1. RADIOACTIVITY is the property of an unstable isotope throwing out or emitting energetic particles and rays from its nucleus. The amount of radioactive material is measured by how many nuclei decay each second. This is called the activity and is measured in units of curies, abbreviated Ci. One microcurie is 1,000,000^th of a Ci and is equal to 37,000 disintegrations per second (DPS's) or 2,220,000 disintegrations per minute (DPM's). The I¹²⁵ used as radioactive labeled antigen in this lab emits gamma rays (photons). The solid sodium iodide crystals in the gamma counters are scintillators, which give off a flash of yellow light when they absorb gamma radiation. A photomultiplier, which is optically coupled to the sodium iodide crystal, produces a pulse every time it "sees" a scintillation. The pulse is then amplified and measured to see if it is of sufficient intensity and of a certain discrete energy level characteristic of the isotope selected to be measured. These measurements from the gamma counter are called counts per minute (CPM's). All three gamma counters in this lab are about 90% efficient, -that is to say, they will "see" and count 90% of the actual DPM's exposed to the sodium iodide crystal.
H³ is also used as radioactive labeled antigen tracer in our lab. It emits a very low energy beta particle. This particle only travels about 1/2 centimeter in air. So, we count these low-energy beta particles using a liquid scintillation counter. The H³ sample is suspended in a cocktail which acts similarly to the sodium iodine crystal. Beta energy is absorbed in the cocktail and produces flashes of blue light (approximately 1 - 3 flashes per keV for H³). This blue light is detected and measured by photomultiplier tubes in a similar manner, and the pulse height represents the energy of the beta.

2. TOTAL COUNT TUBES are tubes that represent the total amount of radioactivity added in an RIA tube. These tubes are not decanted in the separation step. They represent the total amount of tracer aliquoted per tube. When the assay is counted, these tubes will have the highest CPM's. These counts are not used as part of the dose estimate calculation for unknowns, but rather as a quality control comparison to the counts in the 100% tubes. Because the amount of antibody is limiting and tracer is in excess, total count tubes are included to guarantee and document this excess. The degree to excess is expressed as a percent of CPMs in the 100% tubes divided by the CPMs in the total count tubes, often referred to as the % B/T or Bound/Total.

3. NON-SPECIFIC BINDING TUBES (NSB's), N, BACKGROUNDS are tubes that contain labeled antigen, sometimes assay buffer, zero standard or 2nd antibody, but they never have any 1st Antibody. When the assay is counted, these tubes will have the lowest CPM's in a radioimmunoassay system. These counts are considered to be error or background counts and our calculating program subtracts them from the counts of all the other tubes to obtain a more accurate estimate of counts in the bound fraction.

4. B_o, 100%, or MAXIMUM BINDING TUBES are tubes that contain labeled antigen, 1st antibody, may contain assay buffer and 2nd Antibody, but do not have any unlabeled antigen such as unknown samples or standards (except zero standard). After separating the free from the bound fraction, these tubes will have the highest CPM's, other than the total count tubes.

5. STANDARDS, REFERENCE PREPARATION, KNOWNS, DOSE or CALIBRATORS are assay tubes that contain a known amount of the compound that you want to measure. Most of our assay calculations count the bound fraction, and therefore, those tubes with increasing amount of standard have decreasing CPM's. It is by comparison to these tubes that the amount of compound in the unknown sample is determined. Standard units can be nanograms, picograms, international units, or microliters of some in-house reference preparation solution. Radioimmunoassay standard curves typically contain five, six or seven different amounts or doses within an assay, and each amount is run in duplicate or triplicate.

6. INTERASSAY POOLS, IAPs, POOLS, CONTROL SAMPLES, QCS, QUALITY CONTROLS, or CHECKS are samples taken from a large pool, aliquots of which are assayed as unknowns in each assay done. When possible, the pools should be of identical composition to the material that is being assayed, and from the same species and medium. IAPs are used to validate an assay and to measure variation within and between assays. The number of interassay pools required in an assay can be determined with our QC-Validator® software program, which takes into consideration the total allowable error acceptable in the assay and the stable analytical error and imprecision observed from method evaluation tests.

7. UNKNOWNS are assay tubes containing the material in which the compound you wish to measure is contained. Some types of unknown samples that have been assayed in this lab are blood serum & plasma, urine, fecal extracts, saliva and cerebrospinal fluid. We routinely run unknown samples in duplicate whenever sufficient sample amount is available.

8. The BOUND FRACTION of an assay tube is that part of the labeled and unlabeled antigen immunologically bound to the 1st antibody. This is the fraction that we usually count in the gamma counter after the incubation period. The FREE FRACTION is that part of the labeled and unlabeled antigen that has not bound to the 1st antibody. After incubation, it is usually the fraction that is discarded and not counted.

9. LABEL, TRACE, TRACER, HOT, or LABELED ANTIGEN is the name given to the radioactive compound which competes with the unlabeled antigen (standards & unknowns) for binding sites on the 1st antibody. We use both I¹²⁵ and H³ isotopes as labeled antigen tracers.

10. 1st ANTIBODY is an antibody used in a radioimmunoassay system which will bind to the biological compound you want to measure. It will also bind the labeled trace and the standards. 1st antibody strength or concentration is most often expressed as a ratio between 1 mL of whole undiluted serum and milliliters of assay buffer. For example, if 1 mL of whole 1st anti-serum were added to 99 mL of an assay buffer, the antibody concentration would then be 1:100. The storage concentration is the concentration at which the 1st antibody is kept in the freezer. Aliquots are made so that one aliquot taken from the freezer is of sufficient strength when diluted, to do one assay. Repeated freezing and thawing of 1st antibody reduces the 1st antibody's immuno-potency. The pipetting or working concentration of 1st antibody is the concentration that is actually pipetted into an assay tube in a radioimmunoassay. When the 1st antibody is in the assay tube with the buffer, sample or standard, EDTA, and trace, and before the 2nd Antibody has been added, the 1st antibody is said to be at its incubating or final concentration. For example, if the 1st antibody is stored at a concentration of 1:400 in 450 µL aliquots, this is the storage concentration. When this 450µL is thawed and diluted up to 67.5 mL with assay buffer, it is then at its pipetting or working concentration of 1:60,000. When 200 µL of this 1:60,000 1st antibody is added to an assay tube with 400 µL of assay buffer, 100 µL of EDTA, 100 µL of sample, and 200 microliters of trace, the 1st antibody is then said to be at its final or incubating concentration of 1:300,000.

11. 2nd ANTIBODY is an antibody added at the end of a double antibody type radioimmunoassay which is directed at the immuno-globulin contained in the 1st antibody. It binds to the 1st antibody/antigen complex (bound fraction) and after centrifugation, is precipitated to the bottom of the assay tube. After the free liquid fraction, or supernatant, of the assay tube is decanted and thrown away, the assay tube and the precipitate or pellet are counted in the gamma counter. 2nd Antibody is generally pipetted in microliters of whole serum, but in some assays, it may be used in diluted forms (expressed as a ratio between 1 mL of whole 2nd Antibody serum and milliliters of assay buffer). A PRECIPITATING AGENT, added at the end of a double antibody type RIA, also precipitates the antibody/antigen complex in the assay tube, but does this in a chemical way. It does not bind immunologically to the 1st antibody/antigen complex. The most common precipitating agent used in RIA's is polyethylene glycol (PEG). Some 2nd antibody used may be a mixture of the 2nd antibody and PEG.

12. A 1st ANTIBODY TITER is a preliminary radioimmunoassay test done to determine what concentration of 1st antibody in a particular RIA system should be used. The amounts of trace and 2nd Antibody remain constant between groups of NSB's and 100% tubes, and only the concentration of 1st antibody in the 100% tubes varies. Typically, we use three totals, three NSB's and three 100%'s for each concentration of 1st antibody tested. The concentration selected depends on the sensitivity desired in the assay. The less 1st antibody used, the lower the standard curve doses can be reliably used.
See 1st Ab Titer Graph

13. A 2nd ANTIBODY TITER is a preliminary radioimmunoassay test performed to determine what volume or concentration of 2nd Antibody maximally precipitates the bound fraction of the 1st Antibody/antigen complex. In this test, the amount of trace and 1st antibody remain constant while only the 2nd Antibody amount or concentration varies. We typically use three total count tubes, and for each different amount of 2nd Antibody, three NSBs and three 100% tubes.
See 2nd Ab Titer Graph

14. INTERASSAY DRIFT or END OF ASSAY VARIATION is a condition in a radioimmunoassay system where tubes with identical amounts of reagents (that is sample, 1st antibody, Trace, and 2nd Antibody) are not calculated to have identical amounts of compound that you are trying to measure. This generally is a result of the time difference between the first and the last tubes in an assay. Assays with antibody coated tubes or assays using PEG (polyethylene glycol) as an assisting precipitating agent are sources for this type of error. Duplicate sample pools should be placed at the beginning and end of an assay to determine within a particular assay system if interassay drift is a problem. It may be necessary to limit the number of tubes in one assay to say, one centrifuge spin to minimize this problem.

15. SPECIFIC ACTIVITY is a measurement of efficiency in labeling antigen for trace. It reflects how much iodine-125 is incorporated into the antigen during the radioiodination procedure. Generally, the more iodine-125 incorporated the better, although over iodinated antigen may reduce the labeled antigen's ability to bind with the primary antibody and reduce the time that the tracer is stable. Variables such as time and reagent concentration during radioiodination are adjusted to obtain maximum specific activity and tracer stability. Specific activity is usually expressed in units of microcuries of I¹²⁵/micrograms hormone labeled.

16. CROSS-REACTION is the binding or interference in the binding of the 1st antibody by some agent other than the compound chosen to be measured. These interfering agents could be in the buffer, trace, 2nd Antibody or the sample itself. Specification sheets accompanying antibody shipments usually supply known cross-reactivity information in table form for other materials typically present in the measured sample material that may cross-react with the 1st antibody.

17. PARALLELISM tests are performed to help validate a new assay system or new sample material. A serial dilution (varying volumes) of a single pooled sample are compared to the varying standard curve doses within the same assay. Lines of equal slope demonstrate that there is no significant proportional analytical error within those ranges. This allows investigators to assay their unknown samples at various sample volumes with the confidence that the dose estimate per/mL will not have significant error. This is particularly important when unknown samples are run together in the same assay at different volumes. Samples anticipated with higher concentrations of hormone can be assayed at lower sample volumes and those unknown samples anticipated to have lower concentration of hormone can be assayed at higher sample volume.
See Parallelism Graph

18. ACCURACY tests are also performed to further validate a new assay system or new sample material. In this test, we add (spike) a fixed sample volume to varying standard curve doses. The assay also includes an un-spiked standard curve and the fixed sample volume run by itself as regular unknown tubes. The mathematical sum of the known standard curve doses plus the dose estimate of the fixed unknown sample is the "expected" value which is compared to what the assay calculates the spiked combination "observed" to be. If the mean percent of expected (recovery) is close to 100, that particular sample material is shown to exhibit little effect on the assay system's ability do estimate the hormone in question. Separate accuracy and parallelism tests are done for each medium assayed, even if the various media come from the same species. It is not safe to assume for example, that measuring luteinizing hormone in urine from a particular species is valid because parallelism and accuracy test previously run on that same specie's serum were valid. See Accuracy spreadsheet.

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