RADIOIMMUNOASSAY LABORATORY: COMPETENCY ASSESSMENT 2008

DIVISION OF CHEMICAL PATHOLOGY GROOTE SCHUUR HOSPITAL

C17 NHLS

Author: David Haarburger Supervisor: Judy King

February 2008

This document is intended for the use of medical technologists, registrars and scientists, and forms part of the training required when using the RIA lab.

RADIOIMMUNOASSAY LABORATORY: COMPETENCY ASSESSMENT 2008 CHEMICAL PATHOLOGY C17 NHLS 1. Radiation Safety/Radiation Protection Training Course

See SOP 1 2. RIA Spillage Monitoring

See SOP 2 3. RIA Gamma Counter Operation/Assay Protocols

See SOP 3 4. RIA Gamma Counter Calibration and Maintenance

See SOP 4 5. RIA Internal Quality Control/Assay Parameters

See SOP 5 6. Radioactive Waste Disposal

See SOP 7 7. RIA Package Inserts

See SOP 8 8. RIA Turnaround Times

See SOP 9 9. RIA Specimen and Kit Storage

See SOP 10 10. RIA Procurement

See SOP 11 11. Describe briefly the assays performed routinely in the RIA Laboratory. Include:

clinical background, type of assay, sample collection, brief methodology, rationale for each step, sensitivity, specificity, intra- and inter-assay coefficient of variation, reference range. Five assays, radioimmuno- (RIA) and immuno-radiometric assays (IRMAs), are routinely run 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 is managed within the RIA lab, with 3 levels of Bio-Rad lyphochek samples analysed in every 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).

He42

13. List the radioisotopes commonly used in biomedical work. Draw up a table

showing the half-life and type of particle emitted for each isotope. Radioisotope Half-life Decay type Decay equation 3H 12,3y β- e0

132

31 HeH −+⎯→⎯

14C 5730y β- e01

147

146 NC −+⎯→⎯

32P 14,3d β- e01

3216

3215 SP −+⎯→⎯

35S 87d β- e01

3517

3516 ClS −+⎯→⎯

51Cr 27,7d EC, γ γ+⎯→⎯+− VCr 5123

01

5124 e

57Co 272d EC, γ γ+⎯→⎯+− FeCo 5726

01

5727 e

58Co 71d EC, β+, γ γ+⎯→⎯+− FeCo 5826

01

5827 e

γ++⎯→⎯ p01

5826

5827 FeCo

59Fe 45d β-, γ γ++⎯→⎯ − e01

5927

5926 CoFe

99Mo 66h β-, γ γ++⎯→⎯ − e01

9943

9942 TcMo

99mTc 6,0h γ γ+⎯→⎯ TcTc 9943

m9943

125I 60h EC, γ γ+⎯→⎯+− TeI 12552

01

12553 e

131I 8,04d β, γ γ++⎯→⎯ − e01

13154

13153 XeI

14. How is radioactivity measured? What does specific activity mean?

Autoradiography, gas ionization detectors and fluorescent scintillation can be used to measure radiation. Autoradiography Autoradiography is a procedure for localizing and recording a radiolabeled compound within a solid sample, which involves the production of an image in a photographic emulsion. The solid sample often consists of size-fractionated DNA or protein samples that are embedded within a dried gel, fixed to the surface of a dried nylon membrane or nitrocellulose filter, or located within fixed chromatin or tissue samples mounted on a glass slide. The photographic material consists of an emulsion layer sandwiched between two gelatin layers. One provides adhesion and the other protection. The photosensitive emulsion layer contains minute crystals of silver halides ranging from 0,07 to 0,40μm in diameter suspended in gelatin. Following passage through the emulsion of a β-particle or a γ-ray emitted by a radionuclide, the Ag+ ions are converted to Ag atoms. This process repeats until a metallic silver grain of increasing size and stability is formed resulting in a latent image. During development, the silver halide is reduced to metallic silver but the process proceeds faster in crystals with latent image silver, hence amplifying the image. Fixing is then done to remove any unexposed silver halide crystals, giving an autoradiographic image which provides a two-dimensional representation of the distribution of the radiolabel in the original sample. Gas ionization detectors This is the most common type of instrument. This instrument works on the principle that as 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 placed between two areas of the gas filled space, the positive ions will be attracted to the cathode of the detector and the free electrons will travel to the anode. These charges are collected by the anode and cathode which then form a very small current in the wires going to the detector. By placing a very sensitive current measuring device between the wires from the cathode and anode, the small current is measured and displayed as a signal. The more radiation which enters the chamber, the more current displayed by the instrument. Many types of gas-filled detectors exist, but the two most common are the ion chamber used for measuring large amounts of radiation and the Geiger-Muller detector used to measure very small amounts of radiation. Fluorescent scintillation In the scintillation process, the absorbed energy produces a flash of light. When a particle passes through the material it collides with atomic electrons, exciting them to higher energy levels. After a very short period of time the electrons fall back to their natural levels, causing emission of light. Two common scintillation detectors are the sodium iodide crystal scintillation detector (γ-counter) and the organic liquid scintillation detector (β-counter). The crystal scintillation detector commonly occurs as a well detector which has a hole drilled 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

through which the blue-violet (420nm) scintillations are detected. The photos of gamma emitters in the sample easily penetrate the specimen tube and the thin, low-density can and enter the crystal where they are absorbed in the thick, high density sodium iodide. A well counter is not suitable for measuring β-radiation, because it can not penetrate the sample 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 atomic number of iodine and the high density of sodium iodide (3,7g/cm3) favour the absorption of γ-radiation. For this reason, a well counter is often referred to as a γ-counter. For a typical well detector, the counting efficiency for 125I is approximately 70%. A liquid scintillation detector measures radioactivity by recording scintillations occurring within 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 pure β-emitters. Counting efficiencies range from between 60% for 3H to 90% for 14C. The liquid scintillator is known as the scintillation cocktail and contains two components, the primary solvent and the primary scintillator. The primary solvent is usually inexpensive and is chosen for its efficiency in absorbing and transferring radiation energy. It is usually one of the aromatic hydrocarbons: toluene, or xylene. The primary scintillator absorbs energy from the primary solvent and converts it into light. A common primary scintillator is 2,5-diphenyl oxazole which emits ultraviolet light of 380nm. Other components may be added to the liquid scintillator such as a secondary solvent to improve solubility or a surfactant to stabilise or emulsify the sample. A secondary scintillator may be added to absorb the ultraviolet photons of the primary scintillator and reemit the energy at a longer wavelength. 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 pure radionuclide. However, usually the pure radionuclide is unavailable and only makes up a small 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). The Curie was originally defined as the radioactivity of one gram of pure radium, but has been redefined 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 not interact with it. The number of decays detected by the detector is called counts and they are related by the equation: Counting efficiency = Count Rate / Decay Rate. The counts are usually expressed as counts per minute. Radiation carries energy, and when it is absorbed by matter, the matter receives this energy. The radiation dose is the amount of energy deposited per unit of mass. The SI unit of radiation dose is the Gray (Gy), which is defined as the dose of one joule of energy absorbed per kilogram of matter. Various kinds of radiation have different effects on living tissue, so a simple measurement of dose as energy received, stated in grays, does not give a clear indication of the probable biological effects of the radiation. The equivalent dose, which is measured in sieverts, is equal to the actual dose, in grays, multiplied by a ‘quality factor’ which is larger for more dangerous forms of radiation. An effective dose of one sievert requires 1 gray of beta or gamma radiation but only 0,05 gray of alpha radiation or 0,1 gray of neutron radiation.

16. How much radioactivity is commonly used in a RIA (say, 50 samples)? What dose

of 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 total radioactivity of 130kBq, which can be used for 50 samples. To treat a patient with Grave’s disease, doses between 370MBq and 550MBq of I-131 are given.

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 to measure an analyte. The analyte in the patient’s serum is mixed with the radiolabelled analyte and allowed to compete for a limited amount of the antibody for a fixed time period. 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 analyte is then determined from a (decreasing sigmoid) calibration curve. In an immunoradiometric assay, the analyte is incubated with a solid-phase antibody and a 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 is removed in a wash step. The antibody-analyte-radiolablled antibody complex is then measured with a gamma counter. The concentration of the analyte is then determined from a (linear) calibration curve. An enzyme-linked immunosorbent assay is similar to an immunoradiometric assay except that the second antibody is not radiolabelled but instead is covalently bound to an enzyme. The two antibodies (fixed and enzyme-bound) and the sample are allowed to incubate, after which the unbound antibody is washed off. A substrate of the enzyme is then added which is converted to a detectable product. The product may be a coloured-dye, a fluorescent product, or a chemical that undergoes chemiluminescence. The amount of product is measured, and the concentration of the analyte can then be determined from a (linear) calibration curve.

18. What factors determine the stability of hormones in plasma? Discuss the specimen

handling precautions which may be necessary. What are the functions of trasylol and 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 are more stable than peptide hormones, and storing at colder temperatures is better than warmer temperatures. Exceptions to this are aldosterone, a steroid which degrades quickly at room temperature, and C-peptide which is stable for more than 5 days throughout a range of laboratory-used temperatures. EDTA is generally considered the best anti-coagulant to use as it has some anti-proteolytic properties. However, with the exception of ACTH, this effect is usually non-significant. Aprotinin (trasylol) is a polypeptide derived from bovine lung tissue that inhibits serine proteases such as trypsin, chymotrypsin, plasmin and kallikrein. It is often used as an anti-proteolytic when collecting blood for unstable peptide hormones such as ACTH or glucagon. Approximately tenfold more prorenin than renin normally circulates in human plasma, with almost 100 times more being seen in some low-renin patients. At temperatures below 25°C, prorenin develops intrinsic renin activity, and the prosequence becomes vulnerable to cleavage by plasma enzymes, resulting in irreversible formation of renin in vitro. This is called cryoactivation. The lower the temperature (short of freezing) the more likely these processes are to occur. Because renin is remarkably stable in plasma at room temperature, to avoid cryoactivation of prorenin, blood samples for renin should be processed at room temperature. Prorenin does not cryoactivate in frozen plasma, or during rapid freezing and thawing. It is for this reason that plasma must be thawed in a 37°C water bath, and not thawed gently on a bench as for most other analytes. The antibody used in the renin IRMA binds to active renin. If cryoactivation occurs, more of the prorenin will be converted to active renin, and a falsely high renin value will be obtained.

19. In a RIA, what is meant by the following: Total Counts, Zero Binding, Nonspecific Binding, Percentage Bound (%B0), %NSB, ED50, ED20, ED80? What type of curve is used?

Total counts - 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 CPMs. 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 B0 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 of excess is expressed as a percent of CPMs in the B0 tubes divided by the CPMs in the total count tubes, often referred to as the %B/T or Bound/Total. This %B/T value should be between thirty and fifty percent. Zero binding (B0) - are tubes that contain labelled antigen, the limiting antibody, possibly assay buffer and the precipitant, 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 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 is counted, these tubes will have the lowest CPMs in a radioimmunoassay system. These counts serve as a record of binding which is not due to the antibody. For example, the labelled antigen may bind to elements of the buffer or to the tube wall. (Generally, plastic or polystyrene tubes absorb more label than glass tubes, which in turn, absorb more label than polypropylene tubes.) These counts are subtracted from the counts of all the other tubes 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 a percentage of the maximum possible bound (B0). Percent non-specific binding (%NSB) – This is the non-specific binding count divided by the total counts and tells you how much of the total radioactivity added, gets bound to non-specific binding sites. Ideally this should be as low as possible. Effective dose ED50, ED20, ED80 – This is the concentration of analyte that corresponds to 50% / 20% / 80% B/B0. These data are often derived from a plot of B/B0 against the analyte concentration. In RIA, data are often presented as a graph with the B/B0 on the y-axis and log of the concentration on the x-axis. This gives a sigmoid shaped graph from where unknown data points can be extrapolated. To make the graph linear, the y-axis is often replaced with 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 IRMA 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 CPMs. These counts are not used as part of the dose estimate calculation for unknowns, but are rather compared to the counts obtained in the highest standard tubes as 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 this excess. 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 the lowest CPMs in the immunoradiometric assay system. These counts serve as a record of antibody binding to sites which are not on the analyte. For example, the labelled antibody may bind to proteins in the buffer or directly to the tube wall. These counts are subtracted

from the counts of all the other tubes to obtain a more accurate estimate of counts in the bound 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) is plotted against the concentration. This should give a linear graph when plotted on either linear or log-log paper.

21. How is cross-reactivity measured?

Cross-reacting substances are those substances which affect binding of antigen by competing for the specific binding site on the antibody. The cross-reactivity of a substance can be reported several ways. General guidelines on interference testing recommend reporting interferences as the maximum effect expected from the interfering substance at a specified concentration of the interfering substance at the medical decision point of the analyte. This method has been accepted as the most useful way of reporting interferences. However, for immunoassays, interferences are often reported as percent 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 at other levels of displacement such as 20% or the ED20. Depending on the slope and shape of the response curve the percent cross-reactivity may be different at different displacement levels. Another method to report cross-reactivity may be to simply report the concentration of cross-reactant required to displace a given amount of labelled antigen. For example, one might report the concentration of cross-reactant required to displace 50% of the label i.e. the ED50.

Cross-reactivity Cross-reactivity Substance Cross-reactivity,

% hCG added (IU/l) Apparent increase

in TSH concentration

(IU/L) hCG 5x10-5 1000 <0,03

10 000 0,4

100 000 5 Two methods of reporting cross-reactivity

Example 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%

22. Describe the terms “sensitivity” and “specificity” in the context of immunoassays. Sensitivity The term sensitivity can have different meanings depending on the context. In its strictest sense, the sensitivity is the change in the response of a measuring instrument divided by the corresponding change in the stimulus, so for an IRMA, the sensitivity would be the change in cpm divided by the change in analyte concentration. The sensitivity can also refer to the detection limit of the assay and can be defined as that concentration of antigen which can be distinguished from zero concentration with a stated degree of probability. Sensitivity is affected by the titre, affinity, and specificity of the reference antibody used in the assay. As such it can be affected by possible differences in antibody affinity for unlabelled and labelled antigen, and the presence of cross-reactive antigens, interfering substances or conditions in the test sample, as well as separation artefacts generated by experimental technique. Specificity Specificity is a characteristic of a laboratory test which describes its ability to distinguish between true (or specific) and non-specific results. With immunoassay methods, interferences which affect specificity can be categorized into two major classes: 1) those which 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 by competing for the specific binding site on the antibody. These ‘specific’ interferences are often referred to as ‘cross-reactants’. The specificity of an immunoassay may be characterized by adding increasing amounts of a potential cross reacting substance to a sample 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 correct result. It is defined as the closeness of agreement between the result of a test and its true value. Unfortunately, the true value for any individual test result produced is usually unknowable, 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 the correctness of the mean of a number of results. This difference is important because a result’s accuracy is influenced by bias and imprecision whereas trueness is only influenced by bias. Practically, accuracy is estimated from a ‘comparison of methods’ experiment where the average difference between results by the method of interest and a reference or comparative method is calculated. Other tests used to give an indication of accuracy include recovery and dilution testing. Immunoassay manufacturers are required to make a claim for the accuracy of their analytic measurement products and that claim typically is based on results from a comparison of methods experiment. Also, laboratories are required to verify a manufacturer’s claim for accuracy, which again would typically be done by data from a ‘comparison of methods’ experiment. Precision is defined as the closeness of agreement between independent test results under prescribed conditions. The degree of precision is usually expressed on the basis of statistical measures of impression such as the standard deviation or CV. The precision is solely related to the random error of measurements and has no relation to the trueness of measurements. Manufacturers are required to make a claim for precision and typically provide estimates within a single run and for many runs performed over a period of one month. Laboratories are required to verify the manufacturer’s claims, which again is typically by performing 20 measurements within a single run and 20 measurements over 20 different runs over 20 different days. Again, both manufacturers and laboratories are customers who have practical applications in characterizing and verifying this performance characteristic.

24. For endocrine hormones, how do you convert IU to mass or moles? How do you convert mass to moles? List the relevant conversion factors for the five routine RIA/IRMA assays.

Wherever an anaylte has been chemically well defined and can be easily synthesised or isolated, it is preferable to express its concentration in Système International (SI) recommended units of moles per litre. However, conventionally in some parts of the world, mass units are in common usage and concentrations are often expressed in grams per litre. Where the analyte is well characterised, it is easy to convert from mass units to mole units by dividing by the molecular weight (MW). When an analyte can not be chemically well defined (for example it occurs in various isoforms or glycosylation states) or the analyte is actually a group of similar but different analytes, then it is preferable to express its concentration in international units. The international unit (IU) is the unitage assigned by the world health organisation (WHO) to an international biological standard. This standard is a reference preparation, prepared by the best methods available at the time, and distributed world wide to be used as a reference calibrant. International units can be converted to mass units by a conversion factor release by the WHO. The WHO reference preparations are updated periodically and conversion factors do change; the conversion factor used must therefore always match 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 Convention

unit Conversion factor

International recommended unit

Conversion standard

Active Renin pg/ml (ng/l) x 1,8 mIU/l WHO 68/356 Growth Hormone ng/ml (μg/l) x 3 mIU/l WHO IS 98/574 Aldosterone pg/ml (ng/l) x 2,774 pmol/l MW: 360,444020 17OH-Progesterone ng/ml (μg/l) x 3,026 nmol/l MW: 330,4611 11-Deoxycortisol ng/ml (μg/l) x 2,886 nmol/l MW: 346,46050 25. What are inter- and intra-assay coefficients of variation (CV), and how are they

determined? Two indicators of precision are the repeatability and reproducibility. Repeatability is defined as the closeness of agreement between results of successive measurements carried out under the same conditions. It is evaluated by performing twenty measurements, within a single run, and calculating the coefficient of variation (the mean divided by the standard deviation). This result is called the intra-assay CV. Reproducibility is the closeness of agreement between results of measurements performed under changed conditions of measurements (for example: time, operators, calibrators and reagent lots). This is determined by performing twenty measurements over twenty different runs over twenty different days and calculating the coefficient of variation. 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 parameters of a radiolabelled ligand with its receptor. The binding data are derived from a ratio of specifically bound to specifically free antigen, plotted against the concentration of specifically bound antigen. This plot gives an estimate of binding affinity and the number of 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 labelled antigen, but none of the antibody in question; and total binding (TB) tubes containing the labelled antigen and the antibody, which are left to equilibrate. After equilibrium (in the

TB and NSB tubes only) the bound fraction is precipitated and separated. Once the assay is completed, three sets of results should be available for each concentration of radioligand – The total counts (T), the non-specific binding counts (NSB) and the total binding counts (TB). The following can now be calculated: Specifically bound (B): B = TB – NSB Free ligand (F): F = T - B These can be converted from counts to concentration units as follows:

)/(activity specific1

)/(1022,21

)/( effeciency)/( counts)/(ion Concentrat 12 molCiCidpmdpmcpm

lcpmlmol ⋅⋅

⋅=

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 axis and Bound on the X axis. This data can be analyzed by linear regression to give a straight line. This is called a Rosenthal Plot.

The equilibrium constant of the antibody-antigen reaction (Kd) can be calculated from the negative reciprocal of the slope of the graph, whereas the x-intercept of the graph gives the total concentration of binding sites (BMax) measurable under assay conditions. The presence of a curve (as opposed to a line) indicates the presence of a mixed population of binding sites. It should be noted that it is more accurate to do binding analysis on the saturation curve analyzed 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. Since this is the variable containing the greatest error, a larger error will be distributed in both directions.

27. In centrifugation, how do you convert rpm to Xg force? What is the conversion

factor for the centrifuge in the RIA Laboratory? The relative centrifugal force (XG) can be calculated by the equation:

22

)()(00090

rpmspeedrotorcmradiusg

X g ⋅⋅=π

where 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 centrifugal force can be calculated using:

6503)( 2cmspeedrotorX g =

28. Summarise the principle of the plasma renin activity assay, and compare it to the

active renin assay. Renin is a proteolytic enzyme involved in blood pressure control. Renin cleaves angiotensinogen to produce the decapeptide angiotensin I. Angiotensin I is then rapidily cleaved to the biologically active angiotensin II by the angiotensin-converting enzyme. Two methods of estimating the amount of active renin are available - an enzyme kinetic assay and a mass assay.

Plasma renin activity This 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 kept at 37°C to allow for the generation of angiotensin I, the other is kept at 4°C to act as a blank. After 90 minutes the reaction is stopped (by cooling to 4°C) and the amount of angiotensin I is measured in each tube using a radioimmunoassay. The angiotensin I generated is then calculated as shown below. This is the plasma renin activity (PRA) and is usually expressed in ng/ml/hr.

Time

factorDilution PRA Volume Plasma

In AngiotensiIn Angiotensi 437 ⋅=

°° −

Active renin assay Historically, the problem with measuring renin directly was that renin exists in both active and inactive forms. The inactive from (prorenin) can account for up to 90% of the total renin in the circulation, making its measurement worthless. Since the discovery of antibodies which are selective for active renin, direct measurements are possible. The active renin assay is a immunoradiometric assay which uses two antibodies. The first antibody is a solid phase monoclonal antibody that recognises both active and inactive renin. The second antibody is a 125 iodine-labelled monoclonal antibody specific for active renin. The active renin is usually expressed in ng/l.

References[1-5]

1. Deupree, J.D.; Tutorial in Receptor Binding Techniques; http://www.unmc.edu/Pharmacology/receptortutorial/; Lurz, Matthew J.; 1998

2. Edwards, R., S. Blincko, and I. Howes, Principles of immunodiagnostic tests and their development; with specific use of radioisotopes as tracers, in Immunodiagnostics : 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 and Definitions Relevant to Clinical Laboratory Sciences; http://www.westgard.com/isoglossary.htm; Westgard, James O; 1999

4. Kricka, L.J., Principles of Immunochemical Techniques, in Tietz textbook of clinical 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 and molecular diagnostics, C.A. Burtis, et al., Editors. 2006, Elsevier Saunders: Philadelphia. p. XXXVI, 2412 s.