...Antibodies and relatedness Producing anti-cow antibodies COW SERUM ALBUMIN RABBIT ANTI-COW ALBUMIN ANTISERUM! (“COW ANTISERUM”) CONTAINS RABBIT ANTI-COW ALBUMIN ANTISERUM ANTI-COW ALBUMIN ANTIBODIES If we were to create anti-human albumin antibodies, why would they interact with serum albumin from animals other than humans? ANTI-HUMAN ALBUMIN ANTIBODIES mixed with serum albumin from.. HUMAN! CHIMP! GORILLA! BABOON STRONG! reaction HORSE! OX! DEER WEAK! reaction CHICKEN! FROG NO! reaction closely related to distantly related to somewhat related to WESTERN BLOT An immunodiagnostic procedure used to detect (with extremely high specificity) particular proteins in a mixed solution, extract, or homogenate. Native proteins are separated via gel electrophoresis, then transferred to a membrane where they are stained and hybridized with specific conjugated antibodies. Also called PROTEIN IMMUNOBLOT procedure. General Procedure 1. Electrophoresis of sample. 2. Preparation of membrane/blotting of gel. 3. Staining of nitrocellulose blot. 4. Primar y antibody reaction. 5. Color development. Preparation of Nitrocelluose Membrane Nitrocellulose membrane Separated Proteins Gelatin Primary antibody Secondary antibody (Color Development Solution) HRP catalyzes a conformational change in chloronaphthol PHLOEM (e.g. chloronaphthol; TMB) Fluorescent: secondary antibody conjugated with a light-emitting fluorochrome...
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...1.0 1.0 1.0 0.02 -- -- -- 0.02 -- -- -- 0.02 Mix well and incubate at 37oC for 5 min. or at R.T. (25oC) for 15 min. Measure the absorbance of the standard (Abs.S), and Test Sample (Abs.T) against the Blank, within 30 min. Calculations Uric Acid in mg/dl = Abs.T X 8 Abs.S 2. MICROALBUMINURIA Albumin refers generally to any protein that is water soluble, which is moderately soluble in concentrated salt solutions, and experiences heat coagulation (protein denaturation). They are unique...
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...The specificity of albumin binding experiment was to determine the binding interactions that occur between serum albumin and three synthetic dyes with the use of electrophoretic procedure. Whole blood, or plasma. Clots upon standing and if the clot is removed, the remaining straw colored fluid is called serum. The major protein in serum is albumin which functions as a carrier molecule for the transport of certain small molecular weight compounds in blood. Molecules that bind to serum albumin are fatty acids, hormones and some synthetic dyes. In this experiment the synthetic dyes used are Bromophenol Blue, Ponceau S and Orange G. we observed that free dyes not bound to albumin migrate faster that albumin or dyes bound to albumin. This separation enabled use to differentiate between albumin-bound dye and free dye not associated with the protein. Our results were compared to Anderson to see where we failed. Results Group 4 This group shows the ligand binding is protein specific. We see that serum albumin binds bromophenol blue while hemoglobin doesn’t. our results corresponds very close to Anderson’s as the mixture with hemoglobin shows no binding occurred, the hemoglobin and the dye are very separated. The mixture with albumin showed binding with the dye. While the others were left as free dye. Tube/lane # | Buffer | Protein | Bromophenol blue | 1 | 20ul | 0 | 5ul | 2 | 10ul | 10ul hemoglobin | 5ul | 3 | 20ul | 0 | 5ul | 4 | 10ul | 10ul BSA | 5ul | Fig4 . the...
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...The human body works best when the levels of fluid and electrolytes are within a normal range. When a patient with a serious liver disease is presented with health problems, the health care providers must understand every aspect of the problems in order to provide the appropriate care. This includes understanding the pathophysiology, assessing the patient correctly, and reaching the goals of the plan. By following these steps, the patient will be provided the care he or she is entitled to. Obvious ascites with 3+ edema Ascites is the collection of fluid within the peritoneal cavity due to increased hydrostatic pressure from portal hypertension. Cirrhosis is the most common cause of ascites whereas ascites is the most common complication...
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...TOPIC: ANTIBODY DETECTION AND IDENTIFICATION TEST FROM A LEUKAEMIA PATIENT’S SAMPLE INTRODUCTION In today’s world where science are ruled by the ever changing clinical laboratory, few mishaps and problems are still at arising which requires deep understandings and solutions to overcome the problems. Some of the problems do required immediate attention and some are just occurring problems which eventually a solution must be provided. However no problems can be more severe than problems and issues arising from a specific department which is one of the most important sections in a clinical laboratory. That department is called the transfusion service department. At times, nothing is more confounding to a laboratory scientist then the solutions to antibody detection and identification problems. Errors from this antibody detection and identification should be resolved and dealt in a organized and logic mannered and immediate effect should be implemented as fast growing of elderly and oncology population receiving supportive transfusions are increasing as time develops. Some antibodies issues can take up to hours for a solutions to be implemented, and when this occurs, pretransfusion testing will be delay and this will definitely caused many severe damages including costly hospital extensions. Antibody detection and identification testing is one of the most complicated testing ever conducted in a clinical laboratory. This is such as many areas of error...
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...TITLE: FOLATE (CH205) I. PRINCIPLE: Principles of the Procedure The Access Folate assay is a competitive binding receptor assay. For the assay of folate in serum or plasma (heparin), no pre-treatment is required. A serum, plasma (heparin) sample is treated to release folate from endogenous binding proteins. Folate binding protein, mouse anti-folate binding protein, folic acid-alkaline phosphatase conjugate, and goat anti-mouse capture antibody coupled to paramagnetic particles are added to the reaction vessel. Folate in the sample competes with the folic acid-alkaline phosphatase conjugate for binding sites on a limited amount of folate binding protein. Resulting complexes bind to the solid phase via mouse anti-folate binding protein. After incubation in a reaction vessel, materials bound to the solid phase are held in a magnetic field while unbound materials are washed away. Then, the chemiluminescent substrate Lumi-Phos* 530 is added to the vessel and light generated by the reaction is measured with a luminometer. The light production is inversely proportional to the concentration of folate in the sample. The amount of analyte in the sample is determined from a stored, multi-point calibration curve. Summary and Explanation Folate is an essential vitamin vital to normal cell growth and DNA synthesis. It is present in a wide variety of foods such as dark, leafy vegetables, citrus fruits, yeast, beans, eggs, and milk. It is absorbed...
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...They were intraperitoneally injected with anesthesia and heparin. A weight was taken on the fetuses, oxygen was immediately delivered to the lungs via an ET tube and CPAP machine, and their body temperature was maintained. Then the fetuses were then disconnected from the CPAP machine to deflate the lungs by aspirating them with 5% bovine serum albumin. This was done slowly and repeated to ensure that the lung fluid and solution was well mixed. The CPAP was reconnected and a sample of the fluid mixture was taken after one hour for protein analysis. Because in normal fetal lungs, the chloride levels in the fetal fluid is relatively high and the protein content is low, it was assumed that the fetuses’ epithelium would be impermeable to large protein molecules like albumin. This knowledge allowed them to determine how much fluid was either secreted or absorbed from the volume and concentration of the serum added and the initial concertation of the lung fluid. It’s a basic dilution factor calculation. The same procedure was also carried out with another group of guinea pig fetuses that were treated with MβCD instead of IL-1β. MβCD has been previously known to restore pulmonary...
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...whole human body. It carries many nutrients from the digestive tract to the cells, oxygen from the lungs to the cells, waste products from the cells to various organs of excretion, and hormones from secreting cells to other parts of the body. It helps in the distribution of heat formed in the active tissues to all parts of the body. Blood also helps to regulate the acid base balance and to protect against infection. Therefore blood is a crucial fluid for our life and health, the average adult’s blood has 8 to 10 pints of blood. Loss of more than 2 pints at any one time leads to a serious condition. Blood is made up of plasma, which is the liquid portion of blood without its cellular elements; serum is the name given to plasma after a blood clot is formed. Blood also has pathological rudiments that include “erythrocytes”, “leukocytes” and “thrombocytes”. Blood disorders can harm any of these three main compositions of blood. Treatments and antibiotics for blood diseases and disorders can vary it all depend on the blood condition that occurs and its impact on the individual’s body. Plasma is described as being a chaff colored, dense fluid, comprising about 55% of the blood amount which contains six substances which are. 1st Water is the liquid that makes up about 92% of the total amount of plasma. 2nd Plasma proteins are three proteins most abundant found in plasma they are fibrinogen, serum albumin and serum globulin. Fibrinogen is vital for blood clotting, albumin is the...
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...|AGGLUTINATION | Agglutination: • Is the clumping of antibody–antigen complex to form insoluble and visible aggregates. The word agglutination comes from the Latin agglutinare, meaning "to glue to." Antigen: Antigen is any molecule that binds specifically to an antibody. Antibody: Antibodies (immunoglobulins) are proteins secreted by the immune system to identify and neutralize foreign objects (antigens), such as bacteria and viruses. |Factors affecting the agglutination reaction in vitro: | 1) Antigen to antibody ratio: • The ratio between antigen and antibody influences the detection of antigen-antibody complexes. • Antigen or antibody excess make invisible reaction. Prozone phenomenon (antibody excess): • There are too many antibodies. • Antibodies saturating all antigen sites • No antibodies forming cross-linkages between cells or particles so, no agglutination appears (false-negative reactions). [pic] Zone of equivalence: • Antibodies and antigens are present in an optimum ratio. • This leads to cross-linkages between acells or particles, so agglutination appers (positive reaction). [pic] Post-zone phenomenon (Antigen excess): • There are too many antigens • Any agglutination is hidden by masses of unagglutinated antigens that gives false-negative reactions. [pic] | ...
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...Trade name: Jadenu Generic name: Deferasirox Application number (NDA): 206910 Approval date: March 30, 2015 Jadenu is a new drug which is approved as an iron chelator and used for the treatment of chronic iron overload due to the transfusion of blood. This drug is approved for reducing the iron concentration in liver and serum ferritin levels. It is used for chronic excessive iron in liver in the patients who are 10 years and older and suffering from Non Transfusion Dependent Thalassemia syndrome in which iron and serum ferritin gets overloaded in the liver and can cause toxicity. It is also used in patient suffering from sickle cell diseases. Approval Details: This drug is manufactured by Novartis Pharmaceutical Cooperation (Oncology Department). The New Drug Application was submitted under section 505b of Federal Food, Drug and Cosmetic Act on May 30th, 2014. FDA approved the product under accelerated approval regulations 21 CFR314.510 and requested further clinical trials and well controlled studies to explain the clinical benefit of this drug. FDA also requested to submit the post marketing requirements under section 505 (o) and the promotional material. Dosage and Administration: Initial dose is 14 mg/kg for transfusion iron overdose and...
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... Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC 4.2.1.1, from human and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with ligands. It thereby provides a rapid, sensitive...
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...The graph above shows the absorbance of the blue protein (Bovine Serum albumin) and the concentration of vitamin B12 against the number of fractions. During gel filtration chromatography, the blue protein – Bovine Serum Albumin (BSA) comes through the media first as the (BSA) protein has larger particles. Whereas, vitamin b12 are smaller proteins and if the particles are made smaller, conductance (or flow) through the column becomes more difficult which is why vitamin B12 takes a while to come through as its tiny particles are unable to come all the way through the media. The peaks show the increasing absorbance rate which later declines during the end. Q4. Below is the structure of Vitamin B12 and the structure of Coomassie Blue. Vitamin B12 Coomassie Blue Vitamin B12 is a generic name for a group of compounds called corrinoids. The structure of Vitamin B12 contains...
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...The test used was Enzyme-linked immunosorbent assay (ELISA) which is a test that uses antibodies to see a change in color to identify an antigen. Fresh venous blood samples were drawn into pyogen-free blood collection tubes without additives, immersed in ice, and allowed to clot before centrifugation. All serum samples were stored at -70°C. Serum PAF-AH levels were measured with ELISA kits. The detection limit of this assay was 0.074 ng/ml.2 The results for this study were that Serum PAF-AH levels were significantly higher in SSc patients (130.4± 69.5 ng/ml) than in healthy individuals (81.6 ± 34.8 ng/ml; P < 0.05 ;). Concerning SSc subgroups, PAF-AH levels in both patients with (diffuse) dSSc (135.5 ± 79.3 ng/ml) and those with (limited) lSSc (125.1 ± 58.6 ng/ml) patients were also increased compared with those in healthy individuals (P < 0.05 for...
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...B5: Sex Prediction and Selection SEX PREDICTION Sex prediction also termed as “prenatal sex discernment”. It is the prenatal testing for discerning the sex of a fetus before birth. A sex prediction test is a test done on or by pregnant women to determine the sex of babies. Methods: * Maternal Blood Sample to analyze the small amount of fetal DNA that can be found within, it provides the earliest post-implantation test. * Chorionic Villus Sampling (CVS) and Amniocentesis are two rather invasive testing procedures. These may, in principle, be formed as early as the 8th and 9th week of pregnancy. The difficulty of these tests and the risk of damage to the fetus, potentially resulting in miscarriage or congenital abnormalities (especially when done early during the pregnancy), make them quite rare during the first trimester. Amniocentesis (also referred to as amniotic fluid test or AFT) is a medical procedure used in prenatal diagnosis of chromosomal abnormalities and fetal infections, in which a small amount of amniotic fluid, which contains fetal tissues, is sampled from the amnion or amniotic sac surrounding a developing fetus, and the fetal DNA is examined for genetic abnormalities. This process can be used for prenatal sex discernment and hence this procedure has legal restrictions in some countries. * Obstetric Ultrasonography, either transvaginally or transabdominally, can check for the sagittal sign as a marker of fetal sex. It can be performed between...
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... & platlets. HICPAC Hospital Infection Control Practices Advisory Committee What can prevent HICPAC Standard precautions, infection control, proper documentation, Common disinfectants - Phenolics, Halogens, Aldehydes Quaternary Amines If there is a fire or electrical shock, chemical spill what is needed? Ventral Relating to or situated on or close to the anterior aspect of the human body What combines with 02 in blood Hemoglobin Serum - The component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed. Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances (e.g., drugs and microorganisms). Plasma - The straw-colored/pale-yellow liquid component of blood that normally holds the blood cells in whole blood in suspension. It makes up about 55% of total blood volume. It is mostly water (93% by volume) and contains dissolved proteins (major proteins are fibrinogens, globulins and albumins), glucose, clotting factors, mineral ions (Na+, Ca++, Mg++, HCO3- Cl- etc.), hormones and carbon dioxide. Plays a vital role in intravascular osmotic effect that keeps electrolyte in balance form and protects the body from infection and other blood disorders. Cholesterol causes Diet, foods such as meats, eggs, cheese. Physical activity Heredity / Age and sex Depth of a puncture in a newborn...
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