the ordinate at b. The slopes a and b are thus constants determined by the Ag-Ab system under consideration. The length of the cylinder of precipitate is proportional to the

 

Cholinesterase Stain

Indoxyl acetate: 5 mg indoxyl acetate is dissolved in 0.5 mL

acetone, to which are added immediately thereafter 22 mL

of a diethylbarbiturate acetate buffer solution pH 8.2, µ =

0.05 (1 volume of buffer µ = 0.1 and one volume of distilled

water) and 2.5 mL of copper acetate solution 0.1 µmol/L

(0.018) g copper acetate in 100 mL distilled water). This

solution must be used within 8 hours after preparation.

After staining for 2 hours, and subsequent washing in tap

water for 1 hour, cholinesterase is indicated by blue color.

Lipid-protein Double Staining

A volume of 0.5% oil red solution in 50% ethanol is

suitable as a lipid stain. Place the slide in the dye mixture

for approximately 2 hours, then wash 50% ethyl alcohol.

A volume of 0.5% light green solution in 5% trichloroacetic

acid is then immediately applied to achieve the

specific protein stain. The staining time for proteins is

approximately one hour. Wash with trichloroacetic acid.

The slides must be photographed after for keeping a

permanent record.

Identification and Interpretation of Precipitin Lines

The analysis of immunoelectrophoretic patterns requires a

certain degree of experience and training. It is imperative

to be able to recognize the various specific precipitin lines.

Various techniques are available to achieve identification

of given precipitin line by experimental means:

1. Employment of specific antisera

2. Specific stains

3. Selective absorption

4. Employment of purified proteins.

Practical Application of Immunoelectrophoresis

Increased immunoglobulin concentrations are indicated

by elongated and thickened precipitin lines of IgG,

IgA and IgM immunoglobulins and antigen excess

result in positions of these precipitin lines closer to the

antibody trough in comparison with those obtained with

normal sera. Although IEP does not lend itself to precise

quantitation of serum proteins, marked deviations from

normal concentrations may be recognized.

Increased IgG, IgA and IgM are frequently associated

with acute and chronic liver diseases. This is the case

in acute hepatitis, in which a particularly impressive

increase in IgM occurs. In infectious mononucleosis,

chronic hepatitis and Laennec’s cirrhosis all three

major immunoglobulins are frequently, but not invariably, increased. In lipoid hepatitis the IgG-globulin

precipitin line may be markedly accentuated. Collagen

diseases, including Sjögren’s syndrome and systemic

lupus erythematosus (SLE), as well as certain infections,

may be associated with hyperimmunoglobulinemia as

indicated by IEP. In trypanosomiasis, the IgM may be

excessively increased. Certain virus diseases, such as

Coxsackie infections, are often associated with increased

immunoglobulins.

In most cases of sarcoidosis IEP has not revealed

abnormal serum protein changes, though definite IgA

increases have been observed by some. IEP revealed

increased immunoglobulin contents in a variety of

additional diseases such as dermatitis herpetiformis and

dermatitis gestationis and pernicious anemia in which

IgA, and mongolism in which the IgA and IgG were

increased. Besides all the causes mentioned above, the

disorders mentioned under hypoalbunemia, monoclonal

and polyclonal gammopathies discussed under serum

protein in clinical chemistry chapter can also be assessed

by using IEP techniques.

Quantitative Determination of Plasma Proteins by

Immunoprecipitation

The reagents used for quantitative determination are

specific antisera which react stoichiometrically with the

proteins to be determined and form precipitates with them.

These immune reactions may occur either in solutions or

in gels which contain the antiserum in even distribution.

In the first case, the quantity of immune precipitate,

measurable at appropriate dilutions as turbidity or by

other means, gives a quantitative measure of the antigen

concentration. By using, as the reaction medium, a gel

containing antiserum, the reagent is arranged in the form

of a stationary phase into which the antigen can penetrate

either by diffusion or under the influence of an electrical

field. The resulting precipitates, which assume different

698 Concise Book of Medical Laboratory Technology: Methods and Interpretations configurations depending on the method used, can be

quantitatively evaluated.

In the four most widely used methods to be described,

reaction conditions have been selected which ensure that

the quantity of specific antiserum is constant and exceeds

the quantity of antigen, while the concentrations of the

antigens to be determined can vary within wide ranges.

FUNDAMENTAL QUANTITATIVE CONSIDERATIONS

Photometric Method

Principle: This method is a modification based on

the determination of the nitrogen content of isolated

immunoprecipitates as described by Heidelberger. In

contrast to the tedious technique, the antigen and antibody

are allowed to react together at high dilution. Under these

conditions, the reaction product of the immune reaction

does not result in the formation of the precipitate which

sediments, but merely in the appearance of tubidity. The

extinction (e.g. at 450 nm) can be used as a measure of

the turbidity. If the quantity of antiserum is kept constant

and the antigen concentration is varied, a curve can be

obtained. When the antigen is present in excess, soluble

reaction products are formed. For this reason, only the

ascending limb of the curve can be used for quantitative

determinations. By reference to this part of the curve, the

concentration of antigen in an unknown solution can be

determined.

Single Linear Immunodiffusion of Oudin

Principle: Gel containing the antiserum is allowed to

solidify in a test tube, and the antigen solution is poured

on top of it. The precipitate formed at the boundary layer

migrates into gel zone. The distance of the front of the

precipitate from the boundary surface is proportional to

the square root of the diffusion time, t.

h = k x √t

As the migration velocity of the antigen

h

k = ——

  √t

is proportional to the logarithm of its concentration CAg

and the distance h through which the precipitate migrates

may be expressed by the following equation:

 h

log CAg = a × k + b = a x —— + b

      √t h

Accordingly, if CAg as a function of ——, is

     √t

plotted in a semilogarithmic system, the result is a

straight line. Its slope is represented by a and it intersects

the ordinate at b. The slopes a and b are thus constants

determined by the Ag-Ab system under consideration. The

length of the cylinder of precipitate is proportional to the

negative logarithm of antibody concentration.

The fundamental difference between the techniques

of single linear immunodiffusion and single radial

immunodiffusion and electroimmunodiffusion (EID)

described below is simply that in the former the antigen

concentration is determined from the speed of migration

of an immunoprecipitate; while in the latter methods, it is

determined by measuring the precipitin area on the length

of a precipitate which remains constant after a certain time

period.