RIA （ PART ⅡA ） Equipment needed Quality control in RIA
Standards labeled antigen specific antibody patient sample
COMPONENTS OF THE RADIOIMMUNOASSAY SYSTEM
As indicated, three basic components form the basis of the radioimmunoassay system : (1) equilibration or incubation , (2) a method of separation of bound from free elements , and (3) counting of the radioactivity in the components.
Standards that are either identical or similar to the unlabeled antigen (ligand) are most important , they must also behave like the unknowns in the system.
Nonidentity with the substances to be measured occur because of (l) natural variations, such as “big” insulin, fragments of hormones, and other substances , (2) species differences, (3) artifact produced by degradation, and (4) synthetic errors in the manufacture of artificial substances. The stability of the antigen used as a standard becomes critical because the standards are often used in assays over a long period of time. There are interfering substances , which may prevent nonidentity of the standards with the unknowns. These include factors such as heparin ( and other drugs ), urea, bilirubin, buffers, temperature effects, and pH(power of hydrogen ).
The second major element of the RIA system is the labeled antigen. Important criteria for this reactant in the RIA system include: (1) high purification of the antigen and (2) its ability to be radiolabeled without loss of immuno-reactivity. Since many antigens are polypeptides that contain tyrosyl residues, they can be fairly easily labeled with iodine by a variety of methods; Often 125 I is used as the tracer, since a higher specific activity* with iodinated antigens can be obtained than when using either tritium or 14 C.
The third major element in the system is the specific antibody. The main criterion for a suitable antibody concerns its specificity for antigen interaction . This specificity is influenced by the heterogeneity of antibodies for the same antigen; that is, cross-reactivity for similar substances .
Since the antibodies are produced in a variety of animals, species variation may be important . it should be emphasized that the radioimmunoassay system depends on an antigen-antibody reaction and not upon the biologic activity of the antigen or ligand. Other influences on the interaction include antigenic damage that occurs with time (as by enzymes) and nonspecific binding of other ligands.
A specific concentration of the antibody must be used in the RIA system. The dilution of antibody is often chosen so that the antibody binds approximately 50% of the labeled antigen. This results in a bound to free ratio (B/F) of the elements is 1:1. Since the antibody is generally used in a very dilute solution, a high titer is not considered important in the selection of the antibody. However, the affinity of the antibody is extremely important.
In addition to the usual gamma-globulin antibody, there has been recent use made of tissue binders, which are also specific for binding certain antigens . Although not true antibodies, in the RIA system they function in a similar manner. These binders generally have high affinity for the antigens and recognize the active portion of the antigen.
The final element is the patient sample, which is really the antigen (unlabeled) to be measured in the serum or plasma, and is analogous or identical to the unlabeled standard antigen discussed above . Certain separation and purification steps may be necessary before the sample antigen is truly representative of the desired substance to be measured.
RIA KITS
For most laboratories, radioimmunoassay is performed with commercially available kits. These kits contain all the reagents needed to perform the RIA. The reagents and standards are generally shipped in lyophilized form and must be reconstituted before use.
I labeled antigen 125
specific antibody
Seperating agent
specification
standard
Equipment needed
In order to operate, an RIA lab needs four basic pieces of equipment: a well type of scintillation counter, a centrifuge, pipetting devices, and a refrigerator. An existing scintillation counter may be used if it is kept clean and located in a place where the background is low (50 to 100 cpm).
Equipment needed
A centrifuge is necessary in an RIA lab for both processing blood specimens and separation of bound and free ligands with certain separation methods. One of a larger centrifuges is adequate for most RIA labs. The centrifuge should have a swinging bucket (not fixed-angle head) rotor with a capacity of at least 50 tubes. Models that meet these specifications can achieve speeds of around 3000rpm, providing the 1000g necessary to achieve good separations.
Equipment needed
Pipetting devices are the most critical pieces of equipment in an RIA laboratory, because pipetting is the single largest cause of error in RIA. For a typical RIA, pipetting is required to dissolve or dilute reagents, mix samples and reagents together, and add a separating agent.
Pipetting device
dial
Equipment needed
A refrigerator is necessary for the RIA laboratory, primarily for storage ofspecimens and reagents. Household refrigerators are quite adequate for this purpose if no flammable things are stored in them. (Food or beverages Should nerver be stored in the same refrigerator with reagents and specimens.)
The reactant
The elements given in are incubated together to allow a reaction to occur. The competition between labeled and unlabeled ligands is allowed to occur simultaneously until equilibrium is reached between the ligands and the binder (antibody). Because of this equilibrium reaction, the RIA method is often referred to as “equilibrium saturation. ” The sensitivity obtained by radioimmunoassay , while dependent somewhat on the specificity of the antibody as well as antigenicity of the antigen , depends primarily upon the equilibrium constant (or affinity constant). This constant characterizes the energy of the antigen-antibody interaction and is usually of such great degree that a high sensitivity results.
SEPARATION OF BOUND FROM FREE REACTANTS
There are several methods used for separating the bound component from the free component. These are often selected to find which produce the best separation with the least effort while maintaining maximal sensitivity. The methods include differential migration by chromatography , electrophoresis , etc . precipitation of the bound form(such as by a double antibody technique), and absorption of the free phase by charcoal, resin, polypropylene tubes, etc.
The separation technique should have no effect on the antigen-antibody reaction . There are some advantages as well as disadvantages of each system for each separation. Since it is often difficult to predict which separatory technique will be best for any single system, empirical trials are usually necessary
MEASUREMENT OF RADIOACTIVITY
The radioactivity of the bound radioactive components and unbound radioactive components is measured by standard techniques. Gamma counting is used in the case of those tracers emitting gamma rays, such as 125 I and 131 I, Liquid scintillation counting is used to count the same components when 3H and 14 C Or other betaemitting radionuclides are used in the assay system. Because of the high specific activity of the labeled antigen or ligand, high count rates are often easily obtainable . This allows even greater precision in the counting statistics.
MANIPULATION
The measured radioactivity reflects the relative amounts of radioactive bound fraction and radioactive unbound fraction; the results are usually plotted as a "dose-response curve ". In each system certain effects and implications are best seen with specific coordinate systems. Generally, these coordinate systems are chosen to produce the maximal sensitivity for the assay when low levels are being measured, and an overall broad range of sensitivity when a wide range of values may be present in the unknowns.
MANIPULATION The specific plot is also selected to give an accurate reflection of the true quantity of substance being measured, especially at the lowest levels . Plots must allow precision for repeated measurements . The ordinate and abscissa may be in various combinations of linear, logarithmic, or other transformations. Attempts at linearization of the curves or alterations to allow computer generation of the curves may increase their utility . While curve manipulation may increase sensitivity, accuracy, and precision of the measurements, it does not truly alter the basic results of the measurement.
Patterns of the standard curve B/T
B/B0
Patterns of the standard curve B/f
logit B/B0 Straight line
Radioimmunoassay (RIA) Part ⅡB Quality control in RIA Some definitions
Usage of controls ★
The results obtained in any type of laboratory must be valid. Proved quality control measures should be routinely used in order to ensure valid results . These measures are doubly important in a clinical laboratory where results will significantly influence patient care. Quality control measures have generally been defined as any and all measures taken to ensure a valid result.
Why quality control ？ Quality control aimed at: 1. Monitor lab Errors. 2. Determine and resolve the unstable. factors which produce errors. 3. Ensure valid assay results and exclude the unreliable assay results. 4. Monitor and evaluate the quality of the RIA kit in order to make a good choice of it. 5. evaluate the quality of RIA between labs.
Factors that can induce errors There are many factors can induce errors ,for example : 1.Errors in sample preparation. 2.Errors in the reagents used.such as poor mixing of material thawed from the frozen state. 3.Inadequate separation of the bound from the free fractions. 4.Equipment errors e.g. statistical bias, counting efficiency, inappropriate setting of the counters and intermittent electronic faults causing radioactivity counting errors. 5.Operator errors due to human failing. 6.Faults in automatic pipettes or other apparatus.
Some definitions
To understand the language of quality control, a few definitions are required. Precision is the ability to reproduce repeatedly the same result on the same sample. Variation of results indicates an imprecise test. Precision is reflected by the magnitude of the standard deviation of the results.
Accuracy relates to how close the measurement is to the true value of the unknown material. A result may be extremely precise but inaccurate.
Sensitivity refers to the minimum amount of unknown that may be detectable. Specificity refers to the ability of a test to identify a particular substance in a mixture of substances
Reproducibility refers to repeatability which indicates the deviation that occurs in sample results when the test results are evaluated over a finite period of time, such as days, weeks, or months.
This assay characteristic depends upon the protein binding sites of the antibody and its respective ability to combine with only very specific receptor sites on the ligand (bound structure).
the specificity forms the cornerstone of the ability of radioimmunoassay techniques to measure specific substances. The specificity of a system is usually estimated by adding known interfering substances and comparing the doseresponse curves.
The different antibody preparations available may also be compared with a known standard to allow characterization of its specificity.
Stability of the reagents (components)
will determine the shelf life (how long an indivadule RIA kit can be used stably). Lyophilized (freeze dried) agents generally provide the longest shelf life. The frequency and number of tests performed influence this factor of kit selection.
COMPONENT EVALUATION
The evaluation of components of the Radioimmunoassay system is an important area of concern , particularly for those who are involved in the design and development of assays for specific substances . The component evaluation is much less important for those who use kit methods, since this evaluation will have been performed by the manufacturer.
RIA Kit Evaluation Some quality control includes: 1.Zero binding rate(B %).reflect the binding ability of 0 antibody and antigen.it must between 35-75%. It can influence Sensitivity and Precision. 2.Non specific binding rate(NSB),must lower than 4%. 3.ED The dose value when B/B0%=50% ,It must 50 maintain stable .It determines the assay range. 4.Coefficient Variation (CV)and Average Batch Coefficient Variation (ABCV) <5%. Is the average of all measurements
Usage of controls
Very
important
A control may be a standard or any known concentration of test material that serves as a comparison between successive runs (inter - assay), whereas the usual standard involves varying concentration used to evaluate individual samples within a single run ( intra-assay).
Controls may vary in concentration ranges so that high, medium, and low ranges are covered . Controls and standards are solutions that contain known amounts of material, but their end uses may differ.
high,=200
SDh=10
medium=50
SDm=5
Usage of quality control samples If the following situationlowoccur s,the assay must be ranges=10 SDl=2 repeated:
1 One value of control sample between high, medium, and low ranges > or < 3SD target value;
2.Two values of control samples between high, medium, and low ranges > or < 2SD target value and at the same direction;
3. Three values of control sample > or < 1SD target and at the same direction.
Class is over! Good bye!best wishes to you all!
程兵 Chengbing
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