ANA, Y 7 IC A, H,O( H*:MISTRY
92. 147- 155 11979)
Autoradiographic Immunoassay (ARIA): A Rapid Technique Semiquantitative Mass Screening of Haptens’
for the
A semi-quantitative radioimmunoassay was developed which allows the mass screening of more than 10” samples per day per person. The assay is performed in multiwell test plates and employs charcoal separation of antibody-bound and free antigen fractions. The radioactivity ofthe charcoal phase is transformed into fluorescent light which exposes a sheet of X-ray film. By this autoradiographic immunoassay (ARIA) technique. both visual and quantitative evaluation of the tests can be accomplished. The applicability of this new method is demonstrated for two haptens. digoxin and nicotine. Correlation coefticients between charcoal radioactivity and film density were found to exceed 0.95. Measuring ranges extend from 0.2 to 50 ng (digoxin) and from 5 to 500 ng (nicotine). Interassay variabilities are about twice as high for ARIA as for radioimmunoassays. By proper choice ofthe sample dilution used for assays. the screening for high or low antigen concentrations is possible.
The classical radioimmunoassay procedure usually includes several reagent additions, sample transfer steps, and further manipulations. Each sample is processed in a test tube, has to be transferred manually to the radioactivity counter, and a counting time of at least 1 to 2 min is usually required for each sample. Although equipment is available for mechanizing the radioimmunoassay. e.g., pipetting devices, large capacity centrifuges. and automatic beta and gamma counters, the sample processing capacity is usually limited by the time required for radioactivity determination. There are many analytical research projects, however, which do not need quantitation of each individual result but rather aim at tlhe identification of samples with high or low concentrations of the compound under investigation. In screening work, especially, many thousands of analyses may often be required within a short period of time. For example, ’ Part 5 in the Plant Science.”
series
“Use
of
Immunoassay
in the breeding of plant varieties with improved chemical characteristics, it was recognized early ( 1) that only easily performed mass screening methods would be of value. We have used radioimmunoassay in the screening of plant material for secondary plant products (2,3) for several years and, having been confronted by the limitations described above, have developed a semiquantitative, large-scale screening technique based on visualization of the results on X-ray film. Consequently. we have named this test “autoradiographic immunoassay” (ARIA).” Similar multiwell test plates, as utilized in our method, have already been employed in the performance of radioimmunoassays for macromolecular antigens (4) or antibodies (4-6) but not yet for haptens. In addition, the detection procedure. especially designed for large-scale screening, is new 2 Abbreviations used: ARIA, autoradiographic immunoassay; HSA. human serum albumin; BSA. bovine serum albumin: PBS. assay buffer containing 0.01 1~ phosphate and 0.15 M saline. pH 7.4; DCC. dextran-coated charcoal: RIA. radioimmunoassay.
in
147
0003.2697/79/010147-09%02.00/O Copyright All
right*
‘600 to 800 Ci/mmol. ““I-labeled digoxin routinely has an immunoreactive level exceeding 75 to 85%’ and is stable for at least 4 to 6 months. “51-labeled nicotine exhibited immunoreactivities of >70% and could be used for more than 2 months. E.vtruct.s. Fresh leaf samples of Nic,otitrtltr trrtmc~rrttl were extracted with water and Dicqitcrlis lottutrr . with 80% ethanol. If necessary, extracts were diluted with deionized water. Twenty-five-microliter aliquots were assayed. Ztrlrtltrtlorr.s.vr~. Quantitative radioimmunoassays were performed as previously described (2), and the protocol for the ARIA is as follows. Twenty-five microliters of diluted extract or standard solution is pipetted into each of the 96 wells of a test plate. One-hundred microliters of the PBS-
AUTORADIOGRAPHIC
buffered solution, containing dilute a.ntibody , approximately 48,000 cpm of tracer. and 0.13% of BSA (preincubated for 15 min at room temperature), is added and the contents are mixed for 1 min on the microshaker. The plates are then covered with a lid to reduce evaporation and incubated for 2 h at room temperature and then for 45 min at 4°C. Fifty microliters of a chilled charcoal suspension is then added to each well. All the above pipetting steps are done with the multichannel pipetting device. ‘The contents are mixed for 1 to 2 set and the plate
11
FIGURE
IA
FIG. I. Intensifying and detection packet pellet; 3, metal shield; 4 and 6. intensifying Film and intensifying screens are protected
IMMUNOASSAY
149
is centrifuged for 1 min at 2000 rpm at 4°C. This treatment causes the charcoal particles to form a uniform, densely packed layer of about a 0.7.mm thickness at the bottom of the wells. The protein concentration of the supernatant proved to be critical in this regard and best results were obtained with a 0.13% BSA content in the incubation mixture. If desired. an aliquot of the supernatant may now be transferred to small counting tube:, and the radioactivity determined, or the supernatant can be immediately decanted with a slight shake of the hand and collected in a waste container. In the latter case, the plates are then sealed with adhesive tape to prevent any contamination and are placed on the film packet for exposure overnight (see Figs. 1A and B). After processing, the films can be evaluated either visually or densitometrically. High concentrations of charcoal-bound antigen will give dense spots on the film
of ARIA. (A) Schematic drawing. screen: 5, film Iunder exposure. from light by a flexible cassette.
I. Sealed test plate: 2. charcoal (B) Photograph of the packet.
150
WEILER
AND
ZENK
I loop
60.
2
60.
-L ,” m co-
20.
0
antigen
Standard
FIG. 2. Typical standard the conditions given under
curves derived from radioactivity Materials and Methods and Table
whereas low concentrations result in light spots.
of antigen will
RESULTS Intensifying
and Detection
Puckt
When the test plates were placed in direct contact with the film, diffuse spots resulted and. due to the low efficiency of absorption, lengthy exposure times or very high radioactivity levels were required. To overcome these problems, the device shown in Fig. 1 was designed. The sealed test plate is placed on an aluminium sheet of 5 mm thickness in which 96 holes, with an inner diameter of 7 mm, had been drilled. This greatly reduces overlapping of the radiation fields of neighboring wells and gives better contrasts on the film. TABLE IMPORTANT
Compound Digoxin Nicotine ‘I Antiserum b Unspecific
I ng/25ull
CHARAVI-ERISIICS
Antiserum titer” I:4240 I : 1375 titers are final dilutions binding.
Maximum total activity applied (cpm) 48.000 48.000
determination I.
in the supernatant
(II = 4). under
The film is sandwiched between two X-ray intensifying screens which efficiently absorb the radiation energy of weak y emitters. The screens contain rare earth metal oxides, in which the radiation absorbed is transformed into fluorescent light: this in turn exposes the film. In comparison to direct exposure of the films to the radiation. the use of intensifying screens results in more than a IO-fold increase in spot density after identical exposure times. The packet shown in Fig. 1 is very simple to use and, because both film and intensifying screens are kept in a light-tight flexible plastic envelope, no special darkroom is needed for exposure. The system allows the detection of approximately 2500 cpm of lz51 after overnight exposure (15 h). However, to obtain the minimum usable contrast I
OF DICOXIN
ANI)
NI~OTINP
ARIA
UB” (“r)
Measuring range (ng)
Maximum tracer binding CC?)
6-8 6-10
0.2-50 T-500
40-50 40-50
AUTORADIOGRAPHIC
Digoxin
FIG. 3. Correlation the charcoal layer.
between film A typical example
density. (digoxin)
151
IMMUNOASSAY
expressed is given.
difference
on a single test plate ( L!?,,,, a range of at least 10,000 to 12,000 cpm should be used. A great.er range results in increased contrast. however. a lesser range severely reduces the sensitivity of the assay. Consequently. the amount of tracer and the antiserum dilution were chosen. so that, after one half-life of the isotope, the activity difference obtained was still wii.hin this range. Therefore no readjustment of the concentrations of the reactants was necessary, however. a standard curve was established for each day tests were made. = 0.5-0.6)
In Fig. 2, typical standard curves for nicotine and digoxin are shown. These were obtained by radioactivity determination in the supernatant (antibody-bound fraction of tracer). In Table I. the most important assay characteristics are given. IFor both substances the intraassay variability of standard BIB,, values (mean of four determinations) throughout the measuring range was 3 to 65: (mean 4.5%). the interassay variability (II = 4) was 2 to 9% (mean 4.7%). The correlation between film density and radioactivity of the charcoal phase was determined several times and. in iall cases. correlation coefficients were >0.95. In a typical experiment (Fig. 3). I’ = 0.973. Thus,
(ng125pl)
as extinction The coefficient
values at 550 nm. and radioactivity of of cowelation for these data is r 2 0.973.
for quantitative comparisons, the determination of radioactivity in the charcoal and the densitometry of the processed films are equivalent. To determine the accuracy of the method, unpurified plant extracts were analyzed by both ARIA (radioactivity determination in supernatant) and RIA (2.6) for their nicotine or digoxin concentrations. The results, shown in Table 2. clearly demonstrate that TABLE COKRL .AS
I A, ION Chl
(“I
rsf I AIf-,
?
N RIA
U’LL ~ROhl
ANIl
ARIA
SIANLMRI>
RFSULI
Extract
RIA
ARIA
No
(ndml)
(ngiml)
Digoxin I
61
44
2 3
49 44
42 49
4 5 6 r z 0.960 Nicotine I 7 3 4 I = 0.995 ” The
sample\
Each value four (nicotine)
used
represents triplicates
were the
4.4 4.0 3.6
12.4 I I.? 8.4
I I34 610 44
I II4 710 84
30
56
unpurified mean of three done on different
s
CURVL.S”
plant
extracts. (digoxin) days.
or
152
WEILER
-digwin
AND
standard
ZENK
*
curve
A B C D E F G H -1cm 1
2
3
4
5
6
7
8
FIG. 4. Two examples of ARlA films. (A) Digoxin: IB) nicotine. For used. The upper row of spots on each film represents the standard dilute extracts were used and high antigen concentrations appear undiluted extracts were used and low nicotine concentrations result
the methods are in close correlation (r = 0.960 and r = 0.995), however. at very low antigen concentrations, higher values were obtained by the ARIA method. Since the ARIA was developed primarily as a mass-screening technique, quantitation variation within the lower concentration ranges is insignificant . Interassay variations (n = 3 for digoxin, tl = 4 for nicotine) at high antigen concentrations (Table 2) were found to be 4 to 10% for the RIA and 8 to 1.5% for the ARIA. whereas, for the low concentrations tested, values of 8 to 20% (RIA) and I5 to 25% (ARIA) were observed. Thus, the coeffi-
9
10
these films the curve (cf. Fig. as dark spots in lighter spots
11
12
extracts from Table 2 were 2). In the case of digoxin. on the film. For nicotine. (arrows).
cients of variations were about twice as high for ARIA as for RIA. Application with high
to the screetzing fbr samplrs or lmz~ ontigetr cotic’etztrutiotis
The data presented here show that this method may be used for quantitation of results both by radioactivity determination or by densitometry of films and compares favorably with the conventional RIA procedure. The visual evaluation of a processed film permits the semiquantitative estimation of values and, due to the reproducibility of the method. samples with either high or low
AUTORADIOGRAPHIC
-nicotine
IMMUNOASSAY
standard
curve
-1cm 1
2
3
4
5
6 FIGURE
antigen concentration can be rapidly identified. Selection of high or low antigen-containing samples can be achieved by dilution of the extract providing that the exposition time (usually 14-16 h overnight) and the processing of the films are kept constant. Thus far, we have applied the visual evaluation method in screening wild populations of D. I~rncttrt plants for digoxin-rich individuals and for nicotine-free plants of N. trrh~c~~r/~. In Fig. 4. two examples of processed films are given. To compensate for any variation in film quality or processing, a standard curve is included on each test plate (Fig. 4). For the purpose of demonstration. the plates used for Fig. 4 were prepared from the extracts described in Table 2. Extracts representing the mean
7
8
9
10
11
12
4B
of the populations were distributed at random on the plates along with three digoxinrich or four low-nicotine extracts. The latter represent those plant individuals being screened for and which can be selected and identified by this method.
The time requirements and sample capacity for this assay were calculated assuming the use of only a single processing apparatus. The transfer of extracts and the addition of reagents requires about 1 mm/plate. Thus, within 2 h. 120 test plates, corresponding to 11,000 samples, may be prepared. The addition of charcoal, centrifugation, decanting, and sealing of the plates is done two plates at a time and each pair requires
1.54
WElLhR
3 min. Therefore. 120 plates may be finished within 3 h. Exposure takes place overnight and the processing of all films can be done in a single batch. Thus, this method permits the visual evaluation of up to 10J analyses per day per person. If we assume 30 set are needed for one radioactive determination by y counting, then fewer than 3000 tests can be completed within 24 h of continuous counting, whereas the visual evaluation of 120 sheets of film will only take about 15 to 30 min. DISCUSSION The essential factor limiting the sample processing capacity of conventional radioimmunoassay is the minimal counting time required per sample and the relatively limited sample capacity of most of the automatic counters available today. Furthermore, the main problem in mass screening is clear and rapid identification of interesting or desirable samples rather than their individual quantitation. In order to overcome these problems, we have developed the autoradiographic immunoassay (ARIA) and have demonstrated its applicability in the screening for the haptens. digoxin and nicotine. In order to obtain high reproducibility, it is essential that a well-defined geometry be used for the exposition of the films and this has been achieved by the following. (I) Sedimenting the radioactive charcoal into a flat layer of even thickness. so densely packed that no disturbance occurs during the decanting step. (2) The quality of the film, especially the contrast discrimination between adjacent spots. is greatly improved by including the aluminium plate in the detection system. The exposure time can be shortened by improving the absorption of the radiation in the X-ray film. This is accomplished by using intensifying screens to absorb the radioactive decay energy and transform it into fluorescent light. Due to the efficiency of this detection system. the sensitivity of the ARIA procedure is comparable to the
AND
ZENK
conventional radioimmunoassay. In the case of digoxin, ARIA is less sensitive than RIA by a factor of IO and for nicotine by a factor of 5. Comparison of data. using both methods, shows a high degree of correlation (r > 0.95) although ARIA tends to give higher values at very low antigen concentrations. The reason for this is not yet known, but for the intended use of this method, these variations should not be critical. It has been shown that the charcoalseparation method may cause stripping of antigen from the antigen-antibody complex (IO), thus this separation step must be carefully timed and conducted in the cold. We have found that extension of the contact time with the DCC up to IS min at 4°C caused no apparent loss of radioactivity in the antibody-bound antigen phase. Since the contact time in the assay described here, including the centrifugation step, takes only 3 min if two plates are processed simultaneously. this step is not likely to cause any significant variance. However, in each plate, a standard curve should be included to identify any variance occurring in the assay. Thus. ARIA provides not only a reliable means for the semiquantitative screening of as many as IO” hapten samples per day, but also avoids the need for expensive counting equipment. If quantitative results are desired, they may be derived by either radioactivity determination of the antibody-bound phase or densitometry measurement. The latter measurement may also be used to quantitate results after the mass of material has been evaluated visually, however, then a standard curve must be included in each plate. Due to the geometric factors, the film density within a single spot is maximum at its center and produces Gauss-shaped densitometer tracings. It has been observed that the peak height gives a reliable measure for the overall spot intensity and, thus, for the antigen concentration (7).
AUTORADIOGRAPHIC
Recently, a short report has appeared (11) describing a similar autoradiographic detection principle. The author uses as doubleantibody technique for thyroxine performed in V-shaped flexible test plates which have to be cut and then measured by direct autoradiography. This technique, however, involves two lengthy centrifugation steps and is less likely to be useful in mass screening. Also, since no precautions were taken to shield the individual radiation fiellds. problems of overlapping are likely to occur. The major advantages of the ARIA are the speed and simplicity of the workup procedure and the increased sensitivity due to the use of intensifying screens and Auorography rather than direct autoradiography. With our test, it is no longer the analytical procedure which is the time-limiting step in a mass-screening program but rather the collection and preparation of the samples to be tested. Using the ARIA technique presented here, it is now possible, with a minimum of analytical effort. to select from very large populations of plants chemotypes which show either an increased concentration of a natural product (in our case digoxin, lanatoside C) or its absence (e.g., nicotine). This method will prove exceedingly useful in breeding and selecting desired chemical variants of differentiated
IMMUNOASSAY
155
plants, plant cell cultures, bial cultures.
as well as micro-
ACKNOWLEDGMENTS Our thanks are due to Professor R. L. Mansell. Tampa, Florida for his kind and considerate help in prepating the manuscript. We also thank Mrs. P. Westekemper for efficient technical assistance. This work as supported by a grant of the Bundesminister fur Forschung und’rechnologie. Bonn, West Germany.
REFERENCES I. Von
Sengbusch.
R. (1942)
Lcrrlclwirt.
Jtrhrh.
91,
723-880.
?. Weiler.
E. W., and Zenk. M. H. (1976) /‘/r~~~~15, l537- 1545. Arens. H.. Stdckigt, J.. Weiler. E. W.. and Zenk. M. H. t 1978) Pl
92. 147- 155 11979)
Autoradiographic Immunoassay (ARIA): A Rapid Technique Semiquantitative Mass Screening of Haptens’
for the
A semi-quantitative radioimmunoassay was developed which allows the mass screening of more than 10” samples per day per person. The assay is performed in multiwell test plates and employs charcoal separation of antibody-bound and free antigen fractions. The radioactivity ofthe charcoal phase is transformed into fluorescent light which exposes a sheet of X-ray film. By this autoradiographic immunoassay (ARIA) technique. both visual and quantitative evaluation of the tests can be accomplished. The applicability of this new method is demonstrated for two haptens. digoxin and nicotine. Correlation coefticients between charcoal radioactivity and film density were found to exceed 0.95. Measuring ranges extend from 0.2 to 50 ng (digoxin) and from 5 to 500 ng (nicotine). Interassay variabilities are about twice as high for ARIA as for radioimmunoassays. By proper choice ofthe sample dilution used for assays. the screening for high or low antigen concentrations is possible.
The classical radioimmunoassay procedure usually includes several reagent additions, sample transfer steps, and further manipulations. Each sample is processed in a test tube, has to be transferred manually to the radioactivity counter, and a counting time of at least 1 to 2 min is usually required for each sample. Although equipment is available for mechanizing the radioimmunoassay. e.g., pipetting devices, large capacity centrifuges. and automatic beta and gamma counters, the sample processing capacity is usually limited by the time required for radioactivity determination. There are many analytical research projects, however, which do not need quantitation of each individual result but rather aim at tlhe identification of samples with high or low concentrations of the compound under investigation. In screening work, especially, many thousands of analyses may often be required within a short period of time. For example, ’ Part 5 in the Plant Science.”
series
“Use
of
Immunoassay
in the breeding of plant varieties with improved chemical characteristics, it was recognized early ( 1) that only easily performed mass screening methods would be of value. We have used radioimmunoassay in the screening of plant material for secondary plant products (2,3) for several years and, having been confronted by the limitations described above, have developed a semiquantitative, large-scale screening technique based on visualization of the results on X-ray film. Consequently. we have named this test “autoradiographic immunoassay” (ARIA).” Similar multiwell test plates, as utilized in our method, have already been employed in the performance of radioimmunoassays for macromolecular antigens (4) or antibodies (4-6) but not yet for haptens. In addition, the detection procedure. especially designed for large-scale screening, is new 2 Abbreviations used: ARIA, autoradiographic immunoassay; HSA. human serum albumin; BSA. bovine serum albumin: PBS. assay buffer containing 0.01 1~ phosphate and 0.15 M saline. pH 7.4; DCC. dextran-coated charcoal: RIA. radioimmunoassay.
in
147
0003.2697/79/010147-09%02.00/O Copyright All
right*
‘600 to 800 Ci/mmol. ““I-labeled digoxin routinely has an immunoreactive level exceeding 75 to 85%’ and is stable for at least 4 to 6 months. “51-labeled nicotine exhibited immunoreactivities of >70% and could be used for more than 2 months. E.vtruct.s. Fresh leaf samples of Nic,otitrtltr trrtmc~rrttl were extracted with water and Dicqitcrlis lottutrr . with 80% ethanol. If necessary, extracts were diluted with deionized water. Twenty-five-microliter aliquots were assayed. Ztrlrtltrtlorr.s.vr~. Quantitative radioimmunoassays were performed as previously described (2), and the protocol for the ARIA is as follows. Twenty-five microliters of diluted extract or standard solution is pipetted into each of the 96 wells of a test plate. One-hundred microliters of the PBS-
AUTORADIOGRAPHIC
buffered solution, containing dilute a.ntibody , approximately 48,000 cpm of tracer. and 0.13% of BSA (preincubated for 15 min at room temperature), is added and the contents are mixed for 1 min on the microshaker. The plates are then covered with a lid to reduce evaporation and incubated for 2 h at room temperature and then for 45 min at 4°C. Fifty microliters of a chilled charcoal suspension is then added to each well. All the above pipetting steps are done with the multichannel pipetting device. ‘The contents are mixed for 1 to 2 set and the plate
11
FIGURE
IA
FIG. I. Intensifying and detection packet pellet; 3, metal shield; 4 and 6. intensifying Film and intensifying screens are protected
IMMUNOASSAY
149
is centrifuged for 1 min at 2000 rpm at 4°C. This treatment causes the charcoal particles to form a uniform, densely packed layer of about a 0.7.mm thickness at the bottom of the wells. The protein concentration of the supernatant proved to be critical in this regard and best results were obtained with a 0.13% BSA content in the incubation mixture. If desired. an aliquot of the supernatant may now be transferred to small counting tube:, and the radioactivity determined, or the supernatant can be immediately decanted with a slight shake of the hand and collected in a waste container. In the latter case, the plates are then sealed with adhesive tape to prevent any contamination and are placed on the film packet for exposure overnight (see Figs. 1A and B). After processing, the films can be evaluated either visually or densitometrically. High concentrations of charcoal-bound antigen will give dense spots on the film
of ARIA. (A) Schematic drawing. screen: 5, film Iunder exposure. from light by a flexible cassette.
I. Sealed test plate: 2. charcoal (B) Photograph of the packet.
150
WEILER
AND
ZENK
I loop
60.
2
60.
-L ,” m co-
20.
0
antigen
Standard
FIG. 2. Typical standard the conditions given under
curves derived from radioactivity Materials and Methods and Table
whereas low concentrations result in light spots.
of antigen will
RESULTS Intensifying
and Detection
Puckt
When the test plates were placed in direct contact with the film, diffuse spots resulted and. due to the low efficiency of absorption, lengthy exposure times or very high radioactivity levels were required. To overcome these problems, the device shown in Fig. 1 was designed. The sealed test plate is placed on an aluminium sheet of 5 mm thickness in which 96 holes, with an inner diameter of 7 mm, had been drilled. This greatly reduces overlapping of the radiation fields of neighboring wells and gives better contrasts on the film. TABLE IMPORTANT
Compound Digoxin Nicotine ‘I Antiserum b Unspecific
I ng/25ull
CHARAVI-ERISIICS
Antiserum titer” I:4240 I : 1375 titers are final dilutions binding.
Maximum total activity applied (cpm) 48.000 48.000
determination I.
in the supernatant
(II = 4). under
The film is sandwiched between two X-ray intensifying screens which efficiently absorb the radiation energy of weak y emitters. The screens contain rare earth metal oxides, in which the radiation absorbed is transformed into fluorescent light: this in turn exposes the film. In comparison to direct exposure of the films to the radiation. the use of intensifying screens results in more than a IO-fold increase in spot density after identical exposure times. The packet shown in Fig. 1 is very simple to use and, because both film and intensifying screens are kept in a light-tight flexible plastic envelope, no special darkroom is needed for exposure. The system allows the detection of approximately 2500 cpm of lz51 after overnight exposure (15 h). However, to obtain the minimum usable contrast I
OF DICOXIN
ANI)
NI~OTINP
ARIA
UB” (“r)
Measuring range (ng)
Maximum tracer binding CC?)
6-8 6-10
0.2-50 T-500
40-50 40-50
AUTORADIOGRAPHIC
Digoxin
FIG. 3. Correlation the charcoal layer.
between film A typical example
density. (digoxin)
151
IMMUNOASSAY
expressed is given.
difference
on a single test plate ( L!?,,,, a range of at least 10,000 to 12,000 cpm should be used. A great.er range results in increased contrast. however. a lesser range severely reduces the sensitivity of the assay. Consequently. the amount of tracer and the antiserum dilution were chosen. so that, after one half-life of the isotope, the activity difference obtained was still wii.hin this range. Therefore no readjustment of the concentrations of the reactants was necessary, however. a standard curve was established for each day tests were made. = 0.5-0.6)
In Fig. 2, typical standard curves for nicotine and digoxin are shown. These were obtained by radioactivity determination in the supernatant (antibody-bound fraction of tracer). In Table I. the most important assay characteristics are given. IFor both substances the intraassay variability of standard BIB,, values (mean of four determinations) throughout the measuring range was 3 to 65: (mean 4.5%). the interassay variability (II = 4) was 2 to 9% (mean 4.7%). The correlation between film density and radioactivity of the charcoal phase was determined several times and. in iall cases. correlation coefficients were >0.95. In a typical experiment (Fig. 3). I’ = 0.973. Thus,
(ng125pl)
as extinction The coefficient
values at 550 nm. and radioactivity of of cowelation for these data is r 2 0.973.
for quantitative comparisons, the determination of radioactivity in the charcoal and the densitometry of the processed films are equivalent. To determine the accuracy of the method, unpurified plant extracts were analyzed by both ARIA (radioactivity determination in supernatant) and RIA (2.6) for their nicotine or digoxin concentrations. The results, shown in Table 2. clearly demonstrate that TABLE COKRL .AS
I A, ION Chl
(“I
rsf I AIf-,
?
N RIA
U’LL ~ROhl
ANIl
ARIA
SIANLMRI>
RFSULI
Extract
RIA
ARIA
No
(ndml)
(ngiml)
Digoxin I
61
44
2 3
49 44
42 49
4 5 6 r z 0.960 Nicotine I 7 3 4 I = 0.995 ” The
sample\
Each value four (nicotine)
used
represents triplicates
were the
4.4 4.0 3.6
12.4 I I.? 8.4
I I34 610 44
I II4 710 84
30
56
unpurified mean of three done on different
s
CURVL.S”
plant
extracts. (digoxin) days.
or
152
WEILER
-digwin
AND
standard
ZENK
*
curve
A B C D E F G H -1cm 1
2
3
4
5
6
7
8
FIG. 4. Two examples of ARlA films. (A) Digoxin: IB) nicotine. For used. The upper row of spots on each film represents the standard dilute extracts were used and high antigen concentrations appear undiluted extracts were used and low nicotine concentrations result
the methods are in close correlation (r = 0.960 and r = 0.995), however. at very low antigen concentrations, higher values were obtained by the ARIA method. Since the ARIA was developed primarily as a mass-screening technique, quantitation variation within the lower concentration ranges is insignificant . Interassay variations (n = 3 for digoxin, tl = 4 for nicotine) at high antigen concentrations (Table 2) were found to be 4 to 10% for the RIA and 8 to 1.5% for the ARIA. whereas, for the low concentrations tested, values of 8 to 20% (RIA) and I5 to 25% (ARIA) were observed. Thus, the coeffi-
9
10
these films the curve (cf. Fig. as dark spots in lighter spots
11
12
extracts from Table 2 were 2). In the case of digoxin. on the film. For nicotine. (arrows).
cients of variations were about twice as high for ARIA as for RIA. Application with high
to the screetzing fbr samplrs or lmz~ ontigetr cotic’etztrutiotis
The data presented here show that this method may be used for quantitation of results both by radioactivity determination or by densitometry of films and compares favorably with the conventional RIA procedure. The visual evaluation of a processed film permits the semiquantitative estimation of values and, due to the reproducibility of the method. samples with either high or low
AUTORADIOGRAPHIC
-nicotine
IMMUNOASSAY
standard
curve
-1cm 1
2
3
4
5
6 FIGURE
antigen concentration can be rapidly identified. Selection of high or low antigen-containing samples can be achieved by dilution of the extract providing that the exposition time (usually 14-16 h overnight) and the processing of the films are kept constant. Thus far, we have applied the visual evaluation method in screening wild populations of D. I~rncttrt plants for digoxin-rich individuals and for nicotine-free plants of N. trrh~c~~r/~. In Fig. 4. two examples of processed films are given. To compensate for any variation in film quality or processing, a standard curve is included on each test plate (Fig. 4). For the purpose of demonstration. the plates used for Fig. 4 were prepared from the extracts described in Table 2. Extracts representing the mean
7
8
9
10
11
12
4B
of the populations were distributed at random on the plates along with three digoxinrich or four low-nicotine extracts. The latter represent those plant individuals being screened for and which can be selected and identified by this method.
The time requirements and sample capacity for this assay were calculated assuming the use of only a single processing apparatus. The transfer of extracts and the addition of reagents requires about 1 mm/plate. Thus, within 2 h. 120 test plates, corresponding to 11,000 samples, may be prepared. The addition of charcoal, centrifugation, decanting, and sealing of the plates is done two plates at a time and each pair requires
1.54
WElLhR
3 min. Therefore. 120 plates may be finished within 3 h. Exposure takes place overnight and the processing of all films can be done in a single batch. Thus, this method permits the visual evaluation of up to 10J analyses per day per person. If we assume 30 set are needed for one radioactive determination by y counting, then fewer than 3000 tests can be completed within 24 h of continuous counting, whereas the visual evaluation of 120 sheets of film will only take about 15 to 30 min. DISCUSSION The essential factor limiting the sample processing capacity of conventional radioimmunoassay is the minimal counting time required per sample and the relatively limited sample capacity of most of the automatic counters available today. Furthermore, the main problem in mass screening is clear and rapid identification of interesting or desirable samples rather than their individual quantitation. In order to overcome these problems, we have developed the autoradiographic immunoassay (ARIA) and have demonstrated its applicability in the screening for the haptens. digoxin and nicotine. In order to obtain high reproducibility, it is essential that a well-defined geometry be used for the exposition of the films and this has been achieved by the following. (I) Sedimenting the radioactive charcoal into a flat layer of even thickness. so densely packed that no disturbance occurs during the decanting step. (2) The quality of the film, especially the contrast discrimination between adjacent spots. is greatly improved by including the aluminium plate in the detection system. The exposure time can be shortened by improving the absorption of the radiation in the X-ray film. This is accomplished by using intensifying screens to absorb the radioactive decay energy and transform it into fluorescent light. Due to the efficiency of this detection system. the sensitivity of the ARIA procedure is comparable to the
AND
ZENK
conventional radioimmunoassay. In the case of digoxin, ARIA is less sensitive than RIA by a factor of IO and for nicotine by a factor of 5. Comparison of data. using both methods, shows a high degree of correlation (r > 0.95) although ARIA tends to give higher values at very low antigen concentrations. The reason for this is not yet known, but for the intended use of this method, these variations should not be critical. It has been shown that the charcoalseparation method may cause stripping of antigen from the antigen-antibody complex (IO), thus this separation step must be carefully timed and conducted in the cold. We have found that extension of the contact time with the DCC up to IS min at 4°C caused no apparent loss of radioactivity in the antibody-bound antigen phase. Since the contact time in the assay described here, including the centrifugation step, takes only 3 min if two plates are processed simultaneously. this step is not likely to cause any significant variance. However, in each plate, a standard curve should be included to identify any variance occurring in the assay. Thus. ARIA provides not only a reliable means for the semiquantitative screening of as many as IO” hapten samples per day, but also avoids the need for expensive counting equipment. If quantitative results are desired, they may be derived by either radioactivity determination of the antibody-bound phase or densitometry measurement. The latter measurement may also be used to quantitate results after the mass of material has been evaluated visually, however, then a standard curve must be included in each plate. Due to the geometric factors, the film density within a single spot is maximum at its center and produces Gauss-shaped densitometer tracings. It has been observed that the peak height gives a reliable measure for the overall spot intensity and, thus, for the antigen concentration (7).
AUTORADIOGRAPHIC
Recently, a short report has appeared (11) describing a similar autoradiographic detection principle. The author uses as doubleantibody technique for thyroxine performed in V-shaped flexible test plates which have to be cut and then measured by direct autoradiography. This technique, however, involves two lengthy centrifugation steps and is less likely to be useful in mass screening. Also, since no precautions were taken to shield the individual radiation fiellds. problems of overlapping are likely to occur. The major advantages of the ARIA are the speed and simplicity of the workup procedure and the increased sensitivity due to the use of intensifying screens and Auorography rather than direct autoradiography. With our test, it is no longer the analytical procedure which is the time-limiting step in a mass-screening program but rather the collection and preparation of the samples to be tested. Using the ARIA technique presented here, it is now possible, with a minimum of analytical effort. to select from very large populations of plants chemotypes which show either an increased concentration of a natural product (in our case digoxin, lanatoside C) or its absence (e.g., nicotine). This method will prove exceedingly useful in breeding and selecting desired chemical variants of differentiated
IMMUNOASSAY
155
plants, plant cell cultures, bial cultures.
as well as micro-
ACKNOWLEDGMENTS Our thanks are due to Professor R. L. Mansell. Tampa, Florida for his kind and considerate help in prepating the manuscript. We also thank Mrs. P. Westekemper for efficient technical assistance. This work as supported by a grant of the Bundesminister fur Forschung und’rechnologie. Bonn, West Germany.
REFERENCES I. Von
Sengbusch.
R. (1942)
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?. Weiler.
E. W., and Zenk. M. H. (1976) /‘/r~~~~15, l537- 1545. Arens. H.. Stdckigt, J.. Weiler. E. W.. and Zenk. M. H. t 1978) Pl
