Transcript radio immunoassay

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