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Quantitative microdensitometric measurements of immunological precipitates in agar gel
ANALYTICAL
BIOCHEMi8TBY
18,
444-452 (1967)
Quantitative Microdensitometric Immunological Precipitates ALEXANDRA The Wenner-Gren
Institute
VON DER for Experimental
Measurements in Agar Gel
of
DECKEN Biology, Stockholm,
Sweden
Received August 23, 1966
In previous studies (l-5) a labeling in vitro of immunologically active proteins was demonstrated. Radioactivity measurements were made after separation and precipitation of the proteins by means of immunoelectrophoresis (6). For this purpose the precipitates were used in part for calorimetric protein analysis and in part for radioactivity determination. In the present investigation a simplified method is introduced for the determination of the relative incorporation of amino acids into immunologically active proteins. The significance of the measurements involving a microdensitometric technique will be shown. The advantages of this method are the direct determination of the protein content of the precipitates in parallel with radioactivity measurements and the possibility of decreasing the scale of incubation considerably. EXPERIMENTAL
~atcil-ials. DL-(14C)-Leucine (20 mc/mmole) was obtained from New England Nuclear Corp., Boston, Mass. and Special Agar-Noble from Difco Laboratories, Detroit, Mich. Preparation of Antisera. Rat serum albumin and its antiserum were prepared as described earlier (1). Experiments with Slices. Nonstarved Sprague-Dawley rats (180 gm) were used. After decapitation the liver was quickly removed and cooled in ice-cold Krebs-Henseleit Ringer solution (7). Slices were obtained by use of a mechanical chopper (8). Two 1.5~gm samples were incubated in 10 ml of Ringer solution with 10 PC nQ4C-leucine. The incubation was for 3 hr at 35” under “carbogen” gas (5% CO, 95% 02). After incubation, duplicates were combined. The slices were then homogenized and centrifuged for 10 min at 14,000g. A small part of the supernatant was precipitated with trichloroacetic acid; the proteins were extracted (9), plated, and counted at 10% efficiency in a Tracerlab counter. The remaining mitochondria-free supernatant was centrifuged for 50 min at 105,090 g. The pellet was suspended in 2 ml of Ringer solution 444
MEASUREMENT
OF
IMMUNOLOGICAL
445
PBECIPITATES
and treated for 2 min with ultrasonic vibrations (10). The suspension was diluted to 11 ml with Ringer solution and centrifuged for 50 min at 105,006 g. The combined supernatants were dialyzed overnight against 0.3% NaCl and concentrated by freeze-drying. The samples were dissolved by adding a small amount of water. Immunoelectrophoresis was carried out in agar as described by Grabar and Williams (6). Several parallel runs were made with the material obtained from one incubation. After electrophoresis the proteins were precipitated by the antisera and after several days of incubation at room temperature the plates were washed (11). The precipitates were cut out and extracted (2) or the plates were dried and exposed to x-ray film (11). Part of the extracted proteins was used for protein determination according to Lowry, Rosebrough, Farr, and Randall (12). Radioactivity was measured on the remaining part in a liquid scintillation counter (Packard Tri-Carb) as described for proteins (10). Microdensitometric Techniqw. A Joyce Loeble’ double-beam automatic recording microdensitometer, Mark III, was used. The settings of the apparatus during the running were as follows: condenser 25 mm, objective X 10 (32 mm), slit height 1 mm, actual light pass through the specimen 1 mm, slit width 10 CL,wedge dependent on the density 0.5, 1.0, or 3.0, speed as slow as possible approximately 1, ratio record/sample 2. The density of the immunological precipitates of the wet agar plates was measured after the plates had been washed for several days. An increased visibility of the immunological precipitates was obtained by applying an additional light source of 4 V from underneath. This light source did not affect the extinction values obtained. The optical density of the precipitates was measured in the longitudinal direction from the, cathode to the anode. Earlier measurements made in the transverse direction of the precipitates did not give satisfactory results. It was found to be of importance to follow the precipitation lines at the highest possible density to obtain reproducible results. All measurements were repeated several times. Usually the variations between the measurments were less than 4y0. The blackening of the autoradiographs was measured in a similar way. Since the instrument was not provided with an integrator, the area on the diagram which corresponded to the density measured was cut out, weighed, and recalculated to square centimeters. With the extinction factor between the wedges known, all data were recalculated to wedge 0.5. This made possible a direct comparison of the data obtained. As will be seen from Figure 1, it is possible to recalculate the density values to micrograms of protein by using as a standard reference rat serum albumin-i/-globulin precipitates. In some experiments the protein content ,’ .‘Joyce.
I,oehl
& Co.
J,td
Princesway,
Team
Valley.
Gateshead
11, England.
446
ALEXANDRA
0
VON
40
DER
DECKEN
80 ,ug
120
1io
PROTEIN
FIO. 1. Amount of protein in immunological precipitates as a function of density. Rat serum diluted S&fold was applied to agar plates in increasing amounts as indicated. After electrophoresis, albumin was precipitated with a specific antigen. The plates were incubated and washed for several days. The extinction of the precipitation lines was measured by means of the microdensitometer. The lines were cut out and the proteins precipitated and extracted with trichloroacetic acid. The amount of protein was determined according to Lowry et al. (12) using bovine albumin as a standard. The area on the diagram which corresponded to the density measured was recalculated to square centimeters at a wedge range of kO.5. The results of three independent experiments are shown: (1) A ; (2) 0 ; (3) 1.
of the precipitates was determined after the microdensitometer readings. For this purpose the precipitation lines were cut out with a double knife, which had a distance between the edges of exactly 1 mm, i.e., the slit height of the microdensitometer, 1 mm. This implies that only the proteins measured by the densitometer were used for protein determina,tion. The precipitates were extracted as described earlier (2) and protein determination was made according to Lowry et al. (12). RESULTS
Determindion
Protein Content of Precip*tation Lines by Microdensitometer
As shown by Figure 1 the optical density of the immunological precipitates as measured by microdensitometry increased in a linear fashion with the protein content up to very high protein concentrations. At high
MEASUREMENT
OF
IMMUNOLOGICAL
447
PRECIPITATES
antigen concentrations the total amount of precipitated proteins reached a maximum. This is in agreement with the results obtained for antigenantibody precipitates after double diffusion in agar gel (13). In the experiments shown in Figure 1 this value corresponded to about 150 pg of protein. It may be pointed out, however, that the measured values do not refer to the total amount of precipitated protein but only to the proteins contained within a band of 1 mm thickness. At least in the case of albumin the precipitation lines were more than 1 mm wide. (A slit height larger than 1 mm but not available for the apparatus used is recommended.) Dilution
of Radioactive
Antigen
with
Unlubeled
Clam&r
Figure 2 shows an experiment in which the dilution of radioactive antigen with unlabeled carrier was studied. Rat serum albumin labeled in vitro was diluted with increasing amounts of nonradioactive rat serum.
60.
40,
20,
0
10
,Ug C+= WLABELLED
20 SERUM
30
40 PROTEIN AWED
50
100 ISOTOPECONC.
IN %
0
OF UNDIL. PROTON
2A 2B Fro 2. Radioactivity measurements of labeled antigen in the presence of various amounts of unlabeled carrier protein. Rat liver slices were incubated with “C-leucine and the extracted soluble proteins were subjected to agar electrophoresis after addition of increasing amounts of unlabeled rat serum as indicated. Albumin was precipitated with specific antiserum. The extinction of the precipitation lines was measured with the microdensitometer; radioactivity of the lines was determined by counting technique. (a) Total radioactivity present in the precipitates at increasing amounts of unlabeled serumprotein (A) ; area of density obtained from precipitation lines as expressed in cm’ at a wedge range of O-O.5 (0). (b) Specific activity expressed 89 counts/min/lOO cm* computed from results of part (a), and plotted against the corresponding dilutions of the labeled antigen. Undiluted antigen = 100%.
448
ALEXANDRA
VON DE% DECKEN
In Figure 2a are shown the density values of the precipitates and the corresponding radioactivity data. With small additions of the carrierantigen the total radioactivity of the precipitates remained unchanged. At further additions the total radioactivity decreased at the same time as the density of the precipitates increased. While the total radioactivity continued to decrease, with increasing concentrations of the carrierantigen, the density of the precipitates remained constant. In Figure 2b the specific activity of the precipitates has been plotted against the isotope concentration. The data were computed from the results shown in Figure 2a. A decrease in percentage of isotope concentration by dilution with the unlabeled carrier shows a proportional decrease in specific activities of the precipitates. In Figure 3 are demonstrated the results of another approach of studying the effect of nonlabeled carrier protein on the radioactivity of labeled antigen. To the in vitro labeled rat serum albumin was added a small amount of rat serum. Immunoelectrophoresis was run with increasing .
50 AREA
IN SQUARE
100 CM
150
FIQ. 3. Radioactivity measurements of labeled antigen in the presence of a constant amount of carrier protein. In vitro labeled proteins were obtained after incubation of liver slices. To 1 ml of the extracted proteins 1 ~1 of rat serum was added. Volumes of 10, 20, 30, 40, 50, 60, and 70 ,a1 were subjected to electrophoresis in agar gel. Several runs were made with each concentration. Albumin was precipitated with specific antiserum. The density of the precipitation lines was measured and ia expressed aa area in cm’ as indicated. The radioactivity of the lines was then determined and is plotted aa total counts (0). Specific activity -was calculated aa counts/min/density unit corresponding to an area of 100 cm’ (0). The mean value of specific activity was 84 counta/min. The standard deviation was * 7.
MEASUREMENT
OF IMMUNOLOGICAL
449
PREXIPITA!CE+S
amounts of the mixture. The densities of the precipitates of rat albumin were measured and the radioactivities were determined by counting technique. As expected, an increased density of the immunological precipitates was followed by an increase in total radioactivity. Recalculation to specific activity expressed as counts/min/density unit (corresponding to 100 cm2) gave a mean value of about 85 counts/mm Use ‘of Microdensitometry for D’etermining the Blackening on Autoradiographs of Precipitation Lines
Intensity
In autoradiography the intensity of the blackening of x-ray film is directly proportional to the radioactivity only at low concentrations of reduced silver grains. As shown in Figure 4 a linear rate of darkening as measured with the densitometer could be obtained during various periods of exposure. The rate of darkening is a function of the radioactivity present. It is apparent from these results that in comparative studies the density should be measured during the linear increase of the blackening.
DAYS
OF
EXPOSURE
F’m; 4:.Blackening of autoradiographs as a function of days of exposure. Rat liver slices were incubated with “C-leucine and the extracted soluble proteins were subjected to agar gel electrophoresis. Albumin was precipitated with a specific antiserum. The washed and dried plates were covered with x-ray film for the time indicated. Blackening on the film above the precipitation lines was measured by means of the microdensitometer. The area on the diagram which corresponded to the density measured -was recalculated to cm* at a wedge range of O-0.5. The results of autoradiography of 4 immunoelectrophoretic plates are shown.
450
ALEXANDRA
Validity
VON
DEE
DECKEN
of Micwdensitome
tric Memasurenwqts for th.e Determination of Specific Activity A number of experiments were carried out to find to what extent density measurements of immunological precipitates can be combined with radioactivity determinations. Radioactivity was measured either by direct counting of the precipitates or by autoradiography as shown in Figure 4. Rat liver slices were incubated with 14C-leucine. An albumin-containing fraction was isolated, concentrated, and subjected to immunoelectrophoresis in agar gel. The results have been summarized in Table 1. Each experiment represents two parallel runs of incubation, called A and B. In experiments 1 and 2 the incubation conditions were similar for the parallel runs. In experiments 3 and 4 the incubation conditions were slightly varied to produce differences in the specific activities of the proteins. In the first column of Table 1 the density of the immunological precipitates is given as the area in square centimeters measured by the microdensitometer. The ratio between A and B was calculated (A/B),,,,.. In the second column the corresponding values of the autoradiographs are shown. The ratios between A and B were calculated from these data TABLE
1
Comparison
of Autoradiography and Counting Technique for Radioactivity Measurements of Immunological Precipitates Bat liver slices were incubated with r4C-leucine in parallel experiments, A and B. The soluble proteins were extracted and subjected to electrophoresis in agar gel. Several parallel runs were made with each experiment. Albumin was precipitated with a specific antiserum. The precipitation lines of the washed agar plates were measured by microdensitometry (column 1) and the ratios (A/B),,,. were calculated. The dried agar plates were exposed to x-ray film and the density of blackening was measured after 2 days of exposure in experiments 1 and 2, and after 1 day of exposure in experiments 3 and 4 (column 2). On parallel immunoelectrophoretic plates the immunological precipitates were cut out, extracted, and dissolved in KOH. Proteins were determined on one part by the method of Lowry et al. (12). The remaining part was counted in a liquid scintillation counter. The specific activities are given in column 3 and the ratio A/B was calculated. The relative ratios of (A/B),t,. per (A/B),,, are shown in the last column. Area of density (antigen-antibody precipitate) Ex t. N”. 1
2 3 4
A
B
55 51 71 63
56 50 68 64
in cm’
(A/B)-
0.98 1.02 1.04 0.99
0 Antigen plus antibody.
Area of density (autoradiograph) A
B
51 92 38 40
54 79 26 28
in cm*
(A/B).uto..
0.94 1.16 1.46 1.43
counts/min/mg
protein’
A
B
2.430 4.750 5.620 4.950
2.380 4.420 3.700 3.530
1.02 1.07 1.51 1.40
0.96 1.14 1.40 1.45
MEASUREMENT
OF
IMMUNOLOGICAL
PRECIPITATES
451
precipitates (A/B)..tor.. The specific activities of the antigen-antibody per milligram protein are shown in t,he last column together with the ratio obtained between A and B (A/B). Radioactivity was measured by the counting technique and part of the samples was used for calorimetric protein estimation (12). The relative ratios between A and B as measured by the densitometer-(A/B)..tOr. per (A/B),,,,.-are comparable with those obtained from the specific activity datas (A/B). The results are in agreement within 10%. DISCUSSIOiS
The application of a microdensitometric technique has made it possible to determine quantitatively the protein content of intact immunological precipitates. The scanning of the precipitates has advantages over calorimetric protein determinations. The proteins used for calorimetric analysis cannot be recovered for radioactivity determination. Proteins used for radioactivity determinations by liquid scintillation counting cannot be recovered for analysis. For these reasons previous experiments had to be carried out on a scale large enough to allow for both radioact.ivity and protein determinations. As far as liver is concerned, enough material is available. It will be possible now to use small organs for similar investigations of the specific labeling of immunologically active proteins. After the application of microdensitometry for protein analysis, parallel radioactivity determinations can be made on the same precipitation lines. On autoradiographs the blackening intensity above the precipitates can be measured by the microdensitometric procedure. Specific activity values can thereby be obtained. Radioactivity determination by the counting technique is another possibility for obtaining specific activity values. The labeling of specific antigens in biological studies raises the question as to what extent the presence of carrier protein influences the final results. The amount of antigen precipitated by the antibodies after double diffusion soon reaches a maximum. The data shown in Figure 2 indicate that dilution of radioactive antigen by unlabeled carrier decreased the total and specific radioactivity of the precipitates. The specific activity decreased in a linear fashion with dilution by unlabeled carrier. On the other hand, when the ratio of labeled antigen to carrier was kept constant the specific activity values remained unchanged at any amount of antigen precipitated by the antibodies. Therefore, it is strongly recommended that, in comparative studies, the amount of carrier added should be the same. Preferentially, the amount of carrier antigen present in the system may be kept at such a low level as to ensure a total quantitative precipit.ation of the radioactive antigen by the antibodies.
452
ALEXANDRA
VON
DEri
DECKEN
SUMMARY
A microdensitometric technique has been applied for quantitative density measurements of immunological precipitates obtained after electrophoresis in agar gel. A direct correlation was found between the density of the antigen-antibody precipitates and the protein content as determined by calorimetric analysis. Radioactivity of the precipitates was measured by counting technique or by autoradiography using the same microdensitometric technique. The significance of the results obtained is shown in a series of test experiments. ACKNOWLEDGMENT Financial support is acknowledged from the Swedish Natural Science Research Council and the Swedish Medical Research Council. The author is very grateful to Dr. P. Perlmann for the loan of the microdensitometer. Thanks are also due to Miss Chr. Svensson for valuable technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
VON DERDECICEN, A., J.. Cell Bid. 16,471 (1963). VON DERDE&EN, A., Biochem. J. 88,385 (1963). SADDI, R., AND VON DER DECKEN, A., B&ham. Biuphys. Acta 90, 196 (lN4). SADDI, R., AND VON DER DEC~EN, A., Biochim. Biophys. Acta 111, 124 (1965). SADDI, R., AND VON DERDEC~N, A., Ercpetientia 21,577 (1965). GBABAB, P., AND WILLIAMS, C. A., JR., Biochim. Biophys. Acta 17, 67 (1955). HENSELEXT, K., 2. Physiol. Chem. 210,33 (1932). MCILWAIN, H., AND BUDDLE, H. L., Biochem. J. 53,412 (1953). ZAMECNIK, P. C., LOFTFIE~~, R. B., STEPHENSON, M. L., AND STEELE, J. M., Cancer Res. 11, 592 (1951). 10. VON DERDECKEN, A., AND CAMPBELL, P. N., Biochem. J. 91, 195 (1964). 11. MORQAN, W. S., PERLMANN, P., AND HULTIN, T., J. Biophys. Biochem. Cytol. 10, 411 (1961). 12. LOWRY, 0. H., ROSE~~OUQH,N. J., FAN, A. L., AND RANDALL, R. J., J. Bid. Chem. 193, 265 (1951). Immunochemistry,” 2nd ed., 13. KABAT, E. A., AND MAYER, M. M., “Experimental Chap. 8, p. 861, C. C Thomas, Springfield, Ill., 1964.
-.‘-..~.
. ,..
.
BIOCHEMi8TBY
18,
444-452 (1967)
Quantitative Microdensitometric Immunological Precipitates ALEXANDRA The Wenner-Gren
Institute
VON DER for Experimental
Measurements in Agar Gel
of
DECKEN Biology, Stockholm,
Sweden
Received August 23, 1966
In previous studies (l-5) a labeling in vitro of immunologically active proteins was demonstrated. Radioactivity measurements were made after separation and precipitation of the proteins by means of immunoelectrophoresis (6). For this purpose the precipitates were used in part for calorimetric protein analysis and in part for radioactivity determination. In the present investigation a simplified method is introduced for the determination of the relative incorporation of amino acids into immunologically active proteins. The significance of the measurements involving a microdensitometric technique will be shown. The advantages of this method are the direct determination of the protein content of the precipitates in parallel with radioactivity measurements and the possibility of decreasing the scale of incubation considerably. EXPERIMENTAL
~atcil-ials. DL-(14C)-Leucine (20 mc/mmole) was obtained from New England Nuclear Corp., Boston, Mass. and Special Agar-Noble from Difco Laboratories, Detroit, Mich. Preparation of Antisera. Rat serum albumin and its antiserum were prepared as described earlier (1). Experiments with Slices. Nonstarved Sprague-Dawley rats (180 gm) were used. After decapitation the liver was quickly removed and cooled in ice-cold Krebs-Henseleit Ringer solution (7). Slices were obtained by use of a mechanical chopper (8). Two 1.5~gm samples were incubated in 10 ml of Ringer solution with 10 PC nQ4C-leucine. The incubation was for 3 hr at 35” under “carbogen” gas (5% CO, 95% 02). After incubation, duplicates were combined. The slices were then homogenized and centrifuged for 10 min at 14,000g. A small part of the supernatant was precipitated with trichloroacetic acid; the proteins were extracted (9), plated, and counted at 10% efficiency in a Tracerlab counter. The remaining mitochondria-free supernatant was centrifuged for 50 min at 105,090 g. The pellet was suspended in 2 ml of Ringer solution 444
MEASUREMENT
OF
IMMUNOLOGICAL
445
PBECIPITATES
and treated for 2 min with ultrasonic vibrations (10). The suspension was diluted to 11 ml with Ringer solution and centrifuged for 50 min at 105,006 g. The combined supernatants were dialyzed overnight against 0.3% NaCl and concentrated by freeze-drying. The samples were dissolved by adding a small amount of water. Immunoelectrophoresis was carried out in agar as described by Grabar and Williams (6). Several parallel runs were made with the material obtained from one incubation. After electrophoresis the proteins were precipitated by the antisera and after several days of incubation at room temperature the plates were washed (11). The precipitates were cut out and extracted (2) or the plates were dried and exposed to x-ray film (11). Part of the extracted proteins was used for protein determination according to Lowry, Rosebrough, Farr, and Randall (12). Radioactivity was measured on the remaining part in a liquid scintillation counter (Packard Tri-Carb) as described for proteins (10). Microdensitometric Techniqw. A Joyce Loeble’ double-beam automatic recording microdensitometer, Mark III, was used. The settings of the apparatus during the running were as follows: condenser 25 mm, objective X 10 (32 mm), slit height 1 mm, actual light pass through the specimen 1 mm, slit width 10 CL,wedge dependent on the density 0.5, 1.0, or 3.0, speed as slow as possible approximately 1, ratio record/sample 2. The density of the immunological precipitates of the wet agar plates was measured after the plates had been washed for several days. An increased visibility of the immunological precipitates was obtained by applying an additional light source of 4 V from underneath. This light source did not affect the extinction values obtained. The optical density of the precipitates was measured in the longitudinal direction from the, cathode to the anode. Earlier measurements made in the transverse direction of the precipitates did not give satisfactory results. It was found to be of importance to follow the precipitation lines at the highest possible density to obtain reproducible results. All measurements were repeated several times. Usually the variations between the measurments were less than 4y0. The blackening of the autoradiographs was measured in a similar way. Since the instrument was not provided with an integrator, the area on the diagram which corresponded to the density measured was cut out, weighed, and recalculated to square centimeters. With the extinction factor between the wedges known, all data were recalculated to wedge 0.5. This made possible a direct comparison of the data obtained. As will be seen from Figure 1, it is possible to recalculate the density values to micrograms of protein by using as a standard reference rat serum albumin-i/-globulin precipitates. In some experiments the protein content ,’ .‘Joyce.
I,oehl
& Co.
J,td
Princesway,
Team
Valley.
Gateshead
11, England.
446
ALEXANDRA
0
VON
40
DER
DECKEN
80 ,ug
120
1io
PROTEIN
FIO. 1. Amount of protein in immunological precipitates as a function of density. Rat serum diluted S&fold was applied to agar plates in increasing amounts as indicated. After electrophoresis, albumin was precipitated with a specific antigen. The plates were incubated and washed for several days. The extinction of the precipitation lines was measured by means of the microdensitometer. The lines were cut out and the proteins precipitated and extracted with trichloroacetic acid. The amount of protein was determined according to Lowry et al. (12) using bovine albumin as a standard. The area on the diagram which corresponded to the density measured was recalculated to square centimeters at a wedge range of kO.5. The results of three independent experiments are shown: (1) A ; (2) 0 ; (3) 1.
of the precipitates was determined after the microdensitometer readings. For this purpose the precipitation lines were cut out with a double knife, which had a distance between the edges of exactly 1 mm, i.e., the slit height of the microdensitometer, 1 mm. This implies that only the proteins measured by the densitometer were used for protein determina,tion. The precipitates were extracted as described earlier (2) and protein determination was made according to Lowry et al. (12). RESULTS
Determindion
Protein Content of Precip*tation Lines by Microdensitometer
As shown by Figure 1 the optical density of the immunological precipitates as measured by microdensitometry increased in a linear fashion with the protein content up to very high protein concentrations. At high
MEASUREMENT
OF
IMMUNOLOGICAL
447
PRECIPITATES
antigen concentrations the total amount of precipitated proteins reached a maximum. This is in agreement with the results obtained for antigenantibody precipitates after double diffusion in agar gel (13). In the experiments shown in Figure 1 this value corresponded to about 150 pg of protein. It may be pointed out, however, that the measured values do not refer to the total amount of precipitated protein but only to the proteins contained within a band of 1 mm thickness. At least in the case of albumin the precipitation lines were more than 1 mm wide. (A slit height larger than 1 mm but not available for the apparatus used is recommended.) Dilution
of Radioactive
Antigen
with
Unlubeled
Clam&r
Figure 2 shows an experiment in which the dilution of radioactive antigen with unlabeled carrier was studied. Rat serum albumin labeled in vitro was diluted with increasing amounts of nonradioactive rat serum.
60.
40,
20,
0
10
,Ug C+= WLABELLED
20 SERUM
30
40 PROTEIN AWED
50
100 ISOTOPECONC.
IN %
0
OF UNDIL. PROTON
2A 2B Fro 2. Radioactivity measurements of labeled antigen in the presence of various amounts of unlabeled carrier protein. Rat liver slices were incubated with “C-leucine and the extracted soluble proteins were subjected to agar electrophoresis after addition of increasing amounts of unlabeled rat serum as indicated. Albumin was precipitated with specific antiserum. The extinction of the precipitation lines was measured with the microdensitometer; radioactivity of the lines was determined by counting technique. (a) Total radioactivity present in the precipitates at increasing amounts of unlabeled serumprotein (A) ; area of density obtained from precipitation lines as expressed in cm’ at a wedge range of O-O.5 (0). (b) Specific activity expressed 89 counts/min/lOO cm* computed from results of part (a), and plotted against the corresponding dilutions of the labeled antigen. Undiluted antigen = 100%.
448
ALEXANDRA
VON DE% DECKEN
In Figure 2a are shown the density values of the precipitates and the corresponding radioactivity data. With small additions of the carrierantigen the total radioactivity of the precipitates remained unchanged. At further additions the total radioactivity decreased at the same time as the density of the precipitates increased. While the total radioactivity continued to decrease, with increasing concentrations of the carrierantigen, the density of the precipitates remained constant. In Figure 2b the specific activity of the precipitates has been plotted against the isotope concentration. The data were computed from the results shown in Figure 2a. A decrease in percentage of isotope concentration by dilution with the unlabeled carrier shows a proportional decrease in specific activities of the precipitates. In Figure 3 are demonstrated the results of another approach of studying the effect of nonlabeled carrier protein on the radioactivity of labeled antigen. To the in vitro labeled rat serum albumin was added a small amount of rat serum. Immunoelectrophoresis was run with increasing .
50 AREA
IN SQUARE
100 CM
150
FIQ. 3. Radioactivity measurements of labeled antigen in the presence of a constant amount of carrier protein. In vitro labeled proteins were obtained after incubation of liver slices. To 1 ml of the extracted proteins 1 ~1 of rat serum was added. Volumes of 10, 20, 30, 40, 50, 60, and 70 ,a1 were subjected to electrophoresis in agar gel. Several runs were made with each concentration. Albumin was precipitated with specific antiserum. The density of the precipitation lines was measured and ia expressed aa area in cm’ as indicated. The radioactivity of the lines was then determined and is plotted aa total counts (0). Specific activity -was calculated aa counts/min/density unit corresponding to an area of 100 cm’ (0). The mean value of specific activity was 84 counta/min. The standard deviation was * 7.
MEASUREMENT
OF IMMUNOLOGICAL
449
PREXIPITA!CE+S
amounts of the mixture. The densities of the precipitates of rat albumin were measured and the radioactivities were determined by counting technique. As expected, an increased density of the immunological precipitates was followed by an increase in total radioactivity. Recalculation to specific activity expressed as counts/min/density unit (corresponding to 100 cm2) gave a mean value of about 85 counts/mm Use ‘of Microdensitometry for D’etermining the Blackening on Autoradiographs of Precipitation Lines
Intensity
In autoradiography the intensity of the blackening of x-ray film is directly proportional to the radioactivity only at low concentrations of reduced silver grains. As shown in Figure 4 a linear rate of darkening as measured with the densitometer could be obtained during various periods of exposure. The rate of darkening is a function of the radioactivity present. It is apparent from these results that in comparative studies the density should be measured during the linear increase of the blackening.
DAYS
OF
EXPOSURE
F’m; 4:.Blackening of autoradiographs as a function of days of exposure. Rat liver slices were incubated with “C-leucine and the extracted soluble proteins were subjected to agar gel electrophoresis. Albumin was precipitated with a specific antiserum. The washed and dried plates were covered with x-ray film for the time indicated. Blackening on the film above the precipitation lines was measured by means of the microdensitometer. The area on the diagram which corresponded to the density measured -was recalculated to cm* at a wedge range of O-0.5. The results of autoradiography of 4 immunoelectrophoretic plates are shown.
450
ALEXANDRA
Validity
VON
DEE
DECKEN
of Micwdensitome
tric Memasurenwqts for th.e Determination of Specific Activity A number of experiments were carried out to find to what extent density measurements of immunological precipitates can be combined with radioactivity determinations. Radioactivity was measured either by direct counting of the precipitates or by autoradiography as shown in Figure 4. Rat liver slices were incubated with 14C-leucine. An albumin-containing fraction was isolated, concentrated, and subjected to immunoelectrophoresis in agar gel. The results have been summarized in Table 1. Each experiment represents two parallel runs of incubation, called A and B. In experiments 1 and 2 the incubation conditions were similar for the parallel runs. In experiments 3 and 4 the incubation conditions were slightly varied to produce differences in the specific activities of the proteins. In the first column of Table 1 the density of the immunological precipitates is given as the area in square centimeters measured by the microdensitometer. The ratio between A and B was calculated (A/B),,,,.. In the second column the corresponding values of the autoradiographs are shown. The ratios between A and B were calculated from these data TABLE
1
Comparison
of Autoradiography and Counting Technique for Radioactivity Measurements of Immunological Precipitates Bat liver slices were incubated with r4C-leucine in parallel experiments, A and B. The soluble proteins were extracted and subjected to electrophoresis in agar gel. Several parallel runs were made with each experiment. Albumin was precipitated with a specific antiserum. The precipitation lines of the washed agar plates were measured by microdensitometry (column 1) and the ratios (A/B),,,. were calculated. The dried agar plates were exposed to x-ray film and the density of blackening was measured after 2 days of exposure in experiments 1 and 2, and after 1 day of exposure in experiments 3 and 4 (column 2). On parallel immunoelectrophoretic plates the immunological precipitates were cut out, extracted, and dissolved in KOH. Proteins were determined on one part by the method of Lowry et al. (12). The remaining part was counted in a liquid scintillation counter. The specific activities are given in column 3 and the ratio A/B was calculated. The relative ratios of (A/B),t,. per (A/B),,, are shown in the last column. Area of density (antigen-antibody precipitate) Ex t. N”. 1
2 3 4
A
B
55 51 71 63
56 50 68 64
in cm’
(A/B)-
0.98 1.02 1.04 0.99
0 Antigen plus antibody.
Area of density (autoradiograph) A
B
51 92 38 40
54 79 26 28
in cm*
(A/B).uto..
0.94 1.16 1.46 1.43
counts/min/mg
protein’
A
B
2.430 4.750 5.620 4.950
2.380 4.420 3.700 3.530
1.02 1.07 1.51 1.40
0.96 1.14 1.40 1.45
MEASUREMENT
OF
IMMUNOLOGICAL
PRECIPITATES
451
precipitates (A/B)..tor.. The specific activities of the antigen-antibody per milligram protein are shown in t,he last column together with the ratio obtained between A and B (A/B). Radioactivity was measured by the counting technique and part of the samples was used for calorimetric protein estimation (12). The relative ratios between A and B as measured by the densitometer-(A/B)..tOr. per (A/B),,,,.-are comparable with those obtained from the specific activity datas (A/B). The results are in agreement within 10%. DISCUSSIOiS
The application of a microdensitometric technique has made it possible to determine quantitatively the protein content of intact immunological precipitates. The scanning of the precipitates has advantages over calorimetric protein determinations. The proteins used for calorimetric analysis cannot be recovered for radioactivity determination. Proteins used for radioactivity determinations by liquid scintillation counting cannot be recovered for analysis. For these reasons previous experiments had to be carried out on a scale large enough to allow for both radioact.ivity and protein determinations. As far as liver is concerned, enough material is available. It will be possible now to use small organs for similar investigations of the specific labeling of immunologically active proteins. After the application of microdensitometry for protein analysis, parallel radioactivity determinations can be made on the same precipitation lines. On autoradiographs the blackening intensity above the precipitates can be measured by the microdensitometric procedure. Specific activity values can thereby be obtained. Radioactivity determination by the counting technique is another possibility for obtaining specific activity values. The labeling of specific antigens in biological studies raises the question as to what extent the presence of carrier protein influences the final results. The amount of antigen precipitated by the antibodies after double diffusion soon reaches a maximum. The data shown in Figure 2 indicate that dilution of radioactive antigen by unlabeled carrier decreased the total and specific radioactivity of the precipitates. The specific activity decreased in a linear fashion with dilution by unlabeled carrier. On the other hand, when the ratio of labeled antigen to carrier was kept constant the specific activity values remained unchanged at any amount of antigen precipitated by the antibodies. Therefore, it is strongly recommended that, in comparative studies, the amount of carrier added should be the same. Preferentially, the amount of carrier antigen present in the system may be kept at such a low level as to ensure a total quantitative precipit.ation of the radioactive antigen by the antibodies.
452
ALEXANDRA
VON
DEri
DECKEN
SUMMARY
A microdensitometric technique has been applied for quantitative density measurements of immunological precipitates obtained after electrophoresis in agar gel. A direct correlation was found between the density of the antigen-antibody precipitates and the protein content as determined by calorimetric analysis. Radioactivity of the precipitates was measured by counting technique or by autoradiography using the same microdensitometric technique. The significance of the results obtained is shown in a series of test experiments. ACKNOWLEDGMENT Financial support is acknowledged from the Swedish Natural Science Research Council and the Swedish Medical Research Council. The author is very grateful to Dr. P. Perlmann for the loan of the microdensitometer. Thanks are also due to Miss Chr. Svensson for valuable technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
VON DERDECICEN, A., J.. Cell Bid. 16,471 (1963). VON DERDE&EN, A., Biochem. J. 88,385 (1963). SADDI, R., AND VON DER DECKEN, A., B&ham. Biuphys. Acta 90, 196 (lN4). SADDI, R., AND VON DER DEC~EN, A., Biochim. Biophys. Acta 111, 124 (1965). SADDI, R., AND VON DERDEC~N, A., Ercpetientia 21,577 (1965). GBABAB, P., AND WILLIAMS, C. A., JR., Biochim. Biophys. Acta 17, 67 (1955). HENSELEXT, K., 2. Physiol. Chem. 210,33 (1932). MCILWAIN, H., AND BUDDLE, H. L., Biochem. J. 53,412 (1953). ZAMECNIK, P. C., LOFTFIE~~, R. B., STEPHENSON, M. L., AND STEELE, J. M., Cancer Res. 11, 592 (1951). 10. VON DERDECKEN, A., AND CAMPBELL, P. N., Biochem. J. 91, 195 (1964). 11. MORQAN, W. S., PERLMANN, P., AND HULTIN, T., J. Biophys. Biochem. Cytol. 10, 411 (1961). 12. LOWRY, 0. H., ROSE~~OUQH,N. J., FAN, A. L., AND RANDALL, R. J., J. Bid. Chem. 193, 265 (1951). Immunochemistry,” 2nd ed., 13. KABAT, E. A., AND MAYER, M. M., “Experimental Chap. 8, p. 861, C. C Thomas, Springfield, Ill., 1964.
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