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RADIO IMMUNOASSAY DANIEL CASAL WHAT IS RADIOIMMUNOASSAY • Sensitive method that measures small amounts of substances in the blood • Able to measure drugs, antigens and hormones • Any biological substance where an antibody exists can be measured • Uses include drug detection, blood bank screening for certain viruses and cancers (such as HIV and Hepatitis B) • Immunoassays produces signals in response to binding • Able to be detected by many ways such as it showing a color change, fluoresces under light, or emitting radiation. RADIOIMMUNOASSAY RELEVANCE • Most common method to test for drugs of abuse • Screens presence of drugs in different areas such as forensic toxicology and clinical toxicology • (Dated back from 1970 when soldiers were coming back from the Korean war) • Take a urine or blood sample since its easy to collect and obtain in large quantity • Radioimmunoassay technique isolated Diphenyldantoin from semen, blood stains saliva and dried saliva stains. RADIOIMMUNOASSAY RELEVANCE • Testing Opiates for morphine content is mostly performed in forensics urine drug test laboratories • Doses of hydromorphine, hydrocodone, oxymorphone and oxycodone were administered to individuals. Urine sample were collected before and after the drugs were administered. • The radioimmunoassay testing indicated that 3 of the 4 drugs were detectable by urine • (Low to moderate concentrations of these drugs will go undetected when testing) METHOD • First obtain a substance that contains the antigen (containing antiserum) • Introduce antigen with radioactive chemicals and then incubate it at +4C • (Isotopes used are H3, I 125, I 131, C14 because they emit gamma rays). • Most common is I 125 or I 131 (easier for Iodine atoms to be introduced in tyrosine residue) • Mix the antibodies and the radioactive antigen • Both substances bind with each other and end up forming a new substance • Add unknown substance (Also known as “cold”) • This causes the antigens in the new/unknown substance to bind with the antibodies METHOD • The unknown substance displace the isotope substance that was originally joined together with the antigen. • If there is in increase in the cold antigen, there will be an increase in the radioactive antigen being displaced from the antibody molecule • Able to measure radioactivity and a standard biding curve is shown • Separate the bound from free antigen by precipitating the antigen antibody CHEMISTRY IN RADIOIMMUNOASSAY • Very useful in quantitative analysis of drugs. • Antibodies reacting with specific elements to get a measurement • Detection limit depends if the solution was in a liquid phase, or in a solid phase SAFETY • Always wear goggles, apron, and gloves at all times • Be careful when handling the radioactive isotopes • Stay away from the gamma rays being emitted during the radioimmunoassay process • Dispose of the hazardous material accordingly PROBLEMS/DIFFICULTIES • If your test tube isn’t cleaned then the sample will be contaminated • Plug in wrong data • Properties of specimens difficult to detect • Interference • Malfunction on the instrument used to count the radioactivity • Radioactive isotope • Antibodies and antigens aren’t pure LIMITATIONS • Really sensitive (smallest concentration of a substance that can be measured reliably • Specificity depends on the antigen and the antibody and the isotope • Contaminants affect both the precision and the accuracy of quantitative data • Detection limits depend on the standard curves of the sample and it depends of the substance • Advantages • Really sensitive and precise • Able to detect small trace amounts • Require small concentration of antigens • Large capacity • Disadvantages • Usage of radioactive chemicals are extremely dangerous • Only special trained individuals are allowed to perform the lab • Require lots of arrangements and time to store/dispose radioactive material • Expensive equipment • Require pure antibodies and antigens • Takes a lot of time BIBLIOGRAPHY • http://www.txdps.state.tx.us/CrimeLaboratory/ • http://www.randoxtoxicology.com/immunoassay-drug-testing • http://www.ncbi.nlm.nih.gov/pubmed/7536861 • http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/Radioimmunoassay.html • http://www.slideshare.net/chakravarthyrapolu/radio-immuno-assay • http://www.pharmatutor.org/articles/radiommunoassay-drugs-hormones?page=0,3 • http://www.antibodies-online.com/resources/17/1215/Radioimmunoassay+RIA/ • http://www.slideshare.net/banuman35/analytical-method-development-bypravisankar • http://europepmc.org/abstract/MED/709872