radially, and form, upon confluence, immunoprecipitates. These immunoprecipitates often take the form of a curved line. Because of its numerous possible modifications,

 

Conjugates

¾ Store at recommended temperature

¾ Never store excessively diluted conjugate for use at

some later time

¾ Always make up the working dilution of conjugate just

before you need it

¾ Never leave conjugate on the bench for excessive time.

Addition of Samples

Problems caused by:

¾ Failure to put sample into buffer in well, leaving it on

the side of the plate.

Stopping Reagents

Stopping reagents are added to prevent further enzyme

reaction in ELISA. The stopping is usually made at a

time when the relationship among the enzyme-substrate

product is in the linear phase. Molar concentration of

strong acids or strong bases stops enzyme activity by

quickly denaturing enzymes. Some stopping reagents

are enzyme-specific. Sodium azide is a potent inhibitor

of HRPO, whereas EDTA inhibits alkaline phosphatase

by the chelation of metal ion cofactors. Since addition

of stopping agents may alter the absorption spectrum of

the product, the absorption peak must be known. Thus

H2SO4

–

 stopped ELISAs are read at 492 nm (450 nm before

stop).

Temperature

¾ Bring test reagents to room temperature (22–28°C)

approximately 30 minutes prior to use

¾ Maintain proper incubation temperature:

• Lower temperature can decrease OD values

• Higher temperatures can increase OD values

• Evaporation in wells can cause edging effect.

¾ The optimal temperature for incubation is 22–28°C

¾ Check temperature against calibrated thermometer

¾ Strict adherence to time must be maintained:

¾ Check calibration of timers

¾ Record time of incubation

¾ Read plate with specified time limits of adding stop

solution.

Rotation of Plates While Incubating Reagents

In certain ELISA systems, the plates are rotated during

incubation for better antigen-antibody reaction. The effect

of rotating plates is to mix the reactant completely during

the incubation step. Since the solid-phase limits the

surface area of the absorbed reactant, the mixing ensures

that, potentially reactive molecules are continuously

coming into contact with the solid-phase.

During stationary incubation, mixing only takes place

because of diffusion of reagents. Thus, to allow maximum

reaction from reagents in stationary conditions, greater

times of incubation may be required, than if they are

rotated.

Rotation also allow ELISA to be performed independent

of temperature conditions. The interaction of antigen

and antibodies relies on their closeness, and the kinect

energy provided to the system, which is encourage with

the mixing during rotation. Stationary incubation relies

on the diffusion of molecules and thus is dependent on

temperature.

Laboratory Conditions

The laboratory should be devoid of any acid fumes.

23

Diagnostic Immunology

Nonenzymatic, Quantitative Techniques

(Immunodiffusion, Electrophoresis and Turbidimetry)

C H A P T E R

QUALITATIVE DETERMINATION OF PLASMA

PROTEINS BY IMMUNOPRECIPITATION

The principle of immunoprecipitation was first described

by Kraus. Originally, immunoprecipitation reactions

carried out in test tubes were detected by the fact that

turbidity can be observed following the mixing of antigen

(Ag) and antibody (Ab) solutions. Centrifugation of such

samples results in an insoluble sediment. Layering the

antigen solution on top of the antibody solution in a narrow

test tube, without mixing, results in a ring-like turbidity at

the contact area within a short time. This so-called “ring

test” reveals the presence of Ag-Ab precipitation in the

solution studied. A distinction of characteristics between

the various antigens and antibodies cannot be made.

Immunodiffusion Method of Oudin

With this method, the Ag-Ab precipitation takes place

in a gel medium. Due to the different diffusion rates

of heterogeneous antigens (e.g. the proteins of blood

serum) the differentiation of the various antigens is

rendered possible. The linear (one dimensional) double

diffusion technique, i.e. diffusion of antigens and antibodies from opposite directions, results in the formation

of multilayered precipitations if heterologous Ag solutions

and multivalent antisera are used.

Agar is most frequently employed for the gel because of

its transparency and solidification characteristics.

Double Diffusion Method of Ouchterlony

Agar gel is poured uniformly onto glass plates or into Petri

dishes and allowed to solidify. Holes (wells) are cut into

the agar gel which are filled with Ag and Ab solutions,

respectively. Both the antigens and antibodies diffuse

radially, and form, upon confluence, immunoprecipitates.

These immunoprecipitates often take the form of a curved

line. Because of its numerous possible modifications,

the versatile arrangement of Ag-Ab containing wells,

etc. this technique is widely applied in immunological,

biochemical and medical laboratories.

Grabar and Williams’ Method of Immunoelectrophoresis

This represents a combination of physicochemical

and immunochemical techniques. The material to be

examined (e.g. tissue exudates, serum, etc.) is separated

electrophoretically on agar gel and, subsequently,

subjected to the effect of a precipitating antiserum. This is

done by placing antiserum in a trough in the agar parallel

to the electrophoretically separated proteins. Both the

antibodies and antigens diffuse toward one another,

forming, upon confluence, well-defined precipitin lines.