in the msec range with techniques which may have response times of the order of seconds. For example, the measurement of p H (32, 33), 0 2 concentration ( 3 4 ) , or thermal parameters ( 4 ) might be possible by using appropriate cells attached to this flow system. Rapid scan spectroscopy has not been practical for most stopped-flow studies on biological samples because of a lack of sensitiv-
ity and dynamic resolution a t high speed. However, combination of a rapid scanning device with the pulsed-flow technique would be an excellent compromise.
ACKNOWLEDGMENT We would like to thank V. Massey for his constant encouragement and J. Becvar for his suggestion of gasket 2 (Figure 4). The development of the sample tube design was in collaboration with L. Strong and H. Beinert.
(32) L. Rossi-Bernardi and R . L. Berger, J. Bioi. Chem.. 243, 1297 (1968) (33) J Penntslon. J. Soulhard. and D. Green, Arch. Biochem. Biophys.. 142,638 (1971). (34) J. Penniston, Arch. Biochem. Biphys.. 150, 556-65 (1972)
Received for review November 28, 1973. Accepted March 25, 1974. This work was supported by NIH Fellowship GM 39480 (D.B.) and Research Grant GM 12176.
Ion-Electrode Based Immunoassay and Antibody-Antigen Precipitin Reaction Monitoring P. W.
Alexander' and G. A. Rechnitz
Department of Chemistry, State University of New York, Buffalo, N. Y. 14214
We have found a novel approach to immunoassay by using membrane electrodes to monitor proteins involved in antibody-antigen reactions. The sensitivity of electrode measurements coupled with the specificity of antibody-antigen reactions may permit the direct determination of individual proteins in biological fluids without extensive prior separation. In this study, we describe the determination of antigens in the range of 0.5-30 pg/ml and demonstrate the ability of the method to map out antibody-antigen reaction patterns in model systems involving precipitin formation.
Immunochemical analysis has been a well established quantitative analytical technique since Heidelberger and Kendall (1-3) introduced the quantitative precipitin method. This method involves the analysis of a washed specific precipitate (precipitin), formed by an antigen-antibody complex, using Kjeldahl measurement of the nitrogen content of the precipitate. Subsequently, other methods have been developed for the analysis of the precipitin, including the Lowry and biuret colorimetric methods and nephelometry, as reviewed by Kabat and Mayer ( 4 ) ,and Williams and Chase ( 5 ) . Interest continues, however, in finding improved analytical approaches for the quantitative precipitin test. There have been recent reports of automated immunoassays by nephelometry (6, 71, passive hemolysis inhibition (8), quanOn leave f r o m D e p a r t m e n t S o u t h Wales, Australia.
of C h e m i s t r y , U n i v e r s i t y of N e w
(1) M.Heidelberger and F. E. Kendall, J. Exptl. Med., 5 0 , 809 (1929). (2) M. Heidelberger, F. E. Kendall, and C. M. So0 Hoo, J. Expfl. Med., 58, 137 (1933). (3) M. Heideiberger and F. E. Kendall, J. f x p f l . Med., 62, 697 (1935). (4) E. A. Kabat and M. M. Mayer, "Experimental Immunochemistry," 2nd ed., C. C Thomas, Springfield, Ill,, 1961. (5) C. A. Williams and M. W. Chase, Ed., "Methods in Immunology and Immunochemistry," Vol. Ill, Academic Press, New York, N.Y., 1971. (6) I. Eckman, J . E. Robbins, C. J. A. Van den Hamer, J. Lentz. and I. H. Scheinberg, Clin. Chem., 16, 558 (1970).
titation of fluorescence methods (9, I O ) , immunoenzyme ( I I ) , and enzyme-coupled immunoassays (12), as well as a continued development of the use of radioimmunoassay ( 5 , 13) and immunodiffusion methods ( 4 , 5 ) . We now report the development of a procedure using an ion selective crystal membrane electrode of the silver sulfide type to detect specific protein concentration changes due to immunoprecipitin formation. Applications of ion-selective electrodes in bioanalysis are rapidly increasing in number, and include an automated system for sodium, potassium, and chloride ions (Technicon Instruments Corp., Tarrytown, N.Y.), enzyme determinations (14) and determinations of enzyme substrates (15-17), amino acids (18), and proteins (19, 20). In our recent work on protein analysis (19, 20), a sensitive automated method for determination of proteins was described, based on the detection of protein disulfide hydrolysis products with the silver sulfide membrane electrode. This method measures total sulfur-containing protein, however, and is not specific for individual proteins. The present work proposes a means for combining the specificity of antibody-antigen reactions with the earlier method in order to achieve selectivity for individual proteins. We have developed a semiautomated procedure for spe(7)
c. Larson. P. Orenstein. and R . F. Ritchie. in "Advances
in Automated Analysis, Technicon International Congress 1970." Vol. I., E. C. Barton e t a / . ,Ed., Thurman Associates, Miami, Fla., 1971, p 101. P. Sturgeon, M. D. Hili and K. S . Knak, Immunochemistry,6 , 689 (1969). R . D. Spencer, F. B. Toledo, E. T. Williams, and N. L. Yoso, Clin. Chem., 19, 838 (1973). R. C. Aalberse. Clin. Chim. Acta, 48, 109 (1973). W. H. Fishman. J. P. Manning, M. Takeda, D. Angellis, and S. Green, Anal. Biochem., 5 1 (2), 368 (1973). E. Engnall, K. Jonsson, and P. Perlmann, Biochim. Biophys. Acta, 251, 427 (1971). A. Zettner. Clin. Chem., 19, 699 (1973). R. A. Llenado and G. A. Rechnitz, Anal. Chem., 45,826 (1973). R . A , Llenadoand G. A . Rechnitz. Ana/ Chem.. 46, 1109 (1974) R . A. Llenado and G. A. Rechnitz, Anal. Chem., 45, 2165 (1973). H. Thompson and G. A. Rechnitz, Anal. Chem., 46, 246 (1974). M. Matsui and H. Freiser, Anal. Left., 3 , 161 (1970). P. W. Alexander and G. A. Rechnitz, Anal. Chem., 46, 256 (1974). P. W. Alexander and G. A. Rechnitz, Anal. Chem., 46, 860 (1974).
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 9 , A U G U S T 1974
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b
J
L
Figure 1. Typical continuous recording of precipitin supernatants a. Albumin standards 0.15 mg/ml; b. antiserum blank: c.2.4, d. 3.4, e. 6.8,f. 13.3, g. 25.8, h. 31.7, i. 47.5. j. 63.3, k. 79.2, 1. 95.0 pg/ml HSA added to IgG fraction of anti-HSA of potency 2.2 mg/ml
cific determination of protein antigen or antibody by immunoprecipitin formation, followed by analysis of supernatants or washed specific precipitates using electrode potentiometry. T o illustrate the principles of the method, human serum albumin (HSA) was determined in two ways: by the conventional technique using whole goat antiserum to HSA to specifically precipitate HSA followed by analysis of the washed precipitate, and by a novel technique using the immunoglobulin fraction (IgG) of goat antiserum to HSA to precipitate antigen prior to analysis of residual antibody in the supernatant. The final analytical step is achieved by the automated ion electrode method previously described (ZO),either after dissolution of precipitin in dilute alkali or by analysis of residual protein in the supernatant. These procedures permit specific determination of albumin and measurement of the complete shape of the precipitin reaction curve. Potential changes for chosen antibody/ antigen ratios are established, giving highly sensitive determination limits for antigen in the range 0.5-30 wg/ml using as little as 0.1 mg antibody/ml antiserum. Data analysis of the precipitin curve is in good agreement with the theory, reviewed by Kabat and Mayer ( 4 ) , for antigen-antibody equilibria. We expect that the method will be applicable to a wide range of immunoassays.
EXPERIMENTAL Apparatus. The electrode-based autoanalysis system for proteins has been described previously (20), including the continuousflow manifold design and reagents. The electrodes used were an Orion Ag2S Model No. 94-16A indicator electrode and an Orion Model No. 90-02-00 double junction reference electrode. The flow system consisted of Technicon sampling and proportioning pump modules and the potentiometric output of the electrodes was continuously recorded with a Beckman Model 1055 pH/mV recorder. Reagents. Reagents required for the autoanalysis system have also been described previously (20) and include solutions of urea (10M), sodium hydroxide (4M), silver nitrate (1 X 10-4M), and isotonic saline (0.9%). Antisera were obtained from several sources; first from Miles Laboratories Inc., as follows: quantities of IgG fraction of goat antiserum to human albumin (code No. 61-051, lot No. 10, potency 1254
3.6 mg antibody protein/ml antiserum; code No. 61-051, lot No. 04012, potency 2.2 mg/ml; code No. 61-051, lot No. YT03111-F, (0.5 mg/ml potency), and whole goat antiserum t o human albumin (code No. 61-015, lot No. 10, potency 3.6 mg/ml. These samples were certified as monospecific to albumin by immunoelectrophoresis and quantitative precipitin analysis and the IgG fractions were purified by DEAE column chromatography. Other albumin antisera samples used for testing of supernatants for excess antigen were obtained from Nutritional Biochemicals Corporation (lot No. 5042) and Hyland Laboratories (lot No. 8107R002Al). Human serum albumin, Cohn Fraction V, was obtained from Nutritional Biochemicals Corporation. Solutions of HSA were prepared in isotonic saline a t approx 10 mg/ml, and standardized by the biuret method using a standard solution of HSA (10.9 g %) supplied by Miles Laboratories, Inc. (Control No. A8931, code No. 81-017). Human blood serum used was Technicon reference serum (lot No. B2C101) containing 43 mg/ml albumin and 22 mg/ml total globulins. This was diluted by a factor of 100 in isotonic saline for the immunotests of albumin in serum. Procedures. Preliminary qualitative tests using the method of optimal proportions ( 4 ) were performed to find the ratio of antibody (AB) to antigen (AG) in the equivalence zone of the precipitin curve for HSA. T o measure the entire precipitin curve for HSA, a procedure similar to that described by Kabat and Mayer ( 4 ) was used, except for modifications required for electrode determination of the washed precipitates or supernatant antibody content. Increasing volumes (2-100 p l ) of antigen solutions, either HSA or serum, from stock solutions of ea. 0.4 or 4.0 mg/ml in isotonic saline were added to a fixed volume (0.1 mlj of antiserum in 3-ml conical centrifuge tubes, keeping total volume constant at 0.20 ml by addition of saline. The antiserum used was either the whole antiserum to albumin for analysis of washed precipitates or the IgG fraction of antiserum for analysis of supernatants. The immunoprecipitation commenced almost immediately for the solutions near the equivalence zone but much more slowly in the regions of extreme antibody or antigen excess. The precipitates were allowed to incubate for 4 hr with mixing twice in this time by gentle tapping. The tubes were then centrifuged for 20 min, leaving the precipitates firmly packed in the base of the tubes. T o analyze the precipitates by the electrode method, the precipitates were washed three times with ice cold saline solution by adding 1 ml saline and gently mixing the precipitates by tapping, centrifuging, and carefully decanting the supernatants which were discarded after ensuring that no particulate matter was present. After the final washing and decanting, the tubes containing the firmly packed precipitates were inverted on absorbent paper and allowed to drain. The precipitates were then dissolved in exactly 1.0 ml of a solution of sodium hydroxide (0.1M) in saline (0.9%), and thoroughly mixed. The protein content of this final solution was determined, using the automated electrode-based system described previously (201, by aspiration into the continuously-flowing stream using reagent concentrations given above. The sampling rate was 2O/hr with 1:l sample-to-wash ratio, using in this case a wash solution of 0.1M NaOH/saline solution instead of the saline used before (20). Peak heights (mV) for each test solution were recorded and plotted as a function of antigen concentration. For analysis of supernatants, the IgG fraction of the antiserum was used, making it possible to determine unreacted antibody directly in the supernatant without further antigen addition. Precipitation was carried out as described above. Instead of following the above washing procedure, however, 1.0 ml of isotonic saline was added and the solution mixed thoroughly. After centrifuging, the supernatant was immediately sampled with disposable micropipets and aspirated into the AutoAnalyzer flowing-stream, again recording peak height at 20 samples/hr and 1:l sample to wash, but using saline as the wash for this series. An example of recorder output for measurement of the precipitin curve is given in Figure 1.
RESULTS Shape of Precipitin Curves. The entire precipitin reaction curve for human serum albumin was determined by the analysis of supernatants as described above with the IgG fractionated antiserum. The antisera of potency 2.2 and 0.5 mg precipitable antibody/ml antiserum were used in order to establish the potential changes occurring for a given AB/AG ratio. Figure 1 shows a typical set of peak potentials from the AutoAnalyzer output using 0.1 ml of antiserum, 2.2 mg/ml potency, and comparing the antibody
A N A L Y T I C A L CHEMISTRY. VOL. 46, NO. 9, AUGUST 1974
> E
Y
4
l o g [ H S A ~ ,r g / m l
Figure 3. Potential differential between antigenly-globulin solutions and antigen/antibody supernatants
p'
1
0
I
1.0
8
IgG fraction of anti-HSA used with two different potencies, ( 0 )0.5, and ( 0 ) 2.2 rng/ml. showing the three precipitin zones: A . antibody excess, E. equivalence, C. antigen excess
2.0
log[WSAl, W / m l
Figure 2. Dependence of peak potentials of supernatants on total added antigen (HSA) concentration A . Supernatant after precipitin formation, 6. antigen alone, C. antigen plus y-globulin
blank solution-Le., containing no antigen-to test solutions with increasing concentrations of antigen (HSA). Clearly, significant potential changes were detected as the extent of precipitin formation increased, but peak heights increased again with addition of antigen in excess of the precipitable antibody content. A complete curve of peak height potential as a function of added antigen concentration is given in Figure 2 (curve A), showing a minimum a t ca. 30-40 pg/ml HSA. Initially, a progressive decrease in peak heights was observed due to precipitation of antibody, but a t antigen concentrations >40 pg/ml, peak heights began to i.ncrease again. Addition of more antibody to the supernatants in this high antigen concentration range resulted in fresh precipitation, indicating that not all of the antigen was precipitated by the antibody. The increase in peak heights after the minimum was, therefore, due to the presence of unprecipitated antigen but, in addition, to inhibition of precipitation caused by the formation of soluble antigen-antibody complexes of different composition from the precipitate (4,s).Curve B was obtained for calibration of the HS.4 antigen alone, showing that the electrode was capable of detecting HSA concentrations from as low as ca. 5 pglml. Peak heights for curve B were higher than curve A in the equivalence zone. but lower in the antigen excess region, indicating the conversion of insoluble AB/AG complexes to soluble species on addition of excess antigen. If HSA antigen had been incapable of reaction with the antibody, then curve C would be expected. This was ob-
tained by adding HSA to a fixed concentration (0.34 mg/ ml) of y-globulin which was not immunologically active with respect to HSA. Curve C shows the expected additive effect of HSA on non-active globulin solution. Therefore, the minimum in Figure 2 was due to specific and complete precipitation of AB/AG complex, a t which point antigen added in excess began to inhibit precipitin formation. The data in Figure 2 give an alternative way of measuring the true shape of an immunoprecipitin curve. Instead of measuring the precipitated antibody as in the conventional method ( 4 , 5 ) , the quantity of unbound antibody and/or antigen was determined. T o obtain the true shape of the precipitin curve, the potential for total protein (curve C) was subtracted from the potential for unreacted antibody or antigen (curve A). The results are given in Figure 3 for antiserum of two different potencies (2.2 and 0.5 mg/ml, using 0.2 ml antiserum in the latter case). The curve shows the expected three distinct zones for the precipitin curvei.e., the region of antibody excess, the equivalence zone where neither antibody nor antigen are in excess, and the region of antigen excess in which precipitin formation is inhibited. As expected, the potential changes for the more weakly potent antiserum were much smaller than for the high potency sample, with an antibody titer ca. half that of the latter antiserum. The use of ion electrode measurements with IgG fractions, therefore, gives a rapid and simple method of determining the shapes of precipitin curves and of antibodylantigen levels, providing that a large excess of nonspecific protein is not present in the antigen solution. To test the effect of the presence of nonspecific protein in the region of antibody excess, synthetic mixtures of HSA and human y-globulin (y-G) were prepared a t various ratios. Potentials were measured after carrying out the pre-
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tempts were made to analyze the washed precipitates from the reaction of HSA with whole goat antiserum to HSA, using 0.1 ml antiserum of potency 3.6 mgiml. After dissolving the washed precipitate in alkali, peak height potentials for each test solution was recorded automatically. Precipitin curves in the region of antibody excess were determined for a pure solution of HSA and, also, for human serum containing a known concentration of HSA in a multicomponent serum solution. Comparison of the curves was made in order to identify possible nonspecific cross-reactions with antibody homologous to albumin. The curves in Figure 4 show good correlation between calibrations for the simple antigen solutions and for serum, indicating the specificity of the precipitin reaction for albumin and also the high sensitivity of the method, which requires only small quantities of antibody (0.1-0.3 mg) to determine antigen levels in the range 0.5-30 pgiml. The good agreement between these measurements and the curves obtained from supernatant measurements indicates the very low solubility of the albumin-precipitin complex.
DISCUSSION
[HsAl, u s / m l
Figure 4. Calibration of HSA antigen by analysis of washed precipitates
( 0 )HSA in aqueous solution. ( 0 )HSA HSA of potency 3.6 mg.ml
in serum, using whole antiserum to
Table I. Effect of Nonspecific Protein o n Peak Heights of Supernatant Analysisa [HSA],
6.8 6.8 6.8 6.8 13.3 13.3 13.3 13.3 "
inl'
[>-GI,p g m l h
[-,GI [HSA]
Peak helght mV
0 4.8 36.2 60.4 0 9.6 72.5 120.8
0 0.7 5.3 8.9 0 0.7 5.5 9.1
44 43 47 54 23 26 38 46
Antiserum potency 2.2 mg nil.
The results show that the automated electrode method of analysis for protein (20) is applicable as a simple, rapid method for monitoring immunochemical reactions of serum proteins, for development of specific, sensitive methods of analysis for a wide range of antigens, and for study of the properties of immunochemical systems. The observed potential changes were sufficiently large to allow accurate and precise measurement of precipitated antigen or residual antibody in the supernatant to be made by the automated electrode system. Calibration curves constructed in the region of antibody excess can then be used for determination of antigen in the range 0.5-30 pg/ml. It was, therefore, possible to achieve an improvement in sensitivity of ca. a factor of 10 over the earlier electrode method for total protein (20) with the added advantage of specificity due to the AB/AG reaction. The increase in sensitivity can be attributed ( 4 , 5 ) to the fact that only a relatively small quantity of antigen is required to precipitate a much larger quantity of antibody, depending on the combining ratio in the precipitate a t the equivalence zone. The precipitin reaction curve was easily measured for small quantities of antibody in the supernatant giving the shape expected for a typical immunoreaction with three distinct zones as shown in Figure 3. The origin of these x(AG)
Total conceniration in final volume of
+
Y(AB) I -(AG),(AB),,
1.20 ml.
cipitin reaction exactly as for the mono-specific antigen solutions, with results given in Table I for two different HSA concentrations. Interference from the nonspecific globulin started to become serious a t a ratio of 51 of 7-G to HSA where peak heights were greater than for HSA alone. This indicates, first, that the precipitation is specific for HSA, leaving y-G unreacted in the supernatant and, second, that analysis of supernatant cannot be used for specific antigen determination if a large excess of nonspecific protein is present with the antigen in the test solution. However, this approach should be applicable to a simple method of analysis for albumin and the major globulin fractions of serum, using the IgG fraction of antiserum as the source of antibody. Analysis of Washed Precipitates. For completely specific determinations of antigen, analysis of the washed precipitates from the immunoreaction is obviously necessary. T o show that this was possible by an electrode method, at1256
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NO. 9 .
zones has been discussed by many authors (4,5 ) , based on the assumption of a simple chemical equilibrium describing the antibody-antigen reaction: In the region of antigen excess, soluble precipitin complexes are considered ( 4 , 5 ) to form when the combining proportion of the precipitate in the equivalence zone changes. In the region of antibody excess and equivalence zone, it is critical in the calculation of antibody content of a serum that essentially all of the antigen be precipitated-ie., if a system consists of a single antigen and its homologous antibody, the supernatants in the equivalence zone should give negative tests for excess of both AB and AG. Electrode determination of the supernatants of the IgG antiserum are valuable for checking this requirement for the HSA-anti HSA system (Figures 2 and 3 ) , giving an alternative approach to the conventional precipitin method. Heidelberger and Kendall ( 3 )developed a theory for precipitin formation dependent on this assumption that antigen is completely precipitated in the equivalence zone.
AUGUST 1974
Table 11. Analysis of Electrode Potential Data for Precipitin Supernatants - AE, AG added, mg
mV
0 0.0024 0.0034 0.0068 0.0101 0.0133 0.0165 0.0258
66 58 55 44 30 23 19 16
AR'l
A B pptd by
ABIAG
A B pptd
unpptd, mg
difference, mg
ratio in ppt
by calcd, mg
0.336 0.276 0,260 0.204 0.140 0.106 0.088
0,074
-0.3
... 0.060 0,076 0,132 0.196 0.230 0,248 0,262
'& Determined from globulin calibration 120).
25.0 22.3 19.4 19.4 17.3 15.0 10.2
0.055 0.076 0.139 0.188 0,225 0.252 0.267
- 0.2
-a E" 0, a a
C alculated from Equation
2.
They showed that a plot of the ratio ABIAG L I S . added antigen gave a straight line of the general form:
tI Y
0
I
I
0.01
0.02
I
0.03
Io
AG pptd.. rng
where x is the quantity of antigen added and the constants a and b can be evaluated from the intercept on the ordinate ( a ) and the slope ( b ) .The equation then becomes:
AB pptd = ax - bx2
(2 )
This theory was used to check the validity of the electrode determination of unreacted antibody in the supernatants. From the peak height data in Figure 2, unreacted antibody in the supernatant was determined directly from the calibration of a standard globulin given previously (20). The quantity of precipitated antibody was found by difference from the total antibody added, giving the results in Table 11. Calculation of the expected quantity of antibody precipitated was made, from the data in Figure 5 , by plotting the AB/AG ratio us. added antigen to evaluate a and b, and then calculating precipitated antibody from Equation 2. The good agreement between the calculated and measured quantities of precipitated antibody confirms the assumption that the measured potential can be used for determination of antibody levels in the IgG fraction of antisera. Also, plotted in Figure 5 is precipitated AB as a function of added antigen concentration, showing the expected maximum due to saturation of the antibody-combining sites by reaction with antigen. Similarly, the electrode method was found to be capable of measuring the total washed precipitate after redissolu-
Figure 5. Quantitative shape of precipitin curves by analysis of su-
pernatants from the HSA-anti-HSA reaction A. Antibody quantity precipitated: 6.Antibodylantigen ratio in the precipitate
tion in alkali. The data in Figure 4 indicate that as little as 0.5 pg antigen can be quantitatively determined without interference from the other serum proteins. The data also show good agreement with the antigen concentration range found in Figure 2 for analysis of supernatants. The application of this ion electrode methodology could now be made to the wide range of immunoassays possible with commercially available antisera, offering simplification of the usual colorimetric procedures and the possibility of a fully automated system by use of continuous filtration previously described for radio-immunoassay (22 1. Finally, these results suggest interesting ideas for development of an immunoelectrode specific for antigen and/or antibody determinations in biological fluids. Received for review December 18, 1973. Accepted April 23, 1974. We gratefully acknowledge support of a grant from the National Institutes of Health. (21) A Pollard and C. B. Waldron, in "Automation in Analytical Chemistry," Technicon Symposia 1966, Vol. I., Mediad, Inc., White Plains, N.Y., 1967, p 49.
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