Angelo Angelo Immunochemistry. Pergamon Press HEREMANS Research Department of Internal Pathology, Cliniques Universitaires St Pierre, Louvain, Belgium Received 20 January Abstract--When an unknown amount of antigen is allowed to diffuse radially from a well in a uniformly thin layer of antibody-containing agar for a sufficient time to allow all antigen to combine, the final area reached by the precipitate is directly proportional to the amount of antigen employed, and inversely proportional to the concentration of antibody. It is also shown that the temperature at which the plates are incubated has no perceptible influence upon the results. By standardizing the technical conditions of the experiment it is possible to use this principle for the immunochemical determination of antigens. The standard deviation of the antigen determinations was less than 2 per cent of the mean.
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Angelo Angelo Immunochemistry. Pergamon Press HEREMANS Research Department of Internal Pathology, Cliniques Universitaires St Pierre, Louvain, Belgium Received 20 January Abstract--When an unknown amount of antigen is allowed to diffuse radially from a well in a uniformly thin layer of antibody-containing agar for a sufficient time to allow all antigen to combine, the final area reached by the precipitate is directly proportional to the amount of antigen employed, and inversely proportional to the concentration of antibody.
It is also shown that the temperature at which the plates are incubated has no perceptible influence upon the results. By standardizing the technical conditions of the experiment it is possible to use this principle for the immunochemical determination of antigens. The standard deviation of the antigen determinations was less than 2 per cent of the mean. He observed that such cultures became surrounded by one or more ring-shaped precipitates, which might eventually coalesce with similar precipitates formed around neighbouring cultures of the same strain of organisms.
His method was based on the observation that there existed a relationship between the width of the precipitate and the amount of antigen produced by the cultures, and an inverse relationship between the size of the precipitate and the concentration of the antiserum employed. Un- fortunately these studies were not further pursued. Grant No. Am from the U. Department of Health, Education and Welfare. HEREMANS Augustin, 5 and Crowle 6 have described a semi-quantitative modification of the single radial immunodiffusion method, in which use was made of the end-point principle.
Serial dilutions of the same antigen solution were tested in the same antiserum plate, and the highest dilution still giving rise to a visible ring of pre- cipitate around the antigen well was taken as the end-point. The latter technique has also been carried out on cellulose acetate foil soaked in antiserum.
The single radial diffusion method is therefore suitable for very accurate quantita- tive determinations, without any resort to end-point methods and the interpolations they require.
The present paper is concerned with the technique as well the quantitative aspects of single radial immunodiffusion. Preparation of the agar To ml of barbiturate buffer of pH 8.
This suspension is placed in a boiling water- bath and stirred until all the agar has dissolved. Distilled water is added to replace losses due to evaporation. The antiserum-agar mixture is poured, without delay, into the mold hereafter described, using the same heated pipette.
Preparation of the antiserum-agar plates In order to obtain antiserum-agar layers of strictly uniform thickness, use is made of a mold. Upon this is placed a U-shaped frame Fig. The frame is covered by another glass plate of similar dimensions, whose lower surface is siliconized.
The base plate should not be siliconized. The three pieces of the mold are tightly held together by means of clamps Fig.
The frame is now held in a slightly slanting position and the tip of the heated pipette containing the antiserum-agar mixture is applied to the lower corner of the Method of Single Radial Immunodiffusion slit. Filling the mold in this way prevents the inclusion of air bubbles. After solidi- fication min , the clamps are removed and the siliconized top plate is care- fully slid off from the gel, after which the brass frame is in turn removed.
The bottom plate is left to support the gel. Application of the antigen samples Circular wells are punched out in the gel, using a needle of 2 mm bore. The small cylinders of gel cut out by the needle are removed by suction. Preparation of an immunodiffusion plate. The plate is now kept, in a strictly horizontal position, in a moist box, until the antigen solutions have been soaked up by the gel rain.
To prevent drying, the antiserum-agar plates are stored under a 2 cm thick layer of paraffin oil. A few crystals of thymol are dissolved in the oil to prevent the growth of anaerobic micro-organisms. Measurement of the size of the precipitates As will be described under Results, the ring-shaped precipitates which form around the antigen wells grow in size during a few days, after which no further increase in dimensions is observed.
A typical plate with seven antigen wells is illustrated in Fig. Final records are made when the precipitates may be assumed to have reached their final size. After removing the oil by a short rinse in petroleum ether, the magnified silhouettes of the precipitate rings are projected on strong paper and their contours are pencilled out.
The corresponding antigen wells are similarly recorded. The circles are cut out and weighed. This method of recording surfaces has been found advantageous because the form of the precipitates may occasionally depart from the ideal circular shape.
Merely recording the diameters may give erroneous results. Weak precipitates of insufficient contrast can be brought out by staining. This is also advantageous when it is desired to store the preparations for later study. For this purpose the plates are washed with normal saline, in Petri dishes, for two to three days, with several daily changes of the washing fluid.
The preparations are then soaked for one day in distilled water renewed 3 times , in order to remove the salt. The background color is removed by three suc- cessive baths of an aqueous solution containing 50 ml of acetic acid and 5 ml of glycerine per liter.
They are expressed in mg of paper weight, as obtained by cutting out and weighing the disks drawn in pencil on cardboard paper after projection of the magnified silhouettes of the original precipitates. Fundamental quantitative relations 1. Growth of the precipitates as a function of time.
The size of the pre- cipitate ceased to increase after an initial period of rapid growth. As with the linear single diffusion of Oudin, the increase of the precipitate was roughly proportional to the square root of time during the earliest period of diffusion Fig.
This type of growth curve was found with all antigen-antibody systems tested, but marked variation was found as to the time required by the precipitates to reach their final size, as will be discussed in the following sections. Relation between the antigen concentration and the final size of the precipitates. Method of Single Radial Immunodiffusion of the different precipitates were measured repeatedly and the values were plotted as a function of the antigen concentration Fig.
It was thus found that there existed a direct relation between the time required for the attainment of the final size of a precipitate and the corresponding concentration of antigen. In the case illustrated in Fig. Statistical analysis confirmed the absence of any significant non-linear terms in B G. Qag 1 The significance of the intercept So obtained by extrapolating the curve back to zero concentration, and of the slope, K, of the line, will be examined later.
Relation between the concentration of the antiserum, the size of the antigen well, and the final size of the precipitate. In each of these nine plates, twelve antigen wells were punched out. Each set of twelve holes AREA. Relation b e t w e e n area of precipitate and a m o u n t of antigen after different times of diffusion.
Each series of four wells received respectively In summary, each set of antigen dilutions was tested in three types of wells, against three concentrations of antiserum, all tests being carried out in triplicate. The nine regression lines calculated from the data charted in Table 1 are shown in Fig. The origin of the variation of the regression coefficient K in these nine experiments is analyzed in Table 2, whereas Table 3 is concerned with a similar analysis of the intercepts So.
Statistical analysis of the variance of the slope value K demonstrates that this parameter is independent of the diameter of the antigen well Table 2. Method of Single Radial Immunodiffusion I n fact, aside from the small experimental error, the entire variation of K is ascribable to the effect of the antiserum concentration, Cab.
T h e data indicate that the slope K varies inversely with the concentration of antibody, and this relation- ship is well brought out by plotting K against the reciprocal of Cab, as shown in T A B L E 1. T h e linear character of the latter relation is demonstrated by Fig.
Reproducibility of the method 1. Variation due to the use of different plates and different positions within a single plate Experimental error. A volume of 5. Influence of concentration of antibody and size of the antigen well upon the final size of the precipitate. A different brass frame was employed for each plate, so as to provide a test on the reproducibility in function of possible variations in the thickness of the gel layers. Sixteen antigen wells of 2. The pattern of arrangement of the wells was the same for all plates.
Each antigen well received 2 tA of a 0. After 6 days of diffusion, i. TABLE 3. For each plate the data are grouped in blocks of four, corresponding to the four quadrants of the immunodiffusion plates Fig.
This method of presentation was intended to provide a test on the influ- ence of the position of the wells within the plates. Change of the slope of the regression line as a function of the antibody concentration. The statistical analysis of the data is presented in Table 5. The results indicate that: 1 there was a perceptible variation of the results from one plate to another, obviously because of small differences in the thickness of the frames employed; 2 the amount of variation due to the position occupied by the antigen wells in the so Change of the intercept of the regression line as a function of the size of the antigen well.
Unstained immunodiffusion plate. T h e agar contained rabbit anti- serum against human serum-albumin. The antigen wells were filled with serial dilutions of a 1 per cent albumin solution, as indicated on the photo- graph. T h e plate was photographed on a dark background, using indirect light. This picture was taken on the 5th day of the experiment, when diffusion had ceased.
Block-wise arrangement of sixteen antigen wells in a diffusion plate. Photograph of a plate stained after termination of the diffusion. Facing p. Variation due to differences in volume of a solution containing constant amounts of antigen.
One diffusion plate, 1 m m thick, was set up with the mixture, and sixteen antigen wells of 4.
Immunochemical quantitation of antigens by single radial immunodiffusion.
The original versions of these techniques were described respectively by Mancini, Carbonara and Heremans in , and Laurell in Verbruggen4 has recently published an extensive review of quantitative immunoelectrophoretic methods. This process is experimental and the keywords may be updated as the learning algorithm improves. This is a preview of subscription content, log in to check access.
A single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen