Journal of Immunological Methods, 21 (1978) 167--177 © Elsevier/North-Holland Biomedical Press
A MICRO-PROCEDURE
FOR QUANTITATIVE
167
PRECIPITIN
TESTS *
K. MALINOWSKI and W. MANSKI
Departments of Microbiology and Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY, U.S.A. (Received 27 October 1977, accepted 14 November 1977)
A new method for the quantitative analysis of antigens and antibodies has been based on (1) ultrafiltration of the antigen-antibody precipitates through silver membranes of 0.2 pm pore size in a specially designed multisample apparatus, and ( 2 ) s p e c t r o p h o t o metric determination at 210 nm of the amount of proteins in the antigen-antibody precipitates dissolved in 0.01 N HC1. At this wavelength, the = C = O group of the polypeptide chains constitutes the main chromophoric group. In comparisons with the ninhydrin color reaction, protein determination by low UV spectrophotometry, e.g., at 210 nm, was shown to be about 6 times more sensitive, permitting analysis of samples containing from 1.0 to 35.0 pg of antigen. The concentration range of protein solutions in 0.01 N HCI which is measured at low UV can be regulated by a factor of 8--10 by changing absorption between 200 and 230 nm. Comparison of the ultrafiltration microtechnique with the standard quantitative precipitin microtechnique involving centrifugation of precipitates was made in 4 different antigen-antibody systems. The new technique was found to be as accurate as the standard technique. It allows completion of analysis within only 5--6 h. The standard precipitin technique, by contrast, requires a 5--7-day reaction period for completion.
INTRODUCTION The quantitative antigen-antibody precipitin technique first developed by H e i d e l b e r g e r a n d K e n d a l l ( 1 9 2 9 ) is a t t h e f o u n d a t i o n o f m o l e c u l a r i m m u n o l ogy. The theoretical basis of this technique has been reviewed by Kabat ( 1 9 7 6 ) a n d D a y ( 1 9 7 2 ) . I t s m a n y a p p l i c a t i o n s in b a s i c a n d a p p l i e d r e s e a r c h have been described by Kabat and Mayer (1958) and Maurer (1971). The sensitivity and accuracy of different micro-procedures for assaying antigens or antibodies were evaluated by MacDuffy and Kabat (1956). In comparison to the biuret or Folin--Ciocalteau color reaction and to absorption at 277 or 280 nm, the ninhydrin color reaction produced the most sensitive assay, permitting analysis of 6.25--125 pg of protein antigen per ml s a m p l e . H o w e v e r , t h i s p r o c e d u r e is so s e n s i t i v e , e v e n t o s m a l l c h a n g e s in t h e condition of protein hydrolysis or reaction of the hydrolysate with ninh y d r i n t h a t g r e a t c a r e is r e q u i r e d in o r d e r t o s e c u r e r e p r o d u c i b l e r e s u l t s . I n * This investigation was supported by U.S.P.H.S. Research Grant EY 00189.
168
addition, various proteins reacting with ninhydrin produce colors which differ in intensity within a 2-fold range. A problem c o m m o n to all of the procedures evaluated is the long time, 5--7 days, required for the particulation of the antigen-antibody complexes, which then permits their separation in a standard laboratory centrifuge. Two improvements in the quantitative antigen-antibody precipitin tests are described in this report. One improvement involves the separation of antigen-antibody complexes by ultrafittration, which allows completion of quantitative micro-precipitation tests within 5--6 h. The second improvement involves spectrophotometry of the dissolved complexes at low UV (210 nm). Because the carbonyl group of the polypeptide backbone constitutes the main chromophoric group of proteins (Jirgensons, 1973), only relatively small differences occur between the specific absorbance of immunoglobulins and different protein antigens at this wavelength. MATERIALS AND METHODS
A n tigens
Human IgG * used in quantitative precipitin tests was obtained from the New York City Blood Center. Alpha-crystalline was isolated from calf lens cortex as previously described (Malinowski and Manski, 1977). Bovine serum albumin and chicken egg lysozyme were obtained from Nutritional Biochemical Co. (Cleveland, OH). All the antigens tested by immunoelectrophoresis showed a single line in their reaction with the respective homologous antisera. Ovalbumin (Nutritional Biochemical Co., Cleveland, OH), gelatin and fetuin (Sigma Chemical Co., St. Louis, MO), human and rabbit gammaglobulins ** as well as dextran (Pharmacia, Sweden) were used for testing the dependence of absorbance on wavelength and the stability of absorbance in 0.01 N HC1. I m m u n e sera
Chinchilla rabbits (5--6 lb) were injected subcutaneously at 5 different sites with a total of 5 mg antigen in complete Freund's adjuvant (M. butyric u m , Difco Labs., Detroit, MI). The first 3 injections were given at bi-weekly intervals and the other at m o n t h l y intervals. The immune sera were collected under sterile conditions after 7--10 sensitizations, when the antibody level had reached a plateau. For inactivation, the sera were kept in a water bath at 56°C for 30 rain.
* H u m a n IgG a n d h u m a n g a m m a - g l o b u l i n were d o n a t e d by Dr. E. Carlos of T h e Blood Center, N e w Y o r k , NY. ** N o r m a l r a b b i t g a m m a - g l o b u l i n was d o n a t e d by Dr. K o n r a d Hsu, D e p a r t m e n t of Microbiology, College o f Physicians a n d Surgeons, C o l u m b i a University, New York, NY.
169
The quantitative antigen-antibody precipitin reaction Two m e t h o d s were compared. Method 1 used ultrafiltration for the separation o f antigen-antibody complexes and m e t h o d 2 used centrifugation. In b o t h methods, the antigen solutions and the inactivated antisera were first ultrafiltered, using silver filters of 0.2 g m pore size (Selas, Springhouse, PA). Method 1. A plexiglass apparatus with 45 filtration units arranged in 3 rows was designed (Fig. 1). The lower parts of Swinnex filters (Millipore Corp., Bedford, MA) were used as supports for the ultrafilters. Plexiglass cylinders 12 mm in diameter with a 1.5 ml capacity were threaded and equipped with rubber rings to a c c o m m o d a t e the Swinnex filter supports. Plastic tubes 3 cm long were inserted into silicone corks,,and the ultrafilter support units were fitted snugly into the plastic tubes. Each of the filtration units was fitted with a Teflon filter of 10 ~m pore size to support a silver filter and to prevent filtration in the absence o f vacuum. The first 13 units in each of the 3 rows received a constant volume of 0.200 ml of the antiserum dilution and an increasing volume of the antigen 1.9cm
3 cm
EX-13 UNIT )RE CO,
V T
1.3 c m
0 0 0/0 0
0 0 0( 0 0 0 0 0/0 0
4-
46 cm
1.8¢m Fig. 1. Scheme of apparatus designed for micro-procedure for quantitative precipitin test.
170 solution, from 0.025 to 0.325 ml. The final volume in all units was brought to 0.525 ml with 0.85% NaC1 (saline). The antigen control contained 0.325 ml o f the antigen solution and 0.200 ml of the saline. The serum control contained 0.200 ml o f the antiserum dilution and 0.325 ml saline. Two 0.5-ml and one 1-ml aliquantor syringes (Hamilton Co., Reno, NV) were used for the meas ur em e nt of antiserum and antigen, as well as of the saline solutions. All the syringe settings were tested by weighing mercury and were f o u n d to be accurate within _+0.02%. The calibration rings on the antigen and saline syringes were spaced 0.025 ml apart, with the last ring at 0.175 ml. This arrangement per m i t t ed the measurement of reactants with the least refilling o f syringes. In preliminary experiments, the antigen concent rat i on and antiserum dilutions were estimated in order to establish the point of m a x i m u m precipitation close to the middle of the filtration unit, and in order n o t to exceed an absorbance o f a ppr oxi m a t e l y 1.700 at this point, i.e., below the limit of linear responses of the i ns t r um ent at 210 nm. After ultrafiltration units had been filled with antigen, antiserum and saline in triplicate samples, the apparatus, at r o o m temperature, was shaken briefly every 20 min over a 2-h period. The amounts of precipitate formed at this time did n o t change when the reactants were left for 24 h. After 2 h, the precipitates were filtered, then washed with five 0.4-ml portions of ice-cold saline using an E p p e n d o r f pipette. The washed antigen-antibody complexes were dissolved in five 0.4-ml portions of 0.01 N HC1. The filtrate was collected in 2-ml volumetric tubes, accurate within _+0.2% (Fisher Scientific Co., Springfield, NJ) and measured s pe c t r ophot om et ri cal l y. Method 2. Using Hamilton aliquantor syringes, the same volumes and dilutions of serum, antigen and saline were used as in m e t h o d 1 and were placed in 3-ml conical centrifuge tubes. The total volume in each tube was brought to 0.525 with saline. The tubes were kept at 4°C and mixed once daily for 7 days. The antigen-antibody complexes were centrifuged at 2000 rev/min at 4°C in a refrigerated centrifuge (Model PR-2, R o t o r 259; International E q u i p m e n t Co., Boston, MA). The precipitates were then resuspended in 0.5 ml o f cold saline and again centrifuged. This procedure was repeated 3 more times. Finally, the precipitates were dissolved in 2 ml of 0.01 N HC1 and measured s p e c t r o p h o t o m e t r i c a l l y .
Spectrophotometric measurements A Beckman DU-2 s p e c t r o p h o t o m e t e r (Fullerton, CA) equipped with a Gilford a t t a c h m e n t (Gilford I n s t r u m e n t Labs., Oberlin, OH) and a Gilson automatic transferator (Gilson Medical Electronics, Inc., Middleton, WI) was used. Within the limit of O.D. 1.700 the deviation in the linearity of O.D. to c o n cen tr atio n was less than 0.25%.
171 RESULTS One of the problems associated with spectrophotometric determinations of solubilized antigen-antibody complexes at 210 nm is the high absorbance of the solvents used. For example, the O.D. of 0.25 M CH3COOH was 2.570, and that of 0.1 N NaOH was 2.508. In contrast, the O.D. of 0.01 N HC1 was only 0.025 when similarly compared to that of distilled H20. The stability of O.D. at 200, 210 and 230 nm in 0.01 N HC1 was tested by comparing measurements at 0 times and at 24 h. Such a difference did n o t exceed 1.1%. The same stability of O.D. was obtained when H20 or 0.85% NaC1 was used as solvent. Tables la--d contain experimental data on the quantitative precipitation of human IgG, calf alpha-crystalline, bovine serum albumin and egg lysozyme by the homologous antisera. The numbers represent the mean (~) and standard deviation (S.D.) of O.D. readings of the respective antigen-antibody precipitates dissolved in 0.01 N HC1 and measured at 210 nm. Row 2 in each table shows the data obtained from the experimental ultrafiltration procedure (method 1); row 4 shows the data obtained from the standard test (method 2). In the first two systems (Table l a and b), the differences between the ultrafiltration and centrifugation methods at the points of m a x i m u m precipitation were 0.7% and 0.2%, somewhat lower than the differences in the third and fourth systems (Table l c and d) described, 1.5% and 0.2%. In each of the two methods compared, the S.D. at the point of m a x i m u m precipitation was considerably higher than the difference of means between them. The linear relationship between the total precipitate and the antigen added in the antibody excess region described by Heidelberger and Kendall (1935) was evaluated statistically for both methods by a linear regression and correlation coefficient test (Freund, 1962). In the human IgG--anti-human IgG system (Table la), the correlation coefficient between antigen-antibody precipitated and antigen added was 0.993 in the experimental method, as compared to 0.991 in the standard method. The slopes of the linear relationship were 0.125 in the experimental method and 0.127 in the standard method. In the calf crystalline-anti-calf alpha-crystalline system (Table lb), the same comparison showed correlation coefficients of 0.984 vs. 0.990 and the slopes in both procedures were 0.043. In the bovine serum albumin-anti-bovine serum albumin system (Table lc), the correlation coefficients were 0.987 in the experimental m e t h o d and 0.981 in the standard method. The slope of the linear relationship between antigen-antibody precipitated and antigen added was 0.194 in the experimental and 0.195 in the standard method. Correspondingly, in the lysoz y m e - a n t i - l y s o z y m e system (Table l d ) , the correlation coefficients were 0.981 in the experimental method as compared to 0.937 in the standard method. The slope of the linear relationship between total precipitate and
172
TABLE
la
HUMAN IMMUNOGLOBULIN tion of antiserum 1 : 15) Experimental ultrafiltration, = mean
data obtained
G (IgG)--ANTI-HUMAN
from
measurements
(2) centrifugation.
O.D.; S.D. = standard
O.D.
deviation
1. A n t i g e n a d d e d #g
2
at 210
IMMUNOGLOBULIN
of antigen-antibody
G (dilu-
complexes
nm of 0.01 N HCl-dissolved
using (1)
precipitates.
of O.D. 4
6
8
10
12
S.D.
0.023 0.001
0.086 0.002
0,130 0.002
0.174 0.001
0.211 0.001
0.262 0.003
2. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D. Method 1
x S.D.
0.392 0.008
0.682 0.013
0.962 0.018
1.129 0.040
1,324 0.012
1,514 0,010
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. ~g
0.369 19.4
0.596 31.5
0.8.32 43.5
0,955 50.0
1.113 58.0
1,252 65.5
4. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D. Method 2
S.D.
0.393 0.019
0.695 0.007
0.969 0.017
1.128 0.056
1.342 0.013
1.525 0.007
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. pg
0.370 19.4
0,609 32.0
0.839 44.0
0,954 50,0
1.131 59.0
1.263 66.0
O.D.
TABLE
lb
CALF
LENS
(dilution
Experimental ultrafiltration, .x = m e a n .
ALPHA-CRYSTALLINE--ANTI-CALF
of antiserum
data obtained
from
measurements
(2) centrifugation.
O.D.; S.D. = standard
. . . . 1. A n t i g e n a d d e d pg
LENS
ALPHA-CRYSTALLINE
1 : 15)
.
.
O.D.
deviation
at 210
of antigen-antibody
complexes
nm of 0.01 N HCl-dissolved
of O.D.
.
1
5.0
using (1)
precipitates,
7.5
10.0
12.5
15.0
. . 17.5
.
x S.D.
0.086 0.001
0,130 0.002
0.181 0.001
0,218 0.002
0.262 0,003
0.308 0.002
2. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D, Method 1
~ S.D,
0.352 0,027
0.513 0.021
0,663 0,047
0,779 0.036
0,881 0.020
0.943 0.036
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. pg
0,266 14.0
0.383 20.0
0.482 25.0
0.561 29.5
0.619 32.5
0.035 33.5
4. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D. Method 2
x S.D.
0.363 0,019
0.527 0.021
0.651 0.017
0.771 0,017
0.869 0.004
0.955 0.013
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. pg
0.277 14.5
0.397 21.0
0.470 24.5
0.553 29.0
0.607 32.0
0.647 34.0
O.D.
.
173
Controls 14
16
18
20
22
24
26
Serum
0.306
0.350
0.395
0.433
0.481
0.528
0.572
0.002
0.002
0.003
0.003
0.002
0.003
0.002
--
AG --
1.485
1.454
1.354
1.170
0.888
0.771
0.671
0.033
0.021
0.007
0.008
0.016
0.018
0.042
0.053
0.045
0.009
0.002
1.179 62.0
1.104 57.6
0.959 50.5
.
.
.
.
.
.
.
.
.
.
. .
1.464
1.449
1.348
1.181
0.889
0.769
1.671
0.030
0.012
0.019
0.031
0.025
0.025
0.016
0.050
0.036
0.003
0.004
1.158 61.5
1.099 57.5
0.953 50.0
---
Controls 20.0
22.5
25.0
27.5
30.0
32.5
35.0
Serum
AG
0.348 0.004
0.395 0.003
0.438 0.003
0.485 0.003
0.527 0.001
0.572 0.003
0.616 0.001
---
1.007 0.063
1.062 0.019
1.091 0.051
1.073 0.060
1.053 0.027
1.010 0.031
0.972 0.019
0.051 0.013
0.021 0.004
0.659 34.5
0.667 35.0
0.653 34.0
0.588 31.0
0.526 27.5
1.015 0.009
1.057 0.015
1.093 0.012
1.060 0,033
1.041 0.019
0.031 0.013
0.009 0.004
0.667 35.0
0.662 34.5
0.655 34.0
0.575 30.0
0.514 27.0
. .
. .
1.001 0.041
. .
. .
. .
0.958 0.013
. .
. .
---
. .
174 TABLE lc BOVINE SERUM ALBUMIN (BSA)--ANTI-BOVINE antiserum 1 : 15)
S E R U M A L B U M I N (dilution of
Experimental data obtained from m e a s u r e m e n t s of a n t i g e n - a n t i b o d y c o m p l e x e s using (1) ultrafiltration, (2) centrifugation. O.D. at 2 1 0 n m of 0.01 N HCl-dissolved precipitates. = m e a n O.D.; S.D. = standard deviation o f O.D. 1. A n t i g e n a d d e d
pg
1
2
4
3
5
6
x S.D.
0.018 0.001
0.036 0.001
0.047 0.001
0.063 0.002
0.091 0.001
0.110 0.002
2. A n t i g e n - a n t i b o d y precipitated O.D. Method 1
x S.D.
0.238 0.033
0.144 0.078
0.700 0.041
0.948 0.028
1.065 0.051
1.172 0.032
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. pg
0.220 11.5
0.408 21.5
0.653 34.0
0.885 46.5
0.974 51.0
1.062 55.5
4. A n t i g e n - a n t i b o d y precipitated O.D. Method 2
x S.D.
0.249 0.027
0.483 0.063
0.718 0.037
1.011 0.019
1.088 0.043
1.193 0.021
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. ktg
0.231 12.0
0.447 23.5
0.671 35.0
0.948 49.5
0.997 52.0
1.083 56.5
O.D.
TABLE ld EGG WHITE L Y S O Z Y M E - - A N T I - E G G WHITE L Y S O Z Y M E ( d i l u t i o n o f a n t i s e r u m 1 : 5) E x p e r i m e n t a l data o b t a i n e d f r o m m e a s u r e m e n t s o f a n t i g e n - a n t i b o d y c o m p l e x e s using (1) u l t r a f i l t r a t i o n , (2) c e n t r i f u g a t i o n . O.D. at 210 n m o f 0.01 N HCl-dissolved precipitates. = m e a n O.D.; S.D. = s t a n d a r d deviation o f O.D. 1. A n t i g e n a d d e d p g
0.2
0.4
0.6
0.8
1.0
1.2
S,D.
0.003 0.000
0.007 0.001
0.010 0,000
0.015 0,001
0.019 0.001
0.023 0.000
2. A n t i g e n - a n t i b o d y precipitated O.D. Method 1
~S.D.
0.648 0.042
0.942 0.037
1.085 0.039
1.138 0.035
1.192 0,021
1.104 0.015
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. pg
0.645 34.0
0.935 48.0
1.075 56.5
1.123 59.0
1.173 61.5
1.101 57.5
4. A n t i g e n - a n t i b o d y precipitated O.D. Method 2
~ S.D.
0.631 0.029
0.997 0.018
1.076 0.023
1.171 0.041
1.195 0.011
1.187 0.027
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. pg
0.628 33.0
0.990 52.0
1.066 56,0
1.156 60.5
1.176 61.5
1.164 61,0
O.D.
175
Controls
7
8 0.129 0.002
9 0.147 0.003
10 0.165 0.001
11
0.184 0.003
12
0.203 0.002
13
Serum
0.221 0.003
0.258 0.002
---
AG ---
1.305
1.343
1.285
1.139
0.993
0.893
0.713
0.077
0.014
0.039
0.031
0.055
0.057
0.049
0.056
0.043
0.018
0.010
1.176 61.5
1.196 63.0
1.120 58.5
0.956 50.0
0.672 .
0.455 .
0.790 .
.
--
--
.
1.324
1.363
1.298
1.149
1.004
0.854
0.731
0.053
0.029
0.045
0.023
0.031
0.019
0.010
0.014
0.023
0.023
0.011
1.195 62.5
1.216 63.5
1.133 59.5
0.965 50.5
1.4
1.6
0.801 .
0.633 .
0.473 .
--
.
Controls 1.8
2.0
2.4
2.2
2.6
Serum
AG
0.027 0.001
0.031 0.001
0.035 0.000
0.039 0.002
0.043 0.001
0.049 0.001
0.051 0.001
---
---
1.104 0.027
1.048 0.033
0.949 0.098
0.841 0.037
0.731 0.121
0.680 0.043
0.576 0.070
0.023 0.017
0.017 0.009
0,051 0.014
0.015 0.006
1.077 56.5
1.017 53.5
1.102 0.014
1.917 0.070
1.075 56.5
0.986 52.0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0.951 0.047
. .
0.831 0.071
. .
0.716 0.026
. .
. .
0.709 0.025
. .
0.614 0.085
. .
. .
176 antigen added was 1.093 in the experimental method vs. 1.113 in the standard method. The statistical analysis of the data show that the quantitative determination of antigen-antibody complexes by ultrafiltration and spectrop h o t o m e t r y in 0.01 N HC1 at low UV yields the same results as the standard centrifugation procedure. Choosing O.D. 1.700 as the safe upper limit of linear responses of the equipment used, the m a x i m u m protein antigen-antibody concentrations which could be measured at 210 nm was 85 pg/ml. The concentration range of protein determination decreased at 200 nm to 40 pg/ml and at 205 nm to 55 gg/ml. It increased at 215 nm to 200 pg/ml, at 200 nm to 250 gg/ml, at 225 nm to 4 0 0 g g / m l and at 230 nm to 600 gg/ml. The usefulness of measurements at wavelengths lower than 200 nm of dissociated antigen-antibody complexes is limited by the very high O.D. of 0.01 N HC1 in addition to absorption caused by 02 and the necessity to work in a nitrogen atmosphere. However, with increased wavelength, the variation of O.D. of different proteins did increase significantly. At 200--210 nm this variation was found to range from +8.1 to --12.7% of the average value for the 8 proteins tested. At 220 nm this variation increased from +43.4% to --29.2% of the average value and, at 230 nm from +61.2% to --52.9%. By comparison, using equal concentrations at 280 nm, the absorption was 0 for fetuin and gelatin and for the absorbing proteins it varied from +210% to --30% of the average value. DISCUSSION The rapid ultrafiltration m e t h o d described in this report is based on the observation that the initial binding between antigen and antibody occurs during a few seconds (Heidelberger et al., 1940; Mayer and Heidelberger, 1942). It is the complete particulation of the antigen-antibody complexes which enables their separation in a centrifuge at approximately 1000 X g which in the standard procedure requires prolonged time. In control experiments, it was found that the a m o u n t of antigen-antibody complexes ultrafiltered during the first 2 h did n o t increase even after 24 h of interaction. Repeated experimentation with various ultrafilters, such as Nucleopore and Millipore filters, showed t h a t they absorb proteins unspecifically. For this reason, only silver ultrafilters could be used in this method. In the 4 antigen-antibody systems investigated, the differences between the experimental and standard methods at the point of m a x i m u m precipitation was found to be less than the experimental errors of each method. It ranged from --0.2% to --1.5%, with an average of --0.65%. In terms of the measured absorbance, even the highest difference, 1.5% in the BSA--antiBSA system, meant a relatively low O.D. of 0.020, well within the range of standard deviation observed in both procedures. The use of low UV in immunochemical analysis offers the advantage of sensitivity, accuracy, extended range and simplicity. However, differences in
177 a b s o r b a n c e b e t w e e n proteins, even t h a t 10 times l o w e r at 210 n m t h a n at 2 8 0 n m , still do n o t p e r m i t w o r k with an average O.D. per weight unit. The p r o t e i n c o n c e n t r a t i o n yielding an O.D. o f 1 . 7 0 0 can be regulated b y a f a c t o r o f ca. 8 - - 1 0 b y simply c h a n g i n g t h e a b s o r p t i o n b e t w e e n 2 0 0 and 2 3 0 nm. By c o m p a r i s o n , t h e same O.D. o f 1 . 7 0 0 at 2 6 0 n m requires a ca. 40 times and at 2 8 0 n m a ca. 20 times higher c o n c e n t r a t i o n t h a n at 2 0 0 nm. The sensitivity o f the s p e c t r o p h o t o m e t r i c readings at 2 1 0 n m and t h o s e o b t a i n e d b y the n i n h y d r i n m e t h o d was calculated using the e q u a t i o n E = O.D./t~g N in sample. The E values t a k e n f r o m M a c D u f f y and K a b a t ( 1 9 5 6 ) for the n i n h y d r i n m e t h o d using h u m a n IgG were 0 . 0 3 1 7 , 0 . 0 2 1 6 or 0 . 0 1 7 6 , d e p e n d i n g on the p r e p a r a t i o n . O u r p r e p a r a t i o n o f h u m a n IgG, m e a s u r e d b y direct a b s o r b a n c e at 2 1 0 n m and calculated on t h e basis o f a 16% N c o n t e n t , gave a value o f 0 . 1 3 3 1 f o r t h e c o e f f i c i e n t E. Such a c o m p a r i s o n o f the coefficients w o u l d indicate t h a t a b s o r b a n c e at 2 1 0 n m is a p p r o x i m a t e l y 6 times m o r e sensitive t h a n the n i n h y d r i n c o l o r reaction. REFERENCES Day, E.D., 1972, Advanced Immunochemistry (Williams and Wilkins, Baltimore, MD). Freund, J.E., 1962, Mathematical Statistics (Prentice-Hall, New York). Heidelberger, M. and F.E. Kendall, 1929, J. Exp. Med. 50,809. Heidelberger, M. and F.E. Kendall, 1935, J. Exp. Med. 62,697. Heidelberger, M., H.D. Treffers and M. Mayer, 1940, J. Exp. Med. 71,271. Jirgensons, B., 1973, in: Molecular Biology, Biochemistry and Biophysics, 2nd ed., eds. A. Kleinzeller, G.F. Springer and H.G. Wittmann (Springer-Verlag, New York/Heidelberg/Berlin). Kabat, E.A. and M.M. Mayer, 1958, in: Experimental Immunochemistry, Ch. 2 (Thomas, Springfield, IL) p. 18. Kabat, I., 1976, Structural Concept in Immunology and Immunochemistry (Holt, Rinehart and Winston, New York) p. 47. MacDuffy, F.C. and E.A. Kabat, 1956, J. Immunol. 77,193. Malinowski, K. and W. Manski, 1977, Immunochemistry 14,603. Maurer, P.H., 1971, in: Methods in Immunology and Immunochemistry, Vol. III, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 1. Mayer, M. and M. Heidelberger, 1942, J. Biol. Chem. 143,567.
A MICRO-PROCEDURE
FOR QUANTITATIVE
167
PRECIPITIN
TESTS *
K. MALINOWSKI and W. MANSKI
Departments of Microbiology and Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, NY, U.S.A. (Received 27 October 1977, accepted 14 November 1977)
A new method for the quantitative analysis of antigens and antibodies has been based on (1) ultrafiltration of the antigen-antibody precipitates through silver membranes of 0.2 pm pore size in a specially designed multisample apparatus, and ( 2 ) s p e c t r o p h o t o metric determination at 210 nm of the amount of proteins in the antigen-antibody precipitates dissolved in 0.01 N HC1. At this wavelength, the = C = O group of the polypeptide chains constitutes the main chromophoric group. In comparisons with the ninhydrin color reaction, protein determination by low UV spectrophotometry, e.g., at 210 nm, was shown to be about 6 times more sensitive, permitting analysis of samples containing from 1.0 to 35.0 pg of antigen. The concentration range of protein solutions in 0.01 N HCI which is measured at low UV can be regulated by a factor of 8--10 by changing absorption between 200 and 230 nm. Comparison of the ultrafiltration microtechnique with the standard quantitative precipitin microtechnique involving centrifugation of precipitates was made in 4 different antigen-antibody systems. The new technique was found to be as accurate as the standard technique. It allows completion of analysis within only 5--6 h. The standard precipitin technique, by contrast, requires a 5--7-day reaction period for completion.
INTRODUCTION The quantitative antigen-antibody precipitin technique first developed by H e i d e l b e r g e r a n d K e n d a l l ( 1 9 2 9 ) is a t t h e f o u n d a t i o n o f m o l e c u l a r i m m u n o l ogy. The theoretical basis of this technique has been reviewed by Kabat ( 1 9 7 6 ) a n d D a y ( 1 9 7 2 ) . I t s m a n y a p p l i c a t i o n s in b a s i c a n d a p p l i e d r e s e a r c h have been described by Kabat and Mayer (1958) and Maurer (1971). The sensitivity and accuracy of different micro-procedures for assaying antigens or antibodies were evaluated by MacDuffy and Kabat (1956). In comparison to the biuret or Folin--Ciocalteau color reaction and to absorption at 277 or 280 nm, the ninhydrin color reaction produced the most sensitive assay, permitting analysis of 6.25--125 pg of protein antigen per ml s a m p l e . H o w e v e r , t h i s p r o c e d u r e is so s e n s i t i v e , e v e n t o s m a l l c h a n g e s in t h e condition of protein hydrolysis or reaction of the hydrolysate with ninh y d r i n t h a t g r e a t c a r e is r e q u i r e d in o r d e r t o s e c u r e r e p r o d u c i b l e r e s u l t s . I n * This investigation was supported by U.S.P.H.S. Research Grant EY 00189.
168
addition, various proteins reacting with ninhydrin produce colors which differ in intensity within a 2-fold range. A problem c o m m o n to all of the procedures evaluated is the long time, 5--7 days, required for the particulation of the antigen-antibody complexes, which then permits their separation in a standard laboratory centrifuge. Two improvements in the quantitative antigen-antibody precipitin tests are described in this report. One improvement involves the separation of antigen-antibody complexes by ultrafittration, which allows completion of quantitative micro-precipitation tests within 5--6 h. The second improvement involves spectrophotometry of the dissolved complexes at low UV (210 nm). Because the carbonyl group of the polypeptide backbone constitutes the main chromophoric group of proteins (Jirgensons, 1973), only relatively small differences occur between the specific absorbance of immunoglobulins and different protein antigens at this wavelength. MATERIALS AND METHODS
A n tigens
Human IgG * used in quantitative precipitin tests was obtained from the New York City Blood Center. Alpha-crystalline was isolated from calf lens cortex as previously described (Malinowski and Manski, 1977). Bovine serum albumin and chicken egg lysozyme were obtained from Nutritional Biochemical Co. (Cleveland, OH). All the antigens tested by immunoelectrophoresis showed a single line in their reaction with the respective homologous antisera. Ovalbumin (Nutritional Biochemical Co., Cleveland, OH), gelatin and fetuin (Sigma Chemical Co., St. Louis, MO), human and rabbit gammaglobulins ** as well as dextran (Pharmacia, Sweden) were used for testing the dependence of absorbance on wavelength and the stability of absorbance in 0.01 N HC1. I m m u n e sera
Chinchilla rabbits (5--6 lb) were injected subcutaneously at 5 different sites with a total of 5 mg antigen in complete Freund's adjuvant (M. butyric u m , Difco Labs., Detroit, MI). The first 3 injections were given at bi-weekly intervals and the other at m o n t h l y intervals. The immune sera were collected under sterile conditions after 7--10 sensitizations, when the antibody level had reached a plateau. For inactivation, the sera were kept in a water bath at 56°C for 30 rain.
* H u m a n IgG a n d h u m a n g a m m a - g l o b u l i n were d o n a t e d by Dr. E. Carlos of T h e Blood Center, N e w Y o r k , NY. ** N o r m a l r a b b i t g a m m a - g l o b u l i n was d o n a t e d by Dr. K o n r a d Hsu, D e p a r t m e n t of Microbiology, College o f Physicians a n d Surgeons, C o l u m b i a University, New York, NY.
169
The quantitative antigen-antibody precipitin reaction Two m e t h o d s were compared. Method 1 used ultrafiltration for the separation o f antigen-antibody complexes and m e t h o d 2 used centrifugation. In b o t h methods, the antigen solutions and the inactivated antisera were first ultrafiltered, using silver filters of 0.2 g m pore size (Selas, Springhouse, PA). Method 1. A plexiglass apparatus with 45 filtration units arranged in 3 rows was designed (Fig. 1). The lower parts of Swinnex filters (Millipore Corp., Bedford, MA) were used as supports for the ultrafilters. Plexiglass cylinders 12 mm in diameter with a 1.5 ml capacity were threaded and equipped with rubber rings to a c c o m m o d a t e the Swinnex filter supports. Plastic tubes 3 cm long were inserted into silicone corks,,and the ultrafilter support units were fitted snugly into the plastic tubes. Each of the filtration units was fitted with a Teflon filter of 10 ~m pore size to support a silver filter and to prevent filtration in the absence o f vacuum. The first 13 units in each of the 3 rows received a constant volume of 0.200 ml of the antiserum dilution and an increasing volume of the antigen 1.9cm
3 cm
EX-13 UNIT )RE CO,
V T
1.3 c m
0 0 0/0 0
0 0 0( 0 0 0 0 0/0 0
4-
46 cm
1.8¢m Fig. 1. Scheme of apparatus designed for micro-procedure for quantitative precipitin test.
170 solution, from 0.025 to 0.325 ml. The final volume in all units was brought to 0.525 ml with 0.85% NaC1 (saline). The antigen control contained 0.325 ml o f the antigen solution and 0.200 ml of the saline. The serum control contained 0.200 ml o f the antiserum dilution and 0.325 ml saline. Two 0.5-ml and one 1-ml aliquantor syringes (Hamilton Co., Reno, NV) were used for the meas ur em e nt of antiserum and antigen, as well as of the saline solutions. All the syringe settings were tested by weighing mercury and were f o u n d to be accurate within _+0.02%. The calibration rings on the antigen and saline syringes were spaced 0.025 ml apart, with the last ring at 0.175 ml. This arrangement per m i t t ed the measurement of reactants with the least refilling o f syringes. In preliminary experiments, the antigen concent rat i on and antiserum dilutions were estimated in order to establish the point of m a x i m u m precipitation close to the middle of the filtration unit, and in order n o t to exceed an absorbance o f a ppr oxi m a t e l y 1.700 at this point, i.e., below the limit of linear responses of the i ns t r um ent at 210 nm. After ultrafiltration units had been filled with antigen, antiserum and saline in triplicate samples, the apparatus, at r o o m temperature, was shaken briefly every 20 min over a 2-h period. The amounts of precipitate formed at this time did n o t change when the reactants were left for 24 h. After 2 h, the precipitates were filtered, then washed with five 0.4-ml portions of ice-cold saline using an E p p e n d o r f pipette. The washed antigen-antibody complexes were dissolved in five 0.4-ml portions of 0.01 N HC1. The filtrate was collected in 2-ml volumetric tubes, accurate within _+0.2% (Fisher Scientific Co., Springfield, NJ) and measured s pe c t r ophot om et ri cal l y. Method 2. Using Hamilton aliquantor syringes, the same volumes and dilutions of serum, antigen and saline were used as in m e t h o d 1 and were placed in 3-ml conical centrifuge tubes. The total volume in each tube was brought to 0.525 with saline. The tubes were kept at 4°C and mixed once daily for 7 days. The antigen-antibody complexes were centrifuged at 2000 rev/min at 4°C in a refrigerated centrifuge (Model PR-2, R o t o r 259; International E q u i p m e n t Co., Boston, MA). The precipitates were then resuspended in 0.5 ml o f cold saline and again centrifuged. This procedure was repeated 3 more times. Finally, the precipitates were dissolved in 2 ml of 0.01 N HC1 and measured s p e c t r o p h o t o m e t r i c a l l y .
Spectrophotometric measurements A Beckman DU-2 s p e c t r o p h o t o m e t e r (Fullerton, CA) equipped with a Gilford a t t a c h m e n t (Gilford I n s t r u m e n t Labs., Oberlin, OH) and a Gilson automatic transferator (Gilson Medical Electronics, Inc., Middleton, WI) was used. Within the limit of O.D. 1.700 the deviation in the linearity of O.D. to c o n cen tr atio n was less than 0.25%.
171 RESULTS One of the problems associated with spectrophotometric determinations of solubilized antigen-antibody complexes at 210 nm is the high absorbance of the solvents used. For example, the O.D. of 0.25 M CH3COOH was 2.570, and that of 0.1 N NaOH was 2.508. In contrast, the O.D. of 0.01 N HC1 was only 0.025 when similarly compared to that of distilled H20. The stability of O.D. at 200, 210 and 230 nm in 0.01 N HC1 was tested by comparing measurements at 0 times and at 24 h. Such a difference did n o t exceed 1.1%. The same stability of O.D. was obtained when H20 or 0.85% NaC1 was used as solvent. Tables la--d contain experimental data on the quantitative precipitation of human IgG, calf alpha-crystalline, bovine serum albumin and egg lysozyme by the homologous antisera. The numbers represent the mean (~) and standard deviation (S.D.) of O.D. readings of the respective antigen-antibody precipitates dissolved in 0.01 N HC1 and measured at 210 nm. Row 2 in each table shows the data obtained from the experimental ultrafiltration procedure (method 1); row 4 shows the data obtained from the standard test (method 2). In the first two systems (Table l a and b), the differences between the ultrafiltration and centrifugation methods at the points of m a x i m u m precipitation were 0.7% and 0.2%, somewhat lower than the differences in the third and fourth systems (Table l c and d) described, 1.5% and 0.2%. In each of the two methods compared, the S.D. at the point of m a x i m u m precipitation was considerably higher than the difference of means between them. The linear relationship between the total precipitate and the antigen added in the antibody excess region described by Heidelberger and Kendall (1935) was evaluated statistically for both methods by a linear regression and correlation coefficient test (Freund, 1962). In the human IgG--anti-human IgG system (Table la), the correlation coefficient between antigen-antibody precipitated and antigen added was 0.993 in the experimental method, as compared to 0.991 in the standard method. The slopes of the linear relationship were 0.125 in the experimental method and 0.127 in the standard method. In the calf crystalline-anti-calf alpha-crystalline system (Table lb), the same comparison showed correlation coefficients of 0.984 vs. 0.990 and the slopes in both procedures were 0.043. In the bovine serum albumin-anti-bovine serum albumin system (Table lc), the correlation coefficients were 0.987 in the experimental m e t h o d and 0.981 in the standard method. The slope of the linear relationship between antigen-antibody precipitated and antigen added was 0.194 in the experimental and 0.195 in the standard method. Correspondingly, in the lysoz y m e - a n t i - l y s o z y m e system (Table l d ) , the correlation coefficients were 0.981 in the experimental method as compared to 0.937 in the standard method. The slope of the linear relationship between total precipitate and
172
TABLE
la
HUMAN IMMUNOGLOBULIN tion of antiserum 1 : 15) Experimental ultrafiltration, = mean
data obtained
G (IgG)--ANTI-HUMAN
from
measurements
(2) centrifugation.
O.D.; S.D. = standard
O.D.
deviation
1. A n t i g e n a d d e d #g
2
at 210
IMMUNOGLOBULIN
of antigen-antibody
G (dilu-
complexes
nm of 0.01 N HCl-dissolved
using (1)
precipitates.
of O.D. 4
6
8
10
12
S.D.
0.023 0.001
0.086 0.002
0,130 0.002
0.174 0.001
0.211 0.001
0.262 0.003
2. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D. Method 1
x S.D.
0.392 0.008
0.682 0.013
0.962 0.018
1.129 0.040
1,324 0.012
1,514 0,010
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. ~g
0.369 19.4
0.596 31.5
0.8.32 43.5
0,955 50.0
1.113 58.0
1,252 65.5
4. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D. Method 2
S.D.
0.393 0.019
0.695 0.007
0.969 0.017
1.128 0.056
1.342 0.013
1.525 0.007
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. pg
0.370 19.4
0,609 32.0
0.839 44.0
0,954 50,0
1.131 59.0
1.263 66.0
O.D.
TABLE
lb
CALF
LENS
(dilution
Experimental ultrafiltration, .x = m e a n .
ALPHA-CRYSTALLINE--ANTI-CALF
of antiserum
data obtained
from
measurements
(2) centrifugation.
O.D.; S.D. = standard
. . . . 1. A n t i g e n a d d e d pg
LENS
ALPHA-CRYSTALLINE
1 : 15)
.
.
O.D.
deviation
at 210
of antigen-antibody
complexes
nm of 0.01 N HCl-dissolved
of O.D.
.
1
5.0
using (1)
precipitates,
7.5
10.0
12.5
15.0
. . 17.5
.
x S.D.
0.086 0.001
0,130 0.002
0.181 0.001
0,218 0.002
0.262 0,003
0.308 0.002
2. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D, Method 1
~ S.D,
0.352 0,027
0.513 0.021
0,663 0,047
0,779 0.036
0,881 0.020
0.943 0.036
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. pg
0,266 14.0
0.383 20.0
0.482 25.0
0.561 29.5
0.619 32.5
0.035 33.5
4. A n t i g e n - a n t i b o d y p r e c i p i t a t e d O.D. Method 2
x S.D.
0.363 0,019
0.527 0.021
0.651 0.017
0.771 0,017
0.869 0.004
0.955 0.013
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. pg
0.277 14.5
0.397 21.0
0.470 24.5
0.553 29.0
0.607 32.0
0.647 34.0
O.D.
.
173
Controls 14
16
18
20
22
24
26
Serum
0.306
0.350
0.395
0.433
0.481
0.528
0.572
0.002
0.002
0.003
0.003
0.002
0.003
0.002
--
AG --
1.485
1.454
1.354
1.170
0.888
0.771
0.671
0.033
0.021
0.007
0.008
0.016
0.018
0.042
0.053
0.045
0.009
0.002
1.179 62.0
1.104 57.6
0.959 50.5
.
.
.
.
.
.
.
.
.
.
. .
1.464
1.449
1.348
1.181
0.889
0.769
1.671
0.030
0.012
0.019
0.031
0.025
0.025
0.016
0.050
0.036
0.003
0.004
1.158 61.5
1.099 57.5
0.953 50.0
---
Controls 20.0
22.5
25.0
27.5
30.0
32.5
35.0
Serum
AG
0.348 0.004
0.395 0.003
0.438 0.003
0.485 0.003
0.527 0.001
0.572 0.003
0.616 0.001
---
1.007 0.063
1.062 0.019
1.091 0.051
1.073 0.060
1.053 0.027
1.010 0.031
0.972 0.019
0.051 0.013
0.021 0.004
0.659 34.5
0.667 35.0
0.653 34.0
0.588 31.0
0.526 27.5
1.015 0.009
1.057 0.015
1.093 0.012
1.060 0,033
1.041 0.019
0.031 0.013
0.009 0.004
0.667 35.0
0.662 34.5
0.655 34.0
0.575 30.0
0.514 27.0
. .
. .
1.001 0.041
. .
. .
. .
0.958 0.013
. .
. .
---
. .
174 TABLE lc BOVINE SERUM ALBUMIN (BSA)--ANTI-BOVINE antiserum 1 : 15)
S E R U M A L B U M I N (dilution of
Experimental data obtained from m e a s u r e m e n t s of a n t i g e n - a n t i b o d y c o m p l e x e s using (1) ultrafiltration, (2) centrifugation. O.D. at 2 1 0 n m of 0.01 N HCl-dissolved precipitates. = m e a n O.D.; S.D. = standard deviation o f O.D. 1. A n t i g e n a d d e d
pg
1
2
4
3
5
6
x S.D.
0.018 0.001
0.036 0.001
0.047 0.001
0.063 0.002
0.091 0.001
0.110 0.002
2. A n t i g e n - a n t i b o d y precipitated O.D. Method 1
x S.D.
0.238 0.033
0.144 0.078
0.700 0.041
0.948 0.028
1.065 0.051
1.172 0.032
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. pg
0.220 11.5
0.408 21.5
0.653 34.0
0.885 46.5
0.974 51.0
1.062 55.5
4. A n t i g e n - a n t i b o d y precipitated O.D. Method 2
x S.D.
0.249 0.027
0.483 0.063
0.718 0.037
1.011 0.019
1.088 0.043
1.193 0.021
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. ktg
0.231 12.0
0.447 23.5
0.671 35.0
0.948 49.5
0.997 52.0
1.083 56.5
O.D.
TABLE ld EGG WHITE L Y S O Z Y M E - - A N T I - E G G WHITE L Y S O Z Y M E ( d i l u t i o n o f a n t i s e r u m 1 : 5) E x p e r i m e n t a l data o b t a i n e d f r o m m e a s u r e m e n t s o f a n t i g e n - a n t i b o d y c o m p l e x e s using (1) u l t r a f i l t r a t i o n , (2) c e n t r i f u g a t i o n . O.D. at 210 n m o f 0.01 N HCl-dissolved precipitates. = m e a n O.D.; S.D. = s t a n d a r d deviation o f O.D. 1. A n t i g e n a d d e d p g
0.2
0.4
0.6
0.8
1.0
1.2
S,D.
0.003 0.000
0.007 0.001
0.010 0,000
0.015 0,001
0.019 0.001
0.023 0.000
2. A n t i g e n - a n t i b o d y precipitated O.D. Method 1
~S.D.
0.648 0.042
0.942 0.037
1.085 0.039
1.138 0.035
1.192 0,021
1.104 0.015
3. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 2 - - 1)
O.D. pg
0.645 34.0
0.935 48.0
1.075 56.5
1.123 59.0
1.173 61.5
1.101 57.5
4. A n t i g e n - a n t i b o d y precipitated O.D. Method 2
~ S.D.
0.631 0.029
0.997 0.018
1.076 0.023
1.171 0.041
1.195 0.011
1.187 0.027
5. A n t i b o d y p r e c i p i t a t e d ( c a l c u l a t e d 4 - - 1)
O.D. pg
0.628 33.0
0.990 52.0
1.066 56,0
1.156 60.5
1.176 61.5
1.164 61,0
O.D.
175
Controls
7
8 0.129 0.002
9 0.147 0.003
10 0.165 0.001
11
0.184 0.003
12
0.203 0.002
13
Serum
0.221 0.003
0.258 0.002
---
AG ---
1.305
1.343
1.285
1.139
0.993
0.893
0.713
0.077
0.014
0.039
0.031
0.055
0.057
0.049
0.056
0.043
0.018
0.010
1.176 61.5
1.196 63.0
1.120 58.5
0.956 50.0
0.672 .
0.455 .
0.790 .
.
--
--
.
1.324
1.363
1.298
1.149
1.004
0.854
0.731
0.053
0.029
0.045
0.023
0.031
0.019
0.010
0.014
0.023
0.023
0.011
1.195 62.5
1.216 63.5
1.133 59.5
0.965 50.5
1.4
1.6
0.801 .
0.633 .
0.473 .
--
.
Controls 1.8
2.0
2.4
2.2
2.6
Serum
AG
0.027 0.001
0.031 0.001
0.035 0.000
0.039 0.002
0.043 0.001
0.049 0.001
0.051 0.001
---
---
1.104 0.027
1.048 0.033
0.949 0.098
0.841 0.037
0.731 0.121
0.680 0.043
0.576 0.070
0.023 0.017
0.017 0.009
0,051 0.014
0.015 0.006
1.077 56.5
1.017 53.5
1.102 0.014
1.917 0.070
1.075 56.5
0.986 52.0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0.951 0.047
. .
0.831 0.071
. .
0.716 0.026
. .
. .
0.709 0.025
. .
0.614 0.085
. .
. .
176 antigen added was 1.093 in the experimental method vs. 1.113 in the standard method. The statistical analysis of the data show that the quantitative determination of antigen-antibody complexes by ultrafiltration and spectrop h o t o m e t r y in 0.01 N HC1 at low UV yields the same results as the standard centrifugation procedure. Choosing O.D. 1.700 as the safe upper limit of linear responses of the equipment used, the m a x i m u m protein antigen-antibody concentrations which could be measured at 210 nm was 85 pg/ml. The concentration range of protein determination decreased at 200 nm to 40 pg/ml and at 205 nm to 55 gg/ml. It increased at 215 nm to 200 pg/ml, at 200 nm to 250 gg/ml, at 225 nm to 4 0 0 g g / m l and at 230 nm to 600 gg/ml. The usefulness of measurements at wavelengths lower than 200 nm of dissociated antigen-antibody complexes is limited by the very high O.D. of 0.01 N HC1 in addition to absorption caused by 02 and the necessity to work in a nitrogen atmosphere. However, with increased wavelength, the variation of O.D. of different proteins did increase significantly. At 200--210 nm this variation was found to range from +8.1 to --12.7% of the average value for the 8 proteins tested. At 220 nm this variation increased from +43.4% to --29.2% of the average value and, at 230 nm from +61.2% to --52.9%. By comparison, using equal concentrations at 280 nm, the absorption was 0 for fetuin and gelatin and for the absorbing proteins it varied from +210% to --30% of the average value. DISCUSSION The rapid ultrafiltration m e t h o d described in this report is based on the observation that the initial binding between antigen and antibody occurs during a few seconds (Heidelberger et al., 1940; Mayer and Heidelberger, 1942). It is the complete particulation of the antigen-antibody complexes which enables their separation in a centrifuge at approximately 1000 X g which in the standard procedure requires prolonged time. In control experiments, it was found that the a m o u n t of antigen-antibody complexes ultrafiltered during the first 2 h did n o t increase even after 24 h of interaction. Repeated experimentation with various ultrafilters, such as Nucleopore and Millipore filters, showed t h a t they absorb proteins unspecifically. For this reason, only silver ultrafilters could be used in this method. In the 4 antigen-antibody systems investigated, the differences between the experimental and standard methods at the point of m a x i m u m precipitation was found to be less than the experimental errors of each method. It ranged from --0.2% to --1.5%, with an average of --0.65%. In terms of the measured absorbance, even the highest difference, 1.5% in the BSA--antiBSA system, meant a relatively low O.D. of 0.020, well within the range of standard deviation observed in both procedures. The use of low UV in immunochemical analysis offers the advantage of sensitivity, accuracy, extended range and simplicity. However, differences in
177 a b s o r b a n c e b e t w e e n proteins, even t h a t 10 times l o w e r at 210 n m t h a n at 2 8 0 n m , still do n o t p e r m i t w o r k with an average O.D. per weight unit. The p r o t e i n c o n c e n t r a t i o n yielding an O.D. o f 1 . 7 0 0 can be regulated b y a f a c t o r o f ca. 8 - - 1 0 b y simply c h a n g i n g t h e a b s o r p t i o n b e t w e e n 2 0 0 and 2 3 0 nm. By c o m p a r i s o n , t h e same O.D. o f 1 . 7 0 0 at 2 6 0 n m requires a ca. 40 times and at 2 8 0 n m a ca. 20 times higher c o n c e n t r a t i o n t h a n at 2 0 0 nm. The sensitivity o f the s p e c t r o p h o t o m e t r i c readings at 2 1 0 n m and t h o s e o b t a i n e d b y the n i n h y d r i n m e t h o d was calculated using the e q u a t i o n E = O.D./t~g N in sample. The E values t a k e n f r o m M a c D u f f y and K a b a t ( 1 9 5 6 ) for the n i n h y d r i n m e t h o d using h u m a n IgG were 0 . 0 3 1 7 , 0 . 0 2 1 6 or 0 . 0 1 7 6 , d e p e n d i n g on the p r e p a r a t i o n . O u r p r e p a r a t i o n o f h u m a n IgG, m e a s u r e d b y direct a b s o r b a n c e at 2 1 0 n m and calculated on t h e basis o f a 16% N c o n t e n t , gave a value o f 0 . 1 3 3 1 f o r t h e c o e f f i c i e n t E. Such a c o m p a r i s o n o f the coefficients w o u l d indicate t h a t a b s o r b a n c e at 2 1 0 n m is a p p r o x i m a t e l y 6 times m o r e sensitive t h a n the n i n h y d r i n c o l o r reaction. REFERENCES Day, E.D., 1972, Advanced Immunochemistry (Williams and Wilkins, Baltimore, MD). Freund, J.E., 1962, Mathematical Statistics (Prentice-Hall, New York). Heidelberger, M. and F.E. Kendall, 1929, J. Exp. Med. 50,809. Heidelberger, M. and F.E. Kendall, 1935, J. Exp. Med. 62,697. Heidelberger, M., H.D. Treffers and M. Mayer, 1940, J. Exp. Med. 71,271. Jirgensons, B., 1973, in: Molecular Biology, Biochemistry and Biophysics, 2nd ed., eds. A. Kleinzeller, G.F. Springer and H.G. Wittmann (Springer-Verlag, New York/Heidelberg/Berlin). Kabat, E.A. and M.M. Mayer, 1958, in: Experimental Immunochemistry, Ch. 2 (Thomas, Springfield, IL) p. 18. Kabat, I., 1976, Structural Concept in Immunology and Immunochemistry (Holt, Rinehart and Winston, New York) p. 47. MacDuffy, F.C. and E.A. Kabat, 1956, J. Immunol. 77,193. Malinowski, K. and W. Manski, 1977, Immunochemistry 14,603. Maurer, P.H., 1971, in: Methods in Immunology and Immunochemistry, Vol. III, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 1. Mayer, M. and M. Heidelberger, 1942, J. Biol. Chem. 143,567.
