dewiki Digitalis-Antidot; enwiki Digoxin immune fab; eswiki Anticuerpos antidigoxina; plwiki Digitalis-Antidot; shwiki Digoksin imun Fab; srwiki Digoksin imun Fab. In life-threatening situations, antidigoxin antibodies must be used. caciones de los anticuerpos antidigoxina en la intoxicación digitálica. Revisio ́n sistema ́tica sobre la efectividad e indicaciones de los anticuerpos antidigoxina en la intoxicacio ́n digita ́lica. [Systematic review of the effectiveness .
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In general, digoxin is a drug used to treat cardiac disorders. It is a digitalis, i. It has a positive inotropic effect, i. Antidugoxina resulting increase of calcium into the cell causes an increase in the excitability of cardiomyocytes, causing an increase in cardiac contractility. Digoxin has a narrow therapeutic index, with therapeutic level very close to the toxic level.
To combat its toxic effect may be. In a more specific form, the cardiac glycoside digoxin is the most used in the treatment of congestive heart failure and atrial fibrillation Figure 1.
Bidirectional Ventricular Tachycardia due to Digitalis Poisoning
Research and experience with digoxin began over years Withering, and its application in patients with heart failure have been reported only in the 20th century, defining its ability to increase the contractility of the heart.
This membrane protein is responsible for sodium transport to the extracellular milieu and be inhibited, generates an increase snticuerpos intracellular sodium.
This stimulates increased by exchanging the sodium-calcium pump, increasing the concentration of intracellular calcium by contractile proteins used. It is an inexpensive drug, it is important in developing countries where patients do not have access to sophisticated therapies Gheorghiade et al.
The concentration of digoxin in the serum depends not only on the dose administered, but is also related to interactions with other drugs and the patient’s condition Antman, Smith, Digoxin is not antigenic because it is a small molecule InSmith et al. They documented high affinity and specificity of selected antibodies to haptens populations of cardiac glycosides. Fab fragments Fragment antigen binding antibodies specific for digoxin obtained in sheep demonstrated ability to revert digoxin poisoning Smith et al.
A study in dogs showed that intravenous administration of purified Fab portion of the anti -digoxina sheep polyclonal antibodies can quickly reverse the cardiotoxicity, connecting to the free digoxin in plasma and causing a redistribution of the drug from tissues back to blood circulation. Fab fragments are relatively fast excreted in urine, so Fab high affinity, which retain binding with the drug, can provide a route of drug elimination, as well as means to neutralize it Butler et al.
Therapies with potassium and beta-blockers have been used as agents to combat alternative to digitalis intoxication, but the administration of Fab fragments of antibodies is the main therapy to reverse the intoxication ANTMA et al.
The Fab fragment of polyclonal anti-digoxin antibody raised in sheep is being manufactured only by a company Protherics under the name digifab and received FDA approval in The use of Fab fragments in cases of poisoning by digoxin is safe and effective, but the neutralization with Fab fragments is an expensive therapy, is used only when other treatment options seem to fail. Its high cost also limits its availability to all hospitals or countries.
Namely, the antibodies are glycoproteins composed of two light chains LC, light chain identical approximately 24 kDa each and two heavy HC, heavy chain identical kDa each. A light chain is covalently linked to a heavy chain by a disulfide bond, while the two heavy chains are linked together by disulfide bridges. The carboxy terminal regions of the heavy chains perform effector functions.
The light and heavy chains have an amino-terminal variable region VL and VH va iable light, heavy variable which is involved in recognition of antigens. Among these regions are present more conserved framework calls sequences or regions framework: IgG, with molecular weight kDa, is the most abundant in mammalian serum.
The IgGl is most commonly found in human serum Abbas Lichtman, and has well-defined structure with two disulfide bonds linking the heavy chains in the hinge region between CHI and CH2. InKohler and Milstein monoclonal antibodies developed production technology Acmoswhich gave them the Nobel Prize in Physiology and Medicine in Many murine monoclonal antibodies have been produced for treatment or diagnosis of human diseases.
However, its use is limited by having short half-life in serum and due to its high immunogenicity, and may cause a reaction known as human anti-mouse antibody HAMA. Antibody fragments are more advantageous for certain clinical applications that do not rely on whole antibody. Maintain the antigen-binding sites, are less immunogenic than q and whole IgG, the best diffuse interstitial space, is easily eliminated through glomerular filtration and stocks are more stable than IgG Flanagan, Jones, May be obtained by digestion of immunoglobulin with pepsin enzyme that produces two Fab fragments linked by dissulteto bridge, F ab ‘ 2 divalent weight of approximately kDa and fragmented Fc portion or by the enzyme papain to produce two Fab fragments monovalent, having molecular weight of 50 kDa and the entire Fc portion Figure 2.
The anti-digoxin Fab fragments commercially available were obtained by using IgG digestion the enzyme papain. Antibody fragments may also be obtained by expression in Escherichia coli, offering the advantage of obtaining in large quantities at low cost.
Recombinant DNA technology also offers the opportunity to introduce mutations in the gene sequences that can be advantageous.
Phage display technology includes a method widely used in the generation of antibody fragments. It emerged in when George Smith demonstrated that the correlation between genotype and phenotype could be established on bacteriophage phage filamentous Figure 3.
Exogenous peptides exposed on the surface of phage could be selected by affinity for the specific antibody, allowing them to be enriched compared to the original peptide, usually by a process called panning.
InHuse et al reported the generation of a Fab fragment library in lambda phage from the random combination of heavy and light chains of the murine antibody as a method that could replace hybridoma technology.
CARDIOTONICOS by Noemi Martinez on Prezi
The technology allows selection of a phage clone displaying high affinity and specificity antibody fragments for a particular antigen within a phage library constructed from rearrangement of the variable domains Clackson et al. Construction of the antibody phage display technology began with the construction of a library of immunoglobulins which can be generated from immunized animals, immunized or non-synthetic library STRACHAN et al.
The filamentous phage of the Ff class fl, fd and M13 have 11 genes I – XI and of these five encoding capsid proteins that may anidigoxina fused to the recombinant proteins of interest with greater or lesser degrees of success, with pIII the anticuerpis used for phage display. Proteins may also be exposed to phage small particles called phagemids, which are plasmids that carry the recombinant capsid protein gene and contains origin of replication of phage M13 Barbas et al.
The capsid fusion protein is exposed on the phagemid particles containing the helper phage genomes. These particles containing the two genomes may be selected by selection markers Barbas et al.
The helper phage has a defective origin of replication and its genome is packed inefficiently compared to phagemid Barbas et al.
After selection and characterization of monoclonal antibody, the phage display technology allows the manipulation of the antibody genes by the generation of mutants.
Phage display is a handy technology in molecular biology laboratories. It is not, however, commonplace technology and many details need to be worked to arrive at a good result. Due to the advantages of the phage display technology and the importance of anti -digoxina antibodies in cases of poisoning and commercial production is antidiogxina and expensive, applicants believe in the need for the development and production of this drug in Brazil.
A brief survey to databases of patents on the subject “ant i monoclonal -digoxina” resulted in some documents, such as the North American US – The General Hospital Co. DNA genome ; source: It is engineered to digoxin monoclonal human antibody chain used to detect the ajtidigoxina of digoxin concentration in patient serum and detecting the resulting anticuwrpos effect antagonist against digoxin patient poisoned by digoxin. The present patent of invention relates, in a broader view, a production method and obtaining the antibody anti-digoxin Fab clones using phage display technology and characterization of their binding to antigen.
More specifically, the invention has the following objectives:. Were used, materials and methods developed specially for obtaining clones of anti-digoxin antibody Fab fragment anticudrpos the technology of ‘phage display’.
Unlike commercial products imported. Thus, the invention is dedicated, in particular, the development of therapeutic product for use with specific power and more precise antodigoxina to detoxification of patients receiving digoxin.
The complement the present description in order to obtain a better understanding of the characteristics of the present invention and according to a preferred practical embodiment thereof, accompanying description, in the annex, a set of drawings where, exemplified way, although not limiting, it represented its operation:. Figure 2 – Structure of the IgG molecule and fragments thereof which retains antigen-binding sites; immunoglobulin fragments presented: Figure 3 – Schematic representation of the filamentous bacteriophage Ff class and its capsid proteins; presented the capsid proteins annticuerpos be used for expression of recombinant proteins in the form of fusion protein by phage display technology.
Figure 5 – Schematic representation of the main stages of the display and panning phage; the Fab library was transformed into E. Phages not bound were discarded by washing and bound phages were eluted which i and used to re-infect E.
The secondary antibody used was anti-mouse-IgG conjugated with peroxidase. Figure 9 – Products of amplification of the cDNA using oligonucleotides antidigpxina -digoxina anti murine immunoglobulin, where the amplifications were made by PCR and analyzed by electrophoresis on 1.
B Products of amplification of genes using oligonucleotide LC Mok 3 ‘and the six oligonucleotides 5’. Antidogoxina 15 – a symbolic representation of the amino acid sequences deduced from clones of CL 1, 2, 9 and 10, after phage display selected; amino acid residues have been replaced by the symbols and CDR2 regions FR1 to highlight the different sequences.
The anticuer;os regions have identical sequences to each other. Figure 16 – electrophoretic profile of clones 1, 2.
Figure 17 – Western blotting of crude extracts containing Fab fragments of anti -digoxina anticurrpos 1, 2, 9 and. After electrophoresis, the gel was transferred to PVDF membrane. The membrane was incubated with anti-mouse IgG antibody specific for F anntidigoxina ‘ 2 conjugated to peroxidase and detected with the ECL system.
After electrophoresis the gel was stained with silver. Four gels with the same samples used in A were transferred to PVDF membranes and each membrane was incubated with crude extracts of E.
Then the membranes were incubated with anti-mouse IgG antibody specific for F ab ‘ 2 conjugated to peroxidase and developed with ECL system. Figure 19 – Profile of the binding of anti-digoxin Fab expressed in E.
Fab present in crude extracts of E. The secondary antibody used was anti-mouse IgG specific for F ab ‘ 2 conjugated to peroxidase; and. Crude extracts of clones containing Fab expressed in E.
The relative response anticuegpos measured 5 seconds, after the completion of antidigoxima application of anticuerpis analyte, antidigoxinaa the point indicated by the red arrow clone Step 2 – Obtaining monoclonal antibody anti -digoxina; Step 2. Step 3 – Construction of Fab library in phagemid antciuerpos from anti -digoxina hybridomas.
Step 5 – Expression of soluble Fab fragments; Step 5. Step 6 – Characterization of crude extracts containing Fab fragments from the clones obtained by phage display. To better define each step and their respective phase, is described below the method in its comprehensive feature, which can be accompanied by the already related illustrations:.
The protocol was based on the technique described by Erlanger and Beiser After 30 minutes of interaction, 60 ul of 1 M glycerol was added to inactivate excess periodate and the solution was stirred for 5 minutes. The periodate oxidized digoxin the solution was added to the BSA solution slowly and dropwise, keeping the pH between 9. The solution was continued stirring for 1 hour at room temperature, keeping the pH stable for 30 minutes in the range 9.
Was added 6 mg of NaBH 4 dissolved in 2 mL deionized water prepared immediately. To estimate the number of molecules of digoxin conjugated to BSA was used as a reference absorbance at a wavelength of nm from a known concentration of digoxin solution, in duplicate. The reference and sample Dig-BSA were completed with water up to 2 mL, then 10 mL of H2S04 were added and the tubes were kept on standing at room temperature for 4 hours.
Read the absorbance was taken and calculated the number of moles of digoxin present in Dig-BSA conjugate.
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The estimated number of molecules present in the digoxin conjugate was obtained from the ratio between the number anticuer;os Dig-BSA digoxin mole and number of moles BSA added to obtain the conjugate.
The viable cell count was done with the aid of the dye Trypan blue exclusion and a Neubauer chamber. After loading, passed by the column equilibration buffer to remove any non-specific binding and rebalance the spine.