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  1. 1. RADIOIMMUNOASSAY LABORATORY:COMPETENCY ASSESSMENT 2008DIVISION OF CHEMICAL PATHOLOGYGROOTE SCHUUR HOSPITALC17 NHLSAuthor: David HaarburgerSupervisor: Judy KingFebruary 2008This document is intended for the use of medical technologists, registrars and scientists, andforms part of the training required when using the RIA lab.
  2. 2. RADIOIMMUNOASSAY LABORATORY: COMPETENCY ASSESSMENT 2008CHEMICAL PATHOLOGY C17 NHLS1. Radiation Safety/Radiation Protection Training CourseSee SOP 12. RIA Spillage MonitoringSee SOP 23. RIA Gamma Counter Operation/Assay ProtocolsSee SOP 34. RIA Gamma Counter Calibration and MaintenanceSee SOP 45. RIA Internal Quality Control/Assay ParametersSee SOP 56. Radioactive Waste DisposalSee SOP 77. RIA Package InsertsSee SOP 88. RIA Turnaround TimesSee SOP 99. RIA Specimen and Kit StorageSee SOP 1010. RIA ProcurementSee SOP 1111. Describe briefly the assays performed routinely in the RIA Laboratory. Include:clinical background, type of assay, sample collection, brief methodology, rationalefor each step, sensitivity, specificity, intra- and inter-assay coefficient of variation,reference range.Five assays, radioimmuno- (RIA) and immuno-radiometric assays (IRMAs), are routinelyrun in this RIA lab, all employing 125I-labeled material. Four (Aldosterone, 17-OH-Progesterone, Human Growth Hormone and Active Renin) are batched and run bi-weekly, while one (11-Desoxycortisol) is processed quarterly. Internal quality control ismanaged within the RIA lab, with 3 levels of Bio-Rad lyphochek samples analysed inevery run of all assays (except Active Renin – controls come with kit).Details can be found in the kit package inserts.12. What types of particles are emitted during radioactive decay?There are three types of decay. In alpha decay, the nucleus emits an alpha particle( ); in beta decay, the nucleus emits an electron (or positron), and in gamma decay,the nucleus emits gamma particles (high-energy photons).He4213. List the radioisotopes commonly used in biomedical work. Draw up a tableshowing the half-life and type of particle emitted for each isotope.Radioisotope Half-life Decay type Decay equation3H 12,3y β-e013231 HeH −+⎯→⎯14C 5730y β-e01147146 NC −+⎯→⎯32P 14,3d β-e0132163215 SP −+⎯→⎯35S 87d β-e0135173516 ClS −+⎯→⎯51Cr 27,7d EC, γ γ+⎯→⎯+− VCr 5123015124 e57Co 272d EC, γ γ+⎯→⎯+− FeCo 5726015727 e58Co 71d EC, β+, γ γ+⎯→⎯+− FeCo 5826015827 e
  3. 3. γ++⎯→⎯ p0158265827 FeCo59Fe 45d β-, γ γ++⎯→⎯ − e0159275926 CoFe99Mo 66h β-, γ γ++⎯→⎯ − e0199439942 TcMo99mTc 6,0h γ γ+⎯→⎯ TcTc 9943m9943125I 60h EC, γ γ+⎯→⎯+− TeI 125520112553 e131I 8,04d β, γ γ++⎯→⎯ − e011315413153 XeI14. How is radioactivity measured? What does specific activity mean?Autoradiography, gas ionization detectors and fluorescent scintillation can be used tomeasure radiation.AutoradiographyAutoradiography is a procedure for localizing and recording a radiolabeled compoundwithin a solid sample, which involves the production of an image in a photographicemulsion. The solid sample often consists of size-fractionated DNA or protein samplesthat are embedded within a dried gel, fixed to the surface of a dried nylon membrane ornitrocellulose filter, or located within fixed chromatin or tissue samples mounted on aglass slide. The photographic material consists of an emulsion layer sandwiched betweentwo gelatin layers. One provides adhesion and the other protection. The photosensitiveemulsion layer contains minute crystals of silver halides ranging from 0,07 to 0,40μm indiameter suspended in gelatin. Following passage through the emulsion of a β-particle ora γ-ray emitted by a radionuclide, the Ag+ions are converted to Ag atoms. This processrepeats until a metallic silver grain of increasing size and stability is formed resulting in alatent image. During development, the silver halide is reduced to metallic silver but theprocess proceeds faster in crystals with latent image silver, hence amplifying the image.Fixing is then done to remove any unexposed silver halide crystals, giving anautoradiographic image which provides a two-dimensional representation of thedistribution of the radiolabel in the original sample.Gas ionization detectorsThis is the most common type of instrument. This instrument works on the principle thatas radiation passes through air or a specific gas, it gives off energy to orbital electrons,causing ionization and excitation of the gas atoms. When a high voltage is placedbetween two areas of the gas filled space, the positive ions will be attracted to thecathode of the detector and the free electrons will travel to the anode. These charges arecollected by the anode and cathode which then form a very small current in the wiresgoing to the detector. By placing a very sensitive current measuring device between thewires from the cathode and anode, the small current is measured and displayed as asignal. The more radiation which enters the chamber, the more current displayed by theinstrument. Many types of gas-filled detectors exist, but the two most common are theion chamber used for measuring large amounts of radiation and the Geiger-Mullerdetector used to measure very small amounts of radiation.Fluorescent scintillationIn the scintillation process, the absorbed energy produces a flash of light. When aparticle passes through the material it collides with atomic electrons, exciting them tohigher energy levels. After a very short period of time the electrons fall back to theirnatural levels, causing emission of light. Two common scintillation detectors are thesodium iodide crystal scintillation detector (γ-counter) and the organic liquid scintillationdetector (β-counter).The crystal scintillation detector commonly occurs as a well detector which has a holedrilled in the end of the cylindrical crystal to accept a test tube. Because it is hygroscopic,the crystal is sealed in an aluminium can with a transparent quartz window at one end
  4. 4. through which the blue-violet (420nm) scintillations are detected. The photos of gammaemitters in the sample easily penetrate the specimen tube and the thin, low-density canand enter the crystal where they are absorbed in the thick, high density sodium iodide. Awell counter is not suitable for measuring β-radiation, because it can not penetrate thesample container or aluminium lining of the wall.The crystal is usually a circular cylinder machined from a single crystal of sodium iodide,to which a small amount of thallium is added to improve performance. The high atomicnumber of iodine and the high density of sodium iodide (3,7g/cm3) favour the absorptionof γ-radiation. For this reason, a well counter is often referred to as a γ-counter. For atypical well detector, the counting efficiency for 125I is approximately 70%.A liquid scintillation detector measures radioactivity by recording scintillations occurringwithin a transparent vial that contains the unknown sample and liquid scintillator.Because the radionuclide is mixed with the liquid scintillator the technique is ideal for pureβ-emitters. Counting efficiencies range from between 60% for 3H to 90% for 14C. Theliquid scintillator is known as the scintillation cocktail and contains two components, theprimary solvent and the primary scintillator. The primary solvent is usually inexpensiveand is chosen for its efficiency in absorbing and transferring radiation energy. It is usuallyone of the aromatic hydrocarbons: toluene, or xylene. The primary scintillator absorbsenergy from the primary solvent and converts it into light. A common primary scintillatoris 2,5-diphenyl oxazole which emits ultraviolet light of 380nm. Other components may beadded to the liquid scintillator such as a secondary solvent to improve solubility or asurfactant to stabilise or emulsify the sample. A secondary scintillator may be added toabsorb the ultraviolet photons of the primary scintillator and reemit the energy at a longerwavelength.Specific activity refers to the radioactivity per unit mass or unit volume of a substance.The maximum specific activity attainable for each radionuclide is that for the pureradionuclide. However, usually the pure radionuclide is unavailable and only makes up asmall fraction of the substance it represents.15. Units of radioactivity: what are Ci (Curie), cpm, dpm, Sievert, and Becquerels?The Becquerel (Bq) is the SI unit of activity, defined as one decay per second (dps). TheCurie was originally defined as the radioactivity of one gram of pure radium, but has beenredefined as exactly 37GBq.However, not all decays are capable of affecting the scintillator and being recorded.Some photons do not reach the scintillator or the detector, and those that do, may notinteract with it. The number of decays detected by the detector is called counts and theyare related by the equation: Counting efficiency = Count Rate / Decay Rate. The countsare usually expressed as counts per minute.Radiation carries energy, and when it is absorbed by matter, the matter receives thisenergy. The radiation dose is the amount of energy deposited per unit of mass. The SIunit of radiation dose is the Gray (Gy), which is defined as the dose of one joule of energyabsorbed per kilogram of matter.Various kinds of radiation have different effects on living tissue, so a simple measurementof dose as energy received, stated in grays, does not give a clear indication of theprobable biological effects of the radiation. The equivalent dose, which is measured insieverts, is equal to the actual dose, in grays, multiplied by a ‘quality factor’ which is largerfor more dangerous forms of radiation. An effective dose of one sievert requires 1 gray ofbeta or gamma radiation but only 0,05 gray of alpha radiation or 0,1 gray of neutronradiation.16. How much radioactivity is commonly used in a RIA (say, 50 samples)? What doseof I-131 is usually given to patients with Grave’s disease?In RIAs I-125 is commonly used. One vial of labelled I-125 ligand may have a totalradioactivity of 130kBq, which can be used for 50 samples. To treat a patient with Grave’sdisease, doses between 370MBq and 550MBq of I-131 are given.
  5. 5. 17. Describe and illustrate the principles of: Competitive RIA, Double antibody(sandwich type) IRMA, ELISA.In a competitive radioimmunoassay, an antibody and a radiolabelled antigen are used tomeasure an analyte. The analyte in the patient’s serum is mixed with the radiolabelledanalyte and allowed to compete for a limited amount of the antibody for a fixed timeperiod. The unbound analyte is then removed in a wash step, and the bound antibody-analyte is then measured, usually in a gamma counter. The concentration of the analyteis then determined from a (decreasing sigmoid) calibration curve.In an immunoradiometric assay, the analyte is incubated with a solid-phase antibody anda second radiolabelled antibody. An excess of both antibodies should always be present.This is left for a fixed time period, after which all unbound radiolabelled antibody isremoved in a wash step. The antibody-analyte-radiolablled antibody complex is thenmeasured with a gamma counter. The concentration of the analyte is then determinedfrom a (linear) calibration curve.An enzyme-linked immunosorbent assay is similar to an immunoradiometric assay exceptthat the second antibody is not radiolabelled but instead is covalently bound to anenzyme. The two antibodies (fixed and enzyme-bound) and the sample are allowed toincubate, after which the unbound antibody is washed off. A substrate of the enzyme isthen added which is converted to a detectable product. The product may be a coloured-dye, a fluorescent product, or a chemical that undergoes chemiluminescence. Theamount of product is measured, and the concentration of the analyte can then bedetermined from a (linear) calibration curve.18. What factors determine the stability of hormones in plasma? Discuss the specimenhandling precautions which may be necessary. What are the functions of trasyloland EDTA? What is cryoactivation, and why does it affect the Active Renin IRMA?The stability of hormones in samples is determined by the type of hormone, the anti-coagulant used and the storage temperature. As a general rule, steroid hormones aremore stable than peptide hormones, and storing at colder temperatures is better thanwarmer temperatures. Exceptions to this are aldosterone, a steroid which degradesquickly at room temperature, and C-peptide which is stable for more than 5 daysthroughout a range of laboratory-used temperatures. EDTA is generally considered thebest anti-coagulant to use as it has some anti-proteolytic properties. However, with theexception of ACTH, this effect is usually non-significant.Aprotinin (trasylol) is a polypeptide derived from bovine lung tissue that inhibits serineproteases such as trypsin, chymotrypsin, plasmin and kallikrein. It is often used as ananti-proteolytic when collecting blood for unstable peptide hormones such as ACTH orglucagon.Approximately tenfold more prorenin than renin normally circulates in human plasma, withalmost 100 times more being seen in some low-renin patients. At temperatures below25°C, prorenin develops intrinsic renin activity, and the prosequence becomes vulnerableto cleavage by plasma enzymes, resulting in irreversible formation of renin in vitro. This iscalled cryoactivation. The lower the temperature (short of freezing) the more likely theseprocesses are to occur. Because renin is remarkably stable in plasma at roomtemperature, to avoid cryoactivation of prorenin, blood samples for renin should beprocessed at room temperature. Prorenin does not cryoactivate in frozen plasma, orduring rapid freezing and thawing. It is for this reason that plasma must be thawed in a37°C water bath, and not thawed gently on a bench as for most other analytes. Theantibody used in the renin IRMA binds to active renin. If cryoactivation occurs, more ofthe prorenin will be converted to active renin, and a falsely high renin value will beobtained.19. In a RIA, what is meant by the following: Total Counts, Zero Binding, NonspecificBinding, Percentage Bound (%B0), %NSB, ED50, ED20, ED80? What type of curve isused?
  6. 6. Total counts - are tubes that represent the total amount of radioactivity added in an RIAtube. These tubes are not decanted in the separation step. They represent the totalamount of tracer aliquoted per tube. When the assay is counted, these tubes will have thehighest CPMs. These counts are not used as part of the dose estimate calculation forunknowns, but rather as a quality control comparison to the counts in the B0 tubes.Because the amount of antibody is limiting and tracer is in excess, total count tubes areincluded to guarantee and document this excess. The degree of excess is expressed as apercent of CPMs in the B0 tubes divided by the CPMs in the total count tubes, oftenreferred to as the %B/T or Bound/Total. This %B/T value should be between thirty andfifty percent.Zero binding (B0) - are tubes that contain labelled antigen, the limiting antibody, possiblyassay buffer and the precipitant, but do not have any unlabeled antigen such as unknownsamples or standards (except zero standard). After separating the free from the boundfraction, these tubes will have the highest CPMs, other than the total count tubes.Non-specific binding - are tubes that contain labelled antigen, sometimes assay buffer,zero standard or precipitant, but they never have any antibody. When the assay iscounted, these tubes will have the lowest CPMs in a radioimmunoassay system. Thesecounts serve as a record of binding which is not due to the antibody. For example, thelabelled antigen may bind to elements of the buffer or to the tube wall. (Generally, plasticor polystyrene tubes absorb more label than glass tubes, which in turn, absorb more labelthan polypropylene tubes.) These counts are subtracted from the counts of all the othertubes to obtain a more accurate estimate of counts in the bound fraction.Percentage bound of B0 – Data are generally expressed as standards and unknowns as apercentage of the maximum possible bound (B0).Percent non-specific binding (%NSB) – This is the non-specific binding count divided bythe total counts and tells you how much of the total radioactivity added, gets bound tonon-specific binding sites. Ideally this should be as low as possible.Effective dose ED50, ED20, ED80 – This is the concentration of analyte that corresponds to50% / 20% / 80% B/B0. These data are often derived from a plot of B/B0 against theanalyte concentration.In RIA, data are often presented as a graph with the B/B0 on the y-axis and log of theconcentration on the x-axis. This gives a sigmoid shaped graph from where unknowndata points can be extrapolated. To make the graph linear, the y-axis is often replacedwith the logit B/B0 which makes extrapolating the data easier.20. In an IRMA, what is meant by the following: Total Counts, Nonspecific Binding?What type of curve is used?Total counts - are tubes that represent the total amount of radioactivity added in an IRMAtube. These tubes are not decanted in the separation step. They represent the totalamount of tracer aliquoted per tube. When the assay is counted, these tubes will have thehighest CPMs. These counts are not used as part of the dose estimate calculation forunknowns, but are rather compared to the counts obtained in the highest standard tubesas a means of quality control. Because the amount of analyte is limiting and tracer(antibody) is in excess, total count tubes are included to guarantee and document thisexcess.Non-specific binding - are tubes that contain labelled antibody, sometimes assay buffer,zero standard, but no analyte. When the assay is counted, these tubes will have thelowest CPMs in the immunoradiometric assay system. These counts serve as a record ofantibody binding to sites which are not on the analyte. For example, the labelled antibodymay bind to proteins in the buffer or directly to the tube wall. These counts are subtracted
  7. 7. from the counts of all the other tubes to obtain a more accurate estimate of counts in thebound fraction. This tube is equivalent to the zero standard counts.In IRMA data analysis, either the counts per minute or the counts/total counts (B/T) isplotted against the concentration. This should give a linear graph when plotted on eitherlinear or log-log paper.21. How is cross-reactivity measured?Cross-reacting substances are those substances which affect binding of antigen bycompeting for the specific binding site on the antibody. The cross-reactivity of asubstance can be reported several ways. General guidelines on interference testingrecommend reporting interferences as the maximum effect expected from the interferingsubstance at a specified concentration of the interfering substance at the medicaldecision point of the analyte. This method has been accepted as the most useful way ofreporting interferences. However, for immunoassays, interferences are often reported aspercent cross-reactivity – defined as the mass ratio of analyte to interfering substance,each at 50% displacement of label (ED50). Cross-reactivity can also be calculated atother levels of displacement such as 20% or the ED20. Depending on the slope and shapeof the response curve the percent cross-reactivity may be different at differentdisplacement levels. Another method to report cross-reactivity may be to simply reportthe concentration of cross-reactant required to displace a given amount of labelledantigen. For example, one might report the concentration of cross-reactant required todisplace 50% of the label i.e. the ED50.Cross-reactivity Cross-reactivitySubstance Cross-reactivity,%hCG added (IU/l) Apparent increasein TSHconcentration(IU/L)hCG 5x10-51000 <0,0310 000 0,4100 000 5Two methods of reporting cross-reactivityExample calculation of cross-reactivity of hCG in TSH assay. ED50 TSH =10IU/l. ED50 hCG = 100IU/l. hCG cross-reactivity = 10 / 100 x 100% = 10%
  8. 8. 22. Describe the terms “sensitivity” and “specificity” in the context of immunoassays.SensitivityThe term sensitivity can have different meanings depending on the context. In its strictestsense, the sensitivity is the change in the response of a measuring instrument divided bythe corresponding change in the stimulus, so for an IRMA, the sensitivity would be thechange in cpm divided by the change in analyte concentration. The sensitivity can alsorefer to the detection limit of the assay and can be defined as that concentration ofantigen which can be distinguished from zero concentration with a stated degree ofprobability. Sensitivity is affected by the titre, affinity, and specificity of the referenceantibody used in the assay. As such it can be affected by possible differences in antibodyaffinity for unlabelled and labelled antigen, and the presence of cross-reactive antigens,interfering substances or conditions in the test sample, as well as separation artefactsgenerated by experimental technique.SpecificitySpecificity is a characteristic of a laboratory test which describes its ability to distinguishbetween true (or specific) and non-specific results. With immunoassay methods,interferences which affect specificity can be categorized into two major classes: 1) thosewhich affect the binding event between the antibody and an antigen in a general way,such as pH or ionic strength; or 2) those substances which affect binding of antigen bycompeting for the specific binding site on the antibody. These ‘specific’ interferences areoften referred to as ‘cross-reactants’. The specificity of an immunoassay may becharacterized by adding increasing amounts of a potential cross reacting substance to asample and measuring the response in the immunoassay. See previous question.23. Describe the terms “accuracy” and “precision” in measuring hormone levels.Accuracy is usually used to denote the ability of an assay system to generate the correctresult. It is defined as the closeness of agreement between the result of a test and itstrue value. Unfortunately, the true value for any individual test result produced is usuallyunknowable, so this definition of accuracy exists mainly as a theoretical concept.Although accuracy and trueness are often used synonymously, there is a difference.Accuracy strictly refers to the correctness of a single result whereas trueness refers to thecorrectness of the mean of a number of results. This difference is important because aresult’s accuracy is influenced by bias and imprecision whereas trueness is onlyinfluenced by bias. Practically, accuracy is estimated from a ‘comparison of methods’experiment where the average difference between results by the method of interest and areference or comparative method is calculated. Other tests used to give an indication ofaccuracy include recovery and dilution testing. Immunoassay manufacturers are requiredto make a claim for the accuracy of their analytic measurement products and that claimtypically is based on results from a comparison of methods experiment. Also, laboratoriesare required to verify a manufacturer’s claim for accuracy, which again would typically bedone by data from a ‘comparison of methods’ experiment.Precision is defined as the closeness of agreement between independent test resultsunder prescribed conditions. The degree of precision is usually expressed on the basis ofstatistical measures of impression such as the standard deviation or CV. The precision issolely related to the random error of measurements and has no relation to the trueness ofmeasurements. Manufacturers are required to make a claim for precision and typicallyprovide estimates within a single run and for many runs performed over a period of onemonth. Laboratories are required to verify the manufacturer’s claims, which again istypically by performing 20 measurements within a single run and 20 measurements over20 different runs over 20 different days. Again, both manufacturers and laboratories arecustomers who have practical applications in characterizing and verifying thisperformance characteristic.24. For endocrine hormones, how do you convert IU to mass or moles? How do youconvert mass to moles? List the relevant conversion factors for the five routineRIA/IRMA assays.
  9. 9. Wherever an anaylte has been chemically well defined and can be easily synthesised orisolated, it is preferable to express its concentration in Système International (SI)recommended units of moles per litre. However, conventionally in some parts of theworld, mass units are in common usage and concentrations are often expressed in gramsper litre. Where the analyte is well characterised, it is easy to convert from mass units tomole units by dividing by the molecular weight (MW).When an analyte can not be chemically well defined (for example it occurs in variousisoforms or glycosylation states) or the analyte is actually a group of similar but differentanalytes, then it is preferable to express its concentration in international units. Theinternational unit (IU) is the unitage assigned by the world health organisation (WHO) toan international biological standard. This standard is a reference preparation, preparedby the best methods available at the time, and distributed world wide to be used as areference calibrant. International units can be converted to mass units by a conversionfactor release by the WHO. The WHO reference preparations are updated periodicallyand conversion factors do change; the conversion factor used must therefore alwaysmatch the reference samples that the manufactures used to prepare the calibrants.Conversion factors for analytes measured in this lab are given in the table below:Analyte ConventionunitConversionfactorInternationalrecommendedunitConversionstandardActive Renin pg/ml (ng/l) x 1,8 mIU/l WHO 68/356Growth Hormone ng/ml (μg/l) x 3 mIU/l WHO IS 98/574Aldosterone pg/ml (ng/l) x 2,774 pmol/l MW: 360,44402017OH-Progesterone ng/ml (μg/l) x 3,026 nmol/l MW: 330,461111-Deoxycortisol ng/ml (μg/l) x 2,886 nmol/l MW: 346,4605025. What are inter- and intra-assay coefficients of variation (CV), and how are theydetermined?Two indicators of precision are the repeatability and reproducibility. Repeatability isdefined as the closeness of agreement between results of successive measurementscarried out under the same conditions. It is evaluated by performing twentymeasurements, within a single run, and calculating the coefficient of variation (the meandivided by the standard deviation). This result is called the intra-assay CV.Reproducibility is the closeness of agreement between results of measurementsperformed under changed conditions of measurements (for example: time, operators,calibrators and reagent lots). This is determined by performing twenty measurementsover twenty different runs over twenty different days and calculating the coefficient ofvariation. The result of this calculation is called the inter-assay CV.26. Describe and illustrate a Scatchard plot of binding data.Scatchard analysis is a standard method for analysing the equilibrium binding parametersof a radiolabelled ligand with its receptor. The binding data are derived from a ratio ofspecifically bound to specifically free antigen, plotted against the concentration ofspecifically bound antigen. This plot gives an estimate of binding affinity and the numberof available binding sites per volume.The plot is achieved as follows: various concentrations of labelled antigen are prepared.Three tubes are prepared for each concentration of labelled antigen: total tubes (T)containing the labelled antigen; non-specific binding tubes (NSB) containing the labelledantigen, but none of the antibody in question; and total binding (TB) tubes containing thelabelled antigen and the antibody, which are left to equilibrate. After equilibrium (in the
  10. 10. TB and NSB tubes only) the bound fraction is precipitated and separated. Once theassay is completed, three sets of results should be available for each concentration ofradioligand – The total counts (T), the non-specific binding counts (NSB) and the totalbinding counts (TB).The following can now be calculated:Specifically bound (B): B = TB – NSBFree ligand (F): F = T - BThese can be converted from counts to concentration units as follows:)/(activityspecific1)/(1022,21)/(effeciency)/(counts)/(ionConcentrat 12molCiCidpmdpmcpmlcpmlmol ⋅⋅⋅=The bound can now be plotted against the free to achieve a graph as such:The data from the saturation experiment can be plotted with Bound/Free on the Y axisand Bound on the X axis. This data can be analyzed by linear regression to give astraight line. This is called a Rosenthal Plot.
  11. 11. The equilibrium constant of the antibody-antigen reaction (Kd) can be calculated from thenegative reciprocal of the slope of the graph, whereas the x-intercept of the graph givesthe total concentration of binding sites (BMax) measurable under assay conditions. Thepresence of a curve (as opposed to a line) indicates the presence of a mixed populationof binding sites.It should be noted that it is more accurate to do binding analysis on the saturation curveanalyzed by non-linear regression analysis, than by linear analysis on the Rosenthal plot.This is because the Rosenthal Plot contains the bound on both the x- and y-axes. Sincethis is the variable containing the greatest error, a larger error will be distributed in bothdirections.27. In centrifugation, how do you convert rpm to Xg force? What is the conversionfactor for the centrifuge in the RIA Laboratory?The relative centrifugal force (XG) can be calculated by the equation:22)()(00090rpmspeedrotorcmradiusgXg ⋅⋅=πwhere g is the standard gravity equal to exactly 9,80665m/s2.The centrifuge in the RIA laboratory has a radius of 24,5cm so the relative centrifugalforce can be calculated using:6503)( 2cmspeedrotorXg =28. Summarise the principle of the plasma renin activity assay, and compare it to theactive renin assay.Renin is a proteolytic enzyme involved in blood pressure control. Renin cleavesangiotensinogen to produce the decapeptide angiotensin I. Angiotensin I is then rapidilycleaved to the biologically active angiotensin II by the angiotensin-converting enzyme.Two methods of estimating the amount of active renin are available - an enzyme kineticassay and a mass assay.
  12. 12. Plasma renin activityThis method measures the renin (angiotensinogen proteolytic) activity in serum.Plasma is mixed with a buffer (pH 6,0) and a angiotensin-converting enzyme inhibitor(phenylmethylsulfonyl fluoride). The sample is then split into two tubes. One tube is keptat 37°C to allow for the generation of angiotensin I, the other is kept at 4°C to act as ablank. After 90 minutes the reaction is stopped (by cooling to 4°C) and the amount ofangiotensin I is measured in each tube using a radioimmunoassay. The angiotensin Igenerated is then calculated as shown below. This is the plasma renin activity (PRA) andis usually expressed in ng/ml/hr.TimefactorDilutionPRA VolumePlasmaInAngiotensiInAngiotensi 437⋅=°° −Active renin assayHistorically, the problem with measuring renin directly was that renin exists in both activeand inactive forms. The inactive from (prorenin) can account for up to 90% of the totalrenin in the circulation, making its measurement worthless. Since the discovery ofantibodies which are selective for active renin, direct measurements are possible. Theactive renin assay is a immunoradiometric assay which uses two antibodies. The firstantibody is a solid phase monoclonal antibody that recognises both active and inactiverenin. The second antibody is a 125 iodine-labelled monoclonal antibody specific foractive renin. The active renin is usually expressed in ng/l.
  13. 13. References[1-5]1. Deupree, J.D.; Tutorial in Receptor Binding Techniques;; Lurz, Matthew J.; 19982. Edwards, R., S. Blincko, and I. Howes, Principles of immunodiagnostic testsand their development; with specific use of radioisotopes as tracers, inImmunodiagnostics : a practical approach, The Practical approach series ;206, R. Edwards, Editor. 1999, Oxford University Press: Oxford ; New York.p. cm.3. Fuentes-Arderiu, X.; Glossary of ISO Metrological and Realted Terms andDefinitions Relevant to Clinical Laboratory Sciences;; Westgard, James O; 19994. Kricka, L.J., Principles of Immunochemical Techniques, in Tietz textbook ofclinical chemistry and molecular diagnostics, C.A. Burtis, et al., Editors.2006, Elsevier Saunders: Philadelphia. p. XXXVI, 2412 s.5. Linnet, K. and J.C. Boyd, Selection and Analytical Evaluation of Methods -With Statistical Techniques, in Tietz textbook of clinical chemistry andmolecular diagnostics, C.A. Burtis, et al., Editors. 2006, Elsevier Saunders:Philadelphia. p. XXXVI, 2412 s.