Separation Systems in RIA

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Separation Systems in RIARIA 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]

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

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


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FreeThe un-reacted species *Ag, Ag, Ab

BoundIt refers to the [Ag-Ab] complex in RIA

There are two species of complexespresent in RIA i.e. [Ag-Ab] &[ * Ag-Ab]

Separation Systems in RIA Cont..

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 themeasurement

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


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Separation Systems in RIA Cont..

All separation system exploits thephysicochemical difference between theligand (Ag) in its free form and bound tothe 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 fewDaltons to a few KD Eg. T4 and Tg.

Physico-chemical properties areexploited to accomplish

Separation


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Physico-Chemical propertiesT4 + Ab-Ig [T4- Ab-Ig] No Size Gain(777 Da) 165 KD 166KD

(0.77 KD)

Tg + Ab-Ig [Tg-Ab-Ig] Significant(660KD) 165 KD 825 KD Size Gain

T3 + Ab-Ig [T3-Ab-Ig] No Size Gain(650 Da) 165 KD 166(0.65 KD)


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Method ReferencesElectophoresis (Chromatoelectrophoresis,starch gel, Cellulose acetate,polyacrylamide)

Berson et al., 1956; Hunter and Greenwood ,1964

Gel filtration ( Column or batch ) Haber et al., 1965Adsorption (Charcoal, magnetisablecharcoal, sislicates, hydroxiapatite, protein-A- containing Staphylococci)

Herbert et al., Rosselin et al., 1968; Jonsson andKron- vall, 1977; Traffold et al ., 1974: Danes andGardner, 1978

Freactional precipitation(Ethanol,dioxan,polyethylene glycol, sodiumsulphate, ammonium sulphate, trichloro-acetic acid)

Heding, 1966, Thomas and Ferin,1968,Desbuquoisand Aurbach, 1971; Grodsky and Forsham, 1960;Chard et al1971;Mitchell and Byron, 1971.

Partition (Aqueous two phase) Mattiasson, 1980Second antibody precipitation (Stable andsolid phase)

Utiger etal; 1962, Morgan and Lazarow,1963,Hales and Randle. 1963; Den Hollander andScHuurs, 1977.

Solid phase antibody (particles, magneticparticles, tubes, gel entrapment, polymerisedantibody.

Wide and Porath, 1966; Catt and Tregear, 1967;Updike et al1973; Donini and Donini , 1969; Nyeet al, 1976;Halpern and Borden, 1979.

Immunoradiometric assay Miles and Hales, 1968; Woodhead et al 1974.

Methods for the separation of bound and free ligandsin a binding assay.


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Separation Procedure

Efficiency Practicality

Efficiency: The completeness with which the bound and free are separated.

In theory, a perfect separation system would completely divide and separatetwo 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


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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


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Reasons.. Cont..

Practicality

speed / simplicity / applicability / cost


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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)


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Methods of Separation

Physical Means Chemical Means Immuno-Biological Means

Free Adsorption- Actiated Charcoal- Hydroxy Appatites- Particulate Silicates

Bound Adsorption

- Protein A- Protein G- Protein A/G Recomb

Fractionation- Sodium sulphate- Ammonium Sulphate- Ethanol- Dioxane

- PEG

- Protein A- Protein G- Protein A/G Recomb- Anti antibody or

Second Ab


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Physical Means

-Physical Adsorption of Free - Charcoal (Activated/Coated with Dextran)- Particulate Silicates

-What gets separated..! this way..

- Small Peptides/Hapten/Drugs/Steroids..


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Physical MeansPhysical Adsorption of Bound

Protein A/G

[Ag-Ab] Cpx


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Chemical MeansFractional Precipitation

MechanismMolecules remain in solution in a dissolved form because of their

ability to attract the water molecules towards them and this ability forms alayer of water molecule surrounding the solute. Thus forming a shieldingaround 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 andthere by bring about close proximity of dissolved molecules and letting it toaggregate.

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 inertimmunoglobulin (as co-precipitating agent) or by means of addition of anti-antibody (known as second antibody, double antibody etc..,)


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Fractionating

Fractionating:Weather a substance will remain solubleor not at a given concentration of the separatingagent is determined by its own ability to attractwater molecule (than compared to that of theseparating agent); for most biological moleculesthe latter is determined by electrostatic chargeand this in turn by the isoelectric point and thepH of the medium. The grater the gap between

these, the grater is the net charge, which themolecule carries and thus its ability to form water shell.


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Fractionating Cont..

Assays are conducted at around neutral pH;increasing the concentration of salt or organicsolvent will first precipitate those substances withan isoelectric point near neutrality, and then other materials in the order of their isoelectric pointresulting in fractional precipitation.

Agents:- sodium sulphate- ammonium sulphate- ethanol- dioxane- PEG (Poly Ethylene Glycol)


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Solid Phase AssaysImmobilization Stratergies

-Primary antibody Coupling-Second antibody Coupling

Physical adsorptionCovalent 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


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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


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Future Directions of Immunoassays are..

Label Free AssaysLC/MS

SPR AFM

Tunnelling Microscopy

Separation Systems in RIA

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