation of the coagulation and complement system. Adsorbents Atri, Btri and Bdi are available for research use.
Application for depletion of ABO-antibodies from human anti-Rh sera
Polyclonal anti-Rh reagents prepared from human sera contain anti-A and/or anti-B antibodies, depending on the ABO status of the serum donors. Only sera of donors belonging to blood group AB lack anti-A and anti-B and are, therefore, universal (useful for Rh-typing any blood samples, independently of ABO status). Thus, about 3/4 of all Rh reagent donors should be excluded because their sera react with both Rh(D)+ and A/B-erythrocytes. Elimination of anti-A and anti-B with affinity adsorbents is a convenient approach for preparing universal human anti-Rh (as well as anti-Duffy and anti-Kell) reagents.
3. Purification of influenza virus
Application for ABO-incompatible transplantations
ABO blood-group incompatibility between donor and recipient constitutes an obstacle for solid organ and bone marrow transplantation, unless the risk of a hemolytic reaction can be controlled. Hemagglutinins of the recipient's plasma are capable of reacting with A and B antigens present on red blood as well as on endothelial cells of the donor organ. However, it was shown that successful transplantations across ABO-barrier can be performed by using repeated pre- and post-operative plasmapheresis treatment. In the case of bone marrow transplantations the ABO-incompatibility results in a delayed hematopoiesis, which requires sophisticated, long-term supportive care for the patients. An alternative method to plasmapheresis for the removal of isoagglutinins is the immunoadsorption treatment by chemically synthesized affinity sorbents, containing oligosaccharides A and B as active haptens. By making ABO-incompatible kidney transplantation possible, and thus more than doubling the number of potential donors, the therapeutic immunoadsorption using A/B affinity adsorbents offer hope for an independent life to people whose only previous alternative was long-term dialysis.
Syntesome has designed biocompatible adsorbents Allotrans especially for ex vivo antibody removal from human plasma. Biosorbents offer good parameters with respect to non-activation of the coagulation and complement system. Adsorbents Atri, Btri and Bdi are available for research use.
Application for depletion of ABO-antibodies from human anti-Rh sera
Polyclonal anti-Rh reagents prepared from human sera contain anti-A and/or anti-B antibodies, depending on the ABO status of the serum donors. Only sera of donors belonging to blood group AB lack anti-A and anti-B and are, therefore, universal (useful for Rh-typing any blood samples, independently of ABO status). Thus, about 3/4 of all Rh reagent donors should be excluded because their sera react with both Rh(D)+ and A/B-erythrocytes. Elimination of anti-A and anti-B with affinity adsorbents is a convenient approach for preparing universal human anti-Rh (as well as anti-Duffy and anti-Kell) reagents.
Sialic acid-liganded Sepharose FF was tested as an affinity media for isolation and purification of influenza viruses.
Three types of ligand were covalently coupled to Sepharose FF via polyacrylamide:
- (Siaa2-8)5 - ligand resistant to viral neuraminidase
- 3’-SiaLac - ligand specific for avian or egg-adapted viruses
- Neu5Aca-(CH2)3 - the simplest derivative of sialic acid.
Two types of virus were tested:
- Eggs-adapted virus strain X113 (H1N1)
- MDCK-adapted virus ((H1N1).
Fig. 5 represents relative capacity of three adsorbents in respect of the virus binding. The first adsorbent binds only MDCK grown virus, the third one - only egg-adapted virus, 3’SiaLac adsorbent prefers, as expected, egg-adapted strain.
Regeneration, sterilization, storage: see "Xenotran"
Fig. 5
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