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-by-pravisankar

• http://europepmc.org/abstract/MED/709872