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
4
( 2 He ); in beta decay, the nucleus emits an electron (or positron), and in gamma decay,
the nucleus emits gamma particles (high-energy photons).

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

3
H 12,3y β- 3
H⎯
⎯→23 He+ −10e
1
14
C 5730y β- 14
C⎯
⎯→147 N + −10e
6
32
P 14,3d β- P⎯
⎯→32
16 S+ −1 e
32 0
15
35
S 87d β- 35
S⎯
⎯→17
35
Cl + −10e
16
51
Cr 27,7d EC, γ 51
⎯→5123V + γ
Cr + −10e ⎯
24
57
Co 272d EC, γ Co + −10e ⎯ 26 Fe + γ
⎯→ 57
57
27
58
Co 71d EC, β+, γ 58
Co + −10e ⎯
⎯→ 26
58
Fe + γ
27
 58
27 Co ⎯
⎯→ 26
58
Fe + 10p + γ
59 -
Fe 45d β,γ 59
Fe ⎯
⎯→ 27
59
Co + −10e + γ
26
99
Mo 66h β-, γ 99
⎯→9943Tc + −10e + γ
Mo ⎯
42
99m
Tc 6,0h γ 99 m
⎯→9943Tc + γ
Tc ⎯
43
125
I 60h EC, γ 125
⎯→12552Te + γ
I + −10e ⎯
53
131
I 8,04d β, γ I⎯ 54 Xe + −1 e + γ
⎯→131
131 0
53

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)
-5
hCG 5x10 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 Conversion International Conversion

unit factor recommended standard
unit
Active Renin pg/ml (ng/l) x 1,8 mIU/l WHO 68/356
Growth Hormone ng/ml (μg/l) x3 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:

counts (cpm / l ) 1 1
Concentration (mol / l ) = ⋅ ⋅
effeciency (cpm / dpm) 2,22 ⋅ 10 (dpm / Ci ) specific activity(Ci / mol )
12

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:

π2
Xg = ⋅ radius (cm) ⋅ rotor speed (rpm) 2
90 000 g
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:
rotor speed (cm) 2
Xg =
3 650

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.

Angiotensin I 37° − Angiotensin I 4°

⋅ Dilution factor
PRA = Plasma Volume
Time
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.