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Separation Systems in RIA

J . Kumarasamy Radiation Medicine Centre BARC

Separation Systems in RIA

RIA is a Stereo specific Biological reaction between Antigen and its specific Antibody, producing a complex of Antigen-Antibody, expressed as

Ag + Ab Ag-Ab [Ag] + [Ab] [Ag-Ab] [Ag*] [Ag] + [Ab] [Ag*-Ab] [Ag-Ab]

What are we interested in analysing..? ..... it is the Antigen Cold [Ag]...!!!

Separation Systems in RIA Cont..

It refers to the [Ag-Ab] complex in RIA There are two species of complexes present in RIA i.e. [Ag-Ab] &[*Ag-Ab]
The un-reacted species *Ag, Ag, Ab
What do you really want to Measure.!! It is the [Ag] What do you really Measure [ Ag*-Ab] and indirectly infer the Con of [Ag] Then Who is the culprit to be separated and who will interfere in the measurement


It is the [Ag*] that will if not separated completely..

Separation Systems in RIA Cont..

Physico-chemical properties are exploited to accomplish Separation

All separation system exploits the physicochemical difference between the ligand (Ag) in its free form and bound to the antibody (Ag-Ab) Antibody is the primary binder by and large.IgG 165 KD[Receptors/Binding Proteins are exceptions] Ag The analyte size may vary from few Daltons to a few KD Eg. T4 and Tg.

Physico-Chemical properties
(777 Da) (0.77 KD)

+ Ab-Ig [T4- Ab-Ig] No Size Gain

165 KD 166KD


+ Ab-Ig [Tg-Ab-Ig] Significant 165 KD 825 KD Size Gain + Ab-Ig

165 KD

(650 Da) (0.65 KD)

[T3-Ab-Ig] No Size Gain


Methods for the separation of bound and free ligands in a binding assay.
Method Electophoresis (Chromatoelectrophoresis, starch gel, Cellulose acetate, polyacrylamide) Gel filtration ( Column or batch ) Adsorption (Charcoal, magnetisable charcoal, sislicates, hydroxiapatite, proteinA- containing Staphylococci) Freactional precipitation (Ethanol,dioxan,polyethylene glycol, sodium sulphate, ammonium sulphate, trichloroacetic acid) Partition (Aqueous two phase) Second antibody precipitation (Stable and solid phase) References Berson et al., 1956; Hunter and Greenwood ,1964

Haber et al., 1965 Herbert et al., Rosselin et al., 1968; Jonsson and Kron- vall, 1977; Traffold et al ., 1974: Danes and Gardner, 1978 Heding, 1966, Thomas and Ferin,1968,Desbuquois and Aurbach, 1971; Grodsky and Forsham, 1960; Chard et al 1971;Mitchell and Byron, 1971.

Mattiasson, 1980 Utiger etal; 1962, Morgan and Lazarow,1963, Hales and Randle. 1963; Den Hollander and ScHuurs, 1977. Solid phase antibody (particles, magnetic Wide and Porath, 1966; Catt and Tregear, 1967; particles, tubes, gel entrapment, polymerised Updike et al1973; Donini and Donini , 1969; Nye antibody. et al, 1976;Halpern and Borden, 1979.

Immunoradiometric assay

Miles and Hales, 1968; Woodhead et al 1974.

Separation Procedure



The completeness with which the bound and free are separated.

In theory, a perfect separation system would completely divide and separate two components of the assay. But, in practice it is never achieved. - Reasons, - free behaves as bound this effect is know as - assay blank - non-specific binding - misclassification error

Reasons.. Cont..

- Physical trapping amid bound complexes - Adsorption to wall - Incomplete separation of bound complex due to - Impurities like free iodide (either impurity or dissociated) - Separation procedure may lead to dissociation

Reasons.. Cont..

speed / simplicity / applicability / cost

Various Assay Formats..

Liquid Phase Assays Semi solid phase Assays or Mobile Solid Phase Assays - Magnetic Particle (Cellulose Particles impregnated with Iron particles) - Latex Particles / Beads.. Solid Phase Assays.. - Strips / Sticks - Slides / Cover slips - Beads Polystyrene / Glass - Test tubes (PolyStyrene / PolyPropylene)

Methods of Separation
Physical Means
Free Adsorption - Actiated Charcoal - Hydroxy Appatites - Particulate Silicates Bound Adsorption - Protein A - Protein G - Protein A/G Recomb

Chemical Means
Fractionation - Sodium sulphate - Ammonium Sulphate - Ethanol - Dioxane - PEG

ImmunoBiological Means
- Protein A - Protein G - Protein A/G Recomb - Anti antibody or Second Ab

Physical Means
-Physical Adsorption of Free - Charcoal (Activated/Coated with Dextran)
- Particulate Silicates -What gets separated..! this way..

- Small Peptides/Hapten/Drugs/Steroids..

Physical Means
Physical Adsorption of Bound
Protein A/G

[Ag-Ab] Cpx

Chemical Means
Fractional Precipitation
Molecules remain in solution in a dissolved form because of their ability to attract the water molecules towards them and this ability forms a layer of water molecule surrounding the solute. Thus forming a shielding around the solute to let it remain in dissolved form. This salvaging molecules of water is called as the water of hydrating. Mechanism are developed targeting to reduce this water of hydration and there by bring about close proximity of dissolved molecules and letting it to aggregate. These aggregated molecules are packed together by means of centrifugal force to yield a compact pellet The process of aggregation can be enhanced by addition of inert immunoglobulin (as co-precipitating agent) or by means of addition of antiantibody (known as second antibody, double antibody etc..,)

Weather a substance will remain soluble or not at a given concentration of the separating agent is determined by its own ability to attract water molecule (than compared to that of the separating agent); for most biological molecules the latter is determined by electrostatic charge and this in turn by the isoelectric point and the pH of the medium. The grater the gap between these, the grater is the net charge, which the molecule carries and thus its ability to form water shell.

Fractionating Cont..

Assays are conducted at around neutral pH; increasing the concentration of salt or organic solvent will first precipitate those substances with an isoelectric point near neutrality, and then other materials in the order of their isoelectric point resulting in fractional precipitation.
Agents: - sodium sulphate - ammonium sulphate - ethanol - dioxane - PEG (Poly Ethylene Glycol)

Solid Phase Assays Immobilization Stratergies

-Primary antibody Coupling -Second antibody Coupling Physical adsorption Covalent attachment Coupling Chemistries - Amine Reactive Chemistry - Carboxyl reactive Chemistry - Sulphide reactive Chemistry - Formyl group reactive Chemistry -Oriented immobilization - Second Ab Solid phase - Protein A /G

Non Separating Immunoassays

Assays are also developed in the direction of measuring the end products without separating the participating reactants Eg. - Nephlometry - Turbidymetry
Limitations: Not Sensitive in nano Molar concentration

Future Directions of Immunoassays are..

Label Free Assays

LC/MS SPR AFM Tunnelling Microscopy