Outline

Chapter 2.1: Principles of Competitive and Immunometric Assays (Including ELISA) 1.0: 2.0: 3.0: 4.0: Introduction Kinetics of AntibodyAntigen Interactions Immunoassay Design Conclusions

-2-

1.

Introduction

Immunoassays are sensitive analytical tests that harness the unique properties of antibodies They proved to be one of the most productive technological contributions to medicine and fundamental life-science research in the twentieth century Some of these have resulted in more sensitive assays that have opened up new horizons of clinical research and diagnosis, while others have concentrate on simplifying the requirements for support ing technology, rendering it amenable to automation

BioMEMs & Nanoelectrochemistry Lab

-3-

2. Kinetics of AntibodyAntigen Interactions

Scheme: antigen interact with solid immobilize antibody

At the heart of every immunoassay is the binding reaction between the antibody and the analyte. This reaction takes from a few seconds to many hours to achieve equilibrium, depending on a range of different factors.

BioMEMs & Nanoelectrochemistry Lab

-4-

2. Kinetics of AntibodyAntigen Interactions

Figure 2: scatchard plot

linear relationship between the ratio of [bound]/[free] antigen and the con of bound antigen
BioMEMs & Nanoelectrochemistry Lab
-5-

2. Kinetics of AntibodyAntigen Interactions

BioMEMs & Nanoelectrochemistry Lab

-6-

2. Kinetics of AntibodyAntigen interactions

More conveniently expressed as the percentage bound in relation to the total antigen present: FIGURE 10 Influence of antibody concentration.

FIGURE 9: Competitive assay doseresponse curve.

FIGURE 11 Influence of Keq.

-7-

3.0 Immunoassay Design

-8-

3.1 Competitive (reagent-limited) assay

FIGURE 12 Competitive (reagent-limited) immunoassay.
FIGURE 13 Typical calibration (doseresponse) curve.

FIGURE 12 Competitive (reagent-limited) immunoassay.

The concentration of antigen in unknown samples may then be interpolated from the calibration curve
-9-

3.2 Single-site & two-site Immunometric assay

FIGURE 14 Single-site immunometric assay. FIGURE 15 Two-site immunometric assay (reagent excess).

One antigen was just able to bind with one antibody

One antigen can bind with two anti body (because the protein have multiple epitops)

-10-

3.3 Determinants of assay sensitivity

FIGURE 17 Estimation and sensitivity. The sensitivity of any analytical technique is defined as the minimal concentration that can be reliably estimated. Sensitivity therefore depends upon two factors: the increase in signal seen in the presence of analyte and the errors in measurement when no analyte is present

-11-

3.4 Competitive Assay Design

Competitive assay scheme:

The problems which have to study: The relationship between the percentage binding (signal) & other elements (Keq , Conc of capture antibody, antigen & labeled antigen). How to obtain the optimal sensitivity?
-12-

3.4 Competitive Assay Design

Theory of competitive Assay

-13-

3.4 Competitive Assay Design

FIGURE 19: Antibody dilution curves
FIGURE 20: Antigen diluti FIGURE 21: Antigen dilut on curves: Effect of Abt ion curves: effects of Keq .

10-11mol/l

10-9 mol/l

The position of this critical region decreases with decreasing conc of antibody. The use of low antigen conc therefore necessitates the use of correspondingly low antibody conc to maintain the assay in the region where further addition of antigen gives detectable displacement of the labeled antigen binding
-14-

3.5 The sensitivity in competitive assay

FIGURE 22 Effect of labeled antigen TABLE 1 Optimal Assay Characteristics and antibody concentration sensitivity.

The surface plot above shows the predicted sensitivity as a function of the antibody and labeled antigen concentrations Optimal sensitivity is achieved by decreasing the concentration of labeled antigen and increasing the concentration of antibody to raise the specific binding and thus partially compensate for the increase in non-specific binding.
-15-

3.5 The sensitivity in competitive assay

FIGURE 28 Percentage bound. FIGURE 30 Bound/bound at zero dose.

(assay d)

(assay c)

The dose-response relationship in the familiar terms of percentage binding

-16-

3.6 Immunometric Assay Design

With the following the assumptions:

1) How about the antigen bound to the solid phase in the second incubation? 2) The sensitivity of system?
-17-

3.6 Immunometric Assay Design

[Ab2f] is the free labeled antibody concentration. [Ab2b] is the binding labeled antibody concentration.

[Ab2f] = [Ab2t] [Ab2b]

This corresponds to the sensitivity of the assay:

-18-

3.6 Immunometric Assay Design

FIGURE 32 Effect of antibody conc on sensitivity. The first: there is an optimal conc of labeled antibody giving maximal sensitivity. This region is bounded at high conc by the greater rate of increase in non specific binding than specific binding A reduction in signal-to-noise ratio. The second: the sensitivity is governed by the concentration of labeled antibody rather than the capture antibody, which little influence at concentrations greater than 1 109 mol/L

-19-

3.6 Immunometric Assay Design

FIGURE 33 Effect of labeled antibody equilibrium constant on sensitivity This figure shows the influence of the labeled antibody equilibrium constant on the conc of labeled antibody required to attain maximal sensitivity. For any given labeled antibody conc, increasing the equilibrium constant increa ses the initial slope of the doseresponse curve and hence the sensitivity.

-20-

3.7 Solid-Phase Immunoassays

FIGURE 38 Capture bridge assay.

With simultaneous incubations, high-dose hook effects may be problematic, with excessive amounts of antibody swamping the epitops on both the solid phase and labeled antigen and preventing coupling between them.
-21-

3.8 Kinetic Constraints

FIGURE 42 Formation of boundary layer.

FIGURE 42 Formation of boundary layer.

where diffusion is a function only of the size of the molecule and their viscosity The presence of the boundary layer at the solid phase: Here antigen is rapidly depleted by surface-bound antibody, the replenishment of which is limited by diffusion.
-22-

4. conclusions
We study the kinetic of Kinetics of AntibodyAntigen Interactions.

Then amplification to the competitive assay:

And the Immunometric Assay Design:

Finally: we also study about the Immunoassay Design: competitive and non-competitive assay
-23-

Thank you

-24-