Sandwich Assays :

IRMA and ELISA ImmunoRadioMetric Assay Enzyme Linked ImmunoSorbent Assay Need 2 Antibodies To The Same Antigen Capture Antibody Labelled Antibody Antibody is in excess Antigen is in limited concentration

HOW DOES IT WORK ?

HOW DOES IT WORK ?
Antibody bound to solid support (CAPTURE antibody)

Sample Antigen

Labelled Antibody

Sandwich Assays

GPL

SEPARATION
of BOUND (labelled) Antibody from FREE (labelled) Antibody WHY? Because the Detector cannot discriminate between and signal from BOUND label

signal from FREE label

Counting of signal from one fraction

Y Y Y Y Y Y

Y Y Y Y Y Y

Y Y Y Y Y Y

Y Y YY Y Y
+
(Variable Unknown Conc)

Y Y Y

CAPTURE antibody

Incubate

Sandwich Assays

GPL

Y Y Y Y Y Y

Incubate

Sandwich Assays

Y Y

Y Y

GPL

YY

Labelled Antibody

Bound Labelled Antibody (Sandwich)

Free Labelled Antibody

4

CALIBRATION CURVE
Plot Signal against Concentration Straight line graph Useful over several orders of Concentration, but graph doesnt show this well
CALIBRATION CURVE
Plot Signal against Concentration Straight line graph Useful over several orders of Concentration, but graph doesnt show this well
60 50 40 30 20 10 0 0 50 100 150

Modify the plot to spread evenly across x axis Plot log (Signal) against log (Concentration) Straight line graph

Modify the plot to spread evenly across x axis Plot log (Signal) against log (Concentration) Straight line graph
100 10 1 0.1 0.1 1 10 100 1000

Sandwich Assays

GPL

Sandwich Assays

GPL

Noncompetitive Sandwich Assays

Are Suitable For Large Antigens Typically proteins Why are small antigens unsuitable for IRMA, ELISA? Antigen must be large enough to allow two different antibodies to independently bind to it without physical interference from each other

HOW MAY WE CATEGORISE THE IMMUNOCHEMICAL ASSAYS WE HAVE DISCUSSED?

Competitive or NonCompetitive Heterogeneous or Homogeneous ImmunoAssay or Immunometric Assay

(Limited Antibody or excess Antibody?)

(Separation step or no separation step?)

(Labelled antigen or Labelled Antibody?)

RIA FPIA EMIT CEDIA

Competitive Competitive

Heterogeneous Homogeneous

ImmunoAssay ImmunoAssay

IRMA, Sandwich ELISA

NonCompetitive

Heterogeneous

Immunometric Assay

Sandwich Assays

GPL

Polyclonal And Monoclonal Antibodies

Specificity in immunochemical assays is limited by the specificity of the antibodies used Originally only polyclonal antibodies were available : Expensive, limited purity, labour intensive IRMA and ELISA need Monoclonal antibodies 2 different antibodies to the same antigen Antibodies are produced by lymphocytes Each lymphocyte produces only 1 type of antibody Different lymphocytes produce different antibodies Different antibodies to the same antigen must come from different lymphocytes

Preparation of polyclonal antibodies

(i) purify Ag as much as possible (ii) inject Ag into an animal (iii) wait for animal to raise antibodies (iv) bleed animal, or use a lymphocyte rich tissue (e.g. spleen) (v) extract a heterogeneous antibody fraction, purify the Ab of interest by traditional protein purification methods Note: what if the animal doesn't produce antibodies to a substance? Examples : digoxin, paracetamol, T3, T4 haptens

Preparation of monoclonal antibodies

(i) purify Ag as much as possible (ii) inject Ag into an animal (iii) wait for animal to raise antibodies (iv) bleed animal, or use a lymphocyte rich tissue (e.g. spleen)

(v) extract intact lymphocytes (vi) hybridise lymphocytes with immortal lymphoma cells The hybrid cells have : (a) the longevity of the lymphoma parent (b) the specificity of the lymphocyte parent (1 type of antibody per lymphocyte) (vi) plate hybridoma cells and allow colonies to develop from individual cells (vii) select colonies producing desirable antibodies and propogate these lines (forever) by tissue culture methods (viii) scale up culture methods to allow production of antibodies from a line whenever they are required and in whatever quantities are required This allows : Large scale production Of a variety of antibodies To different epitopes Of a single antigen