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   Table of Contents      
Year : 1972  |  Volume : 20  |  Issue : 2  |  Page : 49-54

Immunological techniques

Dept. of Opthalmology Research, McGill University, Montreal, Canada

Correspondence Address:
P D Mehta
Dept. of Opthalmology Research, McGill University, Montreal
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Source of Support: None, Conflict of Interest: None

PMID: 4202991

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How to cite this article:
Mehta P D. Immunological techniques. Indian J Ophthalmol 1972;20:49-54

How to cite this URL:
Mehta P D. Immunological techniques. Indian J Ophthalmol [serial online] 1972 [cited 2023 Dec 10];20:49-54. Available from: https://journals.lww.com/ijo/pages/default.aspx/text.asp?1972/20/2/49/34669

  Immunodiffusion Top

In double diffusion test, antibody and antigen migrate toward each other through the agar gel. As the reagents come in contact with each other, they combine to form a pre­cipitate in the gel. This test offers the unique advantage of not only ennumerating the minimum num­ber of antigen-antibody systems reacting in a given mixture, but also indicating the relationship among various antigens. To deter­mine the complexity of a mixture of the antigens, therefore, it is im­portant to utilize an antiserum containing antibodies to all the antigens involved.

Preparation of Gels

The most commonly used mate­rial for gel diffusion test is agar which is dissolved in physiological salt solutions. In our laboratory the suspension of agar, containing 0.1% of sodium azide as a preser­vative, is heated in the boiling waterbath, till it melts. A layer of about 3 mm thick of melted agar is poured into a petri dish and allowed to solidify so as to form a perfectly level surface. Later on, a template containing cyliderical openings is placed on the top of the dish. The agar plug is sucked out through a flattened end of a pulled glass tubing connected to a vac­uum line. After filling the walls with antigen and antibody solutions the diffusion plates are usually incubated at 25˚C. The interpreta­tion of precipitin patterns obtained in the double diffusion test is des­cribed below. The four basic patterns that can be obtained with an antiserum and preparations of single antigens are illustrated in [Figure - 1]. -

Pattern I- called pattern of identity indicates both antigens have a set of indentical determin­ant groups with respect to the antiserum employed.

Pattern II - Called pattern of nonidentity indicates that the antiserum used does not contain antibodies to any determinant groups common to both antigens.

Pattern III- called the pattern of partial indentity indicates that one antigen reacts more fully with the antibodies employed while other antigen reacts with fewer of these antibodies, thus allowing the formation of a spur.

Pattern IV- indicates that the two antigens have at least one type of determinant group in common and in addition, each has at least one type and probably more types, of antigenic grouping shown by the other.

The sensitivity of the double immunodiffusion test depends to a large extent on the distance bet­ween wells and the relative con­centration of reactants. Sensitivity also depends on the concentration, thickness and the viscosity of the gel employed.

The gel diffusion test is suited for the ennumeration of antigens in a given mixture and for the de­termination of antigenic relation­ships among various antigens and has been used extensively for these purposes in systematic biology.

  Radial Immunodiffusion in Plates Top

Principle of the Method

Simple radial immunodiffusion is the procedure whereby an anti­gen is deposited at a single point (practically in a small cylindrical well) of a thin layer of gel contain­ing a uniform concentration of antibody. As the antigen diffuses into the antibody field, a disc-­shaped immune precipitate is for­med. It has been known for a long time that a quantitative relation­ship exists between the amount of antigen applied to the well and the size of the resulting precipitate.

Recommended Technique

I. Preparation of the Agar

To 100 ml of barbiturate buffer of pH 8.6 and ionic strength 0.1 (made by dissolving 9 gm of sodium diethylbarbiturate, 65 ml of 0.1 NHC1, and 0.5 gm of sodium azide in distilled water and adjust­ing the volume to 1 liter) is added 3 gm of Special Agar-Noble (Difco). The suspension is stirred on a boil­ing waterbath until dissolved, and distilled water is added to replace losses due to evaporation. This stock agar solution is distributed over a number of well-stoppered test tubes, which may be stored for several weeks at 4° .

II. Preparation of the Agar-Anti­serum Mixture

The required amount of solidified 3% agar gel is melted on a boiling waterbath and cooled at 60˚. A suitable dilution of the antiserum (in barbiturate buffer) is warmed to 55˚ and mixed at equal volumes with the molten agar. The mixture is thoroughly stirred (avoiding bubbling) by means of pipette pre­heated to 60˚ in the waterbath. The antiserum-agar mixture is then poured, without delay, into the mould described below, the same heated pipette being used.

To find the appropriate antiserum concentrations one has to set up a few plates with different dilutions of antiserum, the antigen wells being filled with serial dilutions extending over the range contem­plated for practical applications. The antigen must be in significant excess whatever the most useful antibody concentration chosen.

III. Preparation of the Immunodi­ffusion plate

In our laboratory gel of agar mixed with the antiserum is poured into 3 inch diameter petridish. After the agar being solidifed, circular holes of 3 mm diameter are made in the dish. The agar is removed from the well and the solutions of antigen of varying concentration are placed in the holes and the plates are incubated at 25˚C.

IV recording-the Results.

For rough semiquantitative work (i,e.. screening tests), it may be sufficient to read the plate after 1 night of incubation, but as already stated, highly accurate results are obtained if the measurements are postponed until the rings no longer grow in size.

Some workers limit themselves to measuring the diameter of each ring (by holding a comparator scale against the plate under obli­cue illumination), and plotting the diameters on a semilogarithtnic scale (or by plotting the square of the diameter on an ordinary scale).

Recent Applications

The method has also been ad­apted to the qualitative and quanti­tative analysis of antigenic relation ships between different substances and a system has been described by which an antigen related to the immunizing antigen can be titrated without the requirement of a se­parate calibration curve.

  Complement Fixation Top

The complement fixation test depends on two properties of complement :

1. The bind or fixing of comple­ments by antigen-antibody aggre­gates, and

2. The lysis of sensitized erythrocytes.

The test is performed in two steps. First, the serum to be tested for antibody, a specified dose of complement, and an appropriate amount of antigen are allowed to react at a given temperature. Sen­sitized sheep cells are then added and the mixture is incubated at 37° C. for sixty minutes. If the serum contains antibody to the antigen used, the complement is fixed when combination of antigen with antibody occurs. Thus, failure to obtain lysis denotes a positive reaction, while complete hemolysis indicates a negative reaction, meaning that the serum did not contain antibody to the antigen employed.

In the past most complement fixation procedures have been based on the use of 100% units of complement but since 100% he­molysis does not furnish a sharply defined end point for the hemolytic titration of C', in recent years there is increased recognition of the advantage of 50% hemolysis as the end point.

The 50% hemolytic unit of the complement CH 50 is defined as the quantity of complement re­quired for 50% lysis.


The complement system of fresh guinea pig serum has the capacity to combine irreversibly with anti­gen-antibody complexes. If the antigen is associated with the sheep erythrocyte cell surface such a combination may result in lysis of the erythrocyte and, thus, offers an excellent indicator system for C' activity. If two different antigen-antibody systems were added to guniea pig serum, a com­petition for the C' components would result. If, however, C' is allowed to incubate with the anti­gen-antibody system under study, then its combination can be esti­mated by the residual hemolytic activity it possesses when a known quantity of antibody-coated, ie., sensitized, erythrocytes is added at later time.

C-Fixation Procedure

The reagents for the complement fixation test were prepared as des­cribed by MAYER et al. (1948)

To a series of 40-m1 centrifuge tubes in an ice bath is added, in order, 1.0 ml diluted antisera. 3.0 ml diluent, 1.0 ml diluted C' (pre­viously titered), and finally 1.0 ml antigen solution serially diluted 2-fold. Appropriate dilutions of antigen (usually the highest con­centration) and C'. antibody and C', diluent and C', and diluent in total volume of 6.0 ml serve as con­trols and are included in every experiment. After incubation at 2-4° for 16-18 hours. 1.0 ml EA is added and hemolysis allowed to proceed, with occasional swirling. in a waterbath at 35-37° until con­trols are visually estimated to be 90-90% hemolyzed, in about 40-60 minutes. After immersion in an ice­bath to stop the hemolytic reaction, the mixtures are centrifuged for 10 minutes to sediment the unlys­ed EA. and the optical density (OD) of the heme in the supernat­ant fluid is determined at 413 mu. Providing the antigen and anti­body controls do not give evidence of independent pro or anti C' activity. the results may be ex­rressed in either of two ways: (1) as the difference between the OD of the controls (antigen, antibody, and C,) and the OD of the experi­mental tubes (OD), or (2) as the percentage of C' fixed, calculated as (reaction OD/control OD) X100.


The effect of chemical, physical, or enzymatic treatment in altering the immunochemical properties of proteins can be determined by the changes in direct C' fixations. This technique can profitably be used in conjunction with some of the other techniques presently utiliz­ed to investigate conformation changes in proteins. Indeed, the C' fixation assay has proven to be more informative in some cases. For example, exposure of lactic dehydrogenase to urea, followed by dialysis. yields a protein that has regained its original polarization of fluorescence properties. It has, however, refolded by its C' fixation. With proteins possessing no bio­logical activity. the micro-C' fixa­tion assay offers, perhaps, the simplest and most sensitive tool for determining whether renatura­tion has yielded to original mole­cule. It has advantage over other quantitative im.muno-chemi­cal techniaues, commonly used to detect conformational changes. The ability to detect similarities in the same protein from different tissues or different species is of course an obvious use for the assay. The re­nuirement for extremely small amounts of reactants and the ease of performing the analyses. once the system has been standardized, have been mentioned. Since the antibody is highly specific for the protein antigen it is possible to study the conformational changes that the antigen undergoes in the presence of other proteins, provid­ed the latter do not crossreact with the antibody.

  Precipitin Reaction Top

The quantitative estimation of antibody, as developed by HEIDE­LBERGER AND KENDALL is based on the observations that up­on the addition of increasing amounts of soluble antigen to a series of tubes containing a const­ant volume of antisera, the amount of precipitate formed increases to a maximum and then decreases.

Quantitative Precipitin Analyses

Precipitin studies are a very powerful tool that can often give information about the mole ratio of antigen to antibody, and, accord­ingly an estimate (subject to cer­tain reservations) of the number of antigenic areas on the enzyme.

From the qualitative precipitin test the amount of antigen equival­ent to a certain amount of antibody is known, and from the probable valence an estimate of the amount of antibody can be made. Deter­mine the amount of serum or diluted serum required to give an amount of specific precipitate that can be accurately analyzed by mr­cro-Kjeldahl analysis and by Folin analysis. Most other methods re­quire intermediate amounts. The amount of antibody is usually kept constant and duplicate determina­tions at 6 or 7 antigen concentra­tions are set up.

For micro-precipitn reactions, 7 ml conical centrifuge tubes (Labti­cian Products Co., Hollis, New York) are used. It is a little difficult to wash larger precipitates in a conical tube and, for determina­tions by micro-Kjeldahl, 22 X 100 mm round-bottom test tubes may be used. Normally precipitin re­actions are set up at room tempera­ture and after 2 hours are placed in the cold for 48-72 hours. The whole process may be performed at 0˚. Micro precipitin reactions with weak sera are set up in an ice bath and may require 7 days be­fore analysis.

After the precipitin systems have stood for the appropriate in­terval they are centrifuged at 1500 g for 45 minutes. The supernatant solution is poured into clean, label­ed tubes for tests for antigen and antibody, and for examination to make sure that particles of preci­pitate have not been poured off An attempt should be made. The tubes are inverted on clean filter paper for 5 minutes after pouring to remove the last remnants of supernatant. Then the precipitate is suspended in a drop of buffered saline, by holding the tube in the left hand and stroking it with the right. It is especially hard to sus­pend precipitates formed in anti­sen excess. The precipitate should be washed with a volume of solvent (usually saline or buffered saline although buffer may be used). In critical experiments with small amounts of precipitate the condi­tions for washing should be tested to see that appreciable precipitate is not dissolved. Normally preci­nitates are washed at 0˚ and allowed to stand 15-20 minutes before centrifuging again. The centrifugation should be for 30 minutes. After centrifuging the su­nernatants should be, poured off into separate tubes and examined for bits of precipitate, but need not be saved. After another wash the precipitates are ready for analysis. The micro-Kjedahl, Folin, and ninhydrin methods are often used. The washed precipitate may also be dissolved in alkali and the opti­cal denisty of the solution in the ultraviolet region determined.

  Determination of Structural Relationships Among Micromolecules Top

A rough indication of the relative number of cross-reacting groups in the antigens may be obtained from the amount of heterologous anti­gen relative to the amount of homo­logous antigen need to achieve eq­uivalence with the antibody.

Quantitative studies of cross-re­actions between mammalian albu­mins and between mammalian gamma-globulins with rabbit anti­sera have been reported, as well as cross-reactions between sheep. bov­ine, and human thyroglobulins. With rabbit antisera, sheep and bovine Tg appeared closely related, whereas human Tg differed im­munochemically from the sheep and beef Tg preparations.


  [Figure - 1]


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