Electrode-based enzyme immunoassays using urease conjugates

ANALYTICAL

95, 483-493 (1979)

BIOCHEMISTRY

Electrode-Based Enzyme lmmunoassays Using Urease Conjugates M. E. MEYERHOFF Department

of Chemistry,

AND G. A. RECHNITZ

University

of Delaware,

Newark,

DelaH,are

1971 I

Received October 18. 1978 Urease conjugates are employed for competitive-binding enzyme immunoassays (EIA) of a model antigen, bovine serum albumin (BSA), and of cyclic AMP (CAMP). Urease activity bound to a double-antibody solid phase is determined with an ammonia gassensing electrode, after appropriate washing steps. Cyclic AMP analogs coupled to urease are used to determine their effect on the overall response characteristics of the CAMP assay. The use of urease as a label for EIA purposes is shown to yield sensitive assays for both proteins (BSA < 10 t&ml) and haptens (CAMP < lo-* M) with good day-to-day reproducibility.

The use of enzyme labels in place of radioisotopes for the measurement of antigens, antibodies, and haptens has stimulated the new and expanding field of enzyme immunoassay (EIA).’ This technique has been the focus of several recent reviews (l-5) and its merits compared to radioimmunoassay (RIA) have been discussed (6,7). In many cases, EIA can match RIA in terms of sensitivity and selectivity yet has advantages of speed, convenience, and reduced cost. EIA sensitivity and simplicity is, however, dependent on the choice of enzyme label. It is the purpose of this work to introduce urease as a new enzyme label and to demonstrate the applicability of this label to EIA by developing an assay system for a model protein antigen, bovine serum albumin (BSA), and for the biologically important nucleotide, cyclic adenosine 3’,5’monophosphoric acid (CAMP). The basic concepts of competitive-binding solid-phase EIA have been described elsewhere (1,2). The required separation-of antibody from the assay mixture can be accomplished in a variety of ways. Double’ Abbreviations used: EIA, enzyme immunoassay; B(H)SA. bovine (human) serum albumin.

antibody techniques are quite popular and involve the use of a second antibody, an antibody to the principle antibody, to induce separation. Crosslinking the second antibody serum (8,9) with ethyl chloroformate forms an insoluble suspension of small particles which still maintain a high degree of immunoreactivity toward the first antibody. Addition of such particles to an EIA assay mixture pulls the first antibody from solution along with enzymelabeled molecules bound to the antibody. Such a process is depicted in Fig. 1 and employed for all separations in this work. Measurement of enzyme activity in the bound solid phase is desirable because complete purification of the labeled substance is not necessary. A separation step may not always be necessary when measuring low molecular weight haptens by EIA. In some cases, antibody binding to the enzyme-labeled hapten causes complete inhibition of enzyme activity. This may occur when antibody binding either sterically hinders substrate access to the active site of the enzyme or induces enzyme conformational changes. Whatever the cause, a more convenient homogeneous EIA system can result (10,ll).

483

0003-2697/79/080483-l 1$02.00/O Copyright k 1979 by Academic Press, Inc. All rights of reproduction in any iorm rewrved.

484

MEYERHOFF ElA assay mixture. containing first antibody+ snzyms labeled and free molecule*

addition of second antibody suspension

pellet

insolubilizsd antiswum particles

supernatant

FIG. 1. Schematic representation of double-antibody solid-phase EIA using crossIinked second-antibody particles.

Detection limits in EIA are ultimately determined by how low one can measure the label’s concentration via an activity assay. Sensitivity in such a kinetic determination is dependent upon the turnover number of the enzyme molecule and the method employed to detect the product of the catalyzed reaction. Purified urease, obtained from Sigma Chemical Company, has considerably higher activity on a molar basis (I.U. per mole of enzyme) than the best available commercial preparations of some other common enzyme labels such as alkaline phosphatase (13), ,&gaIactosidase (14), peroxidase (13,14), and glucose oxidase (13). This is due to the high molecular weight of urease [480,000 (IS)], and as a result, urease should be an excellent choice for sensitive immunoassays. Activity assays of enzymes bound to solid phases in EIA systems have been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time (1). Kinetic assays of enzyme activity have not been used to date because of the difficulty

AND RECHNITZ

in directly monitoring initial rates of enzyme reactions in a turbid solid-phase suspension. With urease as the label, an ammonia gassensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as a substrate. In this paper we demonstrate that urease can be used as a label for protein antigen-type molecules by employing a ureasebovine serum albumin (BSA) conjugate to carry out competitive binding EIA for BSA. Results here give evidence that urease is a good choice of enzyme label for sensitive and simple assays. We further show that urease can be effectively applied to hapten assays by developing an EIA system for CAMP. Results indicate that with proper choice of enzyme-nucleotide conjugate, a sensitive and selective assay of CAMP in the nanomolar concentration range is possible. EXPERIMENTAL Equipment. All potentiometric measurements were taken on a Coming model 12 research pH meter in conjunction with a Heath-Schlumberger model SR-255B strip chart recorder. An Orion model 95-10 ammonia gas-sensing electrode was used for all assays of urease activity. Potentiometric activity measurements were made at 25°C in a lo-ml glass thermostated cell, with circulation provided by a Haake model Fs water bath/circulator. Separation of double-antibody solid phase was done with a Precision Scientific Universal centrifuge at 1OOOg. Ultraviolet absorption spectra of protein-nucleotide conjugates were taken on a Hitachi model 100-60 UV-Vis spectrophotometer with l-mm pathlength cells. Reagents. Urease, Type VII, highly purified powder from jack beans, was obtained from Sigma Chemical Company, St. Louis,

UREASECONJUGATESINENZYMEIMMUNOASSAYS

Missouri. During the course of this work various lots were obtained with specific activities ranging from 40,000 to 70,000 units/g according to Sigma’s assay procedure (13). All nucleotides, including 02’monosuccinyl cyclic adenosine 3’,5’-monophosphoric acid ( 02’-monosuccinyl-CAMP), 02’-monosuccinyl cyclic guanosine 3’ ,5’monophosphoric acid (O*‘-monosuccinylcGMP), 02’-monosuccinyl cyclic inosine 3’S’-monophosphoric acid (O*‘-monosuccinyl-cIMP), adenosine 5’-monophosphoric acid (AMP), guanosine 5’-monophosphoric acid (GMP), cyclic guanosine 3’S’monophosphoric acid (cGMP), and cyclic adenosine 3’,5’-monophosphoric acid (CAMP), as well as BSA (fraction V) and isobutyl chloroformate, were also products of Sigma. Glutaraldehyde was obtained from J. T. Baker Chemical Company, and ethyl chloroformate from Aldrich Chemical Company. Ultra-pure ammonium sulfate and urea were purchased from Schwarzf Mann, Orangeburg, New York. Antiserum to BSA was a commercial preparation obtained from Miles Laboratories Inc., Elkhart, Indiana, which contained 2.4 mg/ml of anti-BSA antibody. Goat anti-rabbit IgG antiserum was obtained from Calbiochem Laboratories, La Jolla, California. A tris(hydroxymethyl)aminomethanehydrochloric acid buffer, pH 7.5, containing 1 mM ethylenediaminetetraacetic acid (Tris-HCl-EDTA), was used as a working buffer throughout this work. The ionic strength of this buffer was 0.1 M. These specific conditions were chosen based on known pH and ionic strength optimums for urease activity (15). EDTA was added because of its stabilizing effect on the enzyme (16). All other buffers and solutions were prepared with reagent-grade chemicals and distilled-deionized water. Preparation

of urease-BSA

conjugate.

The method of glutaraldehyde conjugation used was based on the previously reported techniques of Avrameas (17) for preparation of enzyme-protein conjugates.

485

Two milligrams of BSA and 2 mg of urease (40,000 W/g) were dissolved in 1.0 ml of 0.1 M phosphate buffer, pH 6.8, and 0.1 ml of 1% aqueous glutaraldehyde was added slowly to the stirred solution. The reaction mixture was stirred for 2 h at room temperature, then dialyzed overnight vs 3 liters of Tris-HCl-EDTA buffer at 4°C. Particulate matter (probably high molecular weight aggregates of urease) were centrifuged out and the resulting supernatant was partially purified on a Sephadex G-200 column equilibrated with working buffer. The ureaseBSA conjugate appeared in the void volume since its molecular weight is well above 500,000. A second protein fraction of much lower molecular weight appeared later and was assumed to be unconjugated BSA or BSA aggregates. The resulting conjugate could be stored at 4°C for several months without significant loss of activity. A 1: 10 dilution of this enzyme conjugate was used in the actual assay of BSA. Preparation of CAMP antibody nucleotide -urease conjugates.

and cyclic

Antibody to CAMP was obtained through the Immunology Department of Roswell Park Memorial Institute, Buffalo, New York. Rabbits were immunized with an 02’monosuccinyl-CAMP-human serum albumin (HSA) conjugate prepared by the carbodiimide reaction method described by Steiner et al. (18). The rabbits were injected subcutaneously with 1.5 mg of conjugate in a 1: 1 mixture of phosphate-buffered saline and complete Freund’s adjuvant. Rabbits were boosted with the same dosage every 2 weeks for the Hurst 2 months, and then monthly after that. The rabbits were usually bled 2 weeks after injections. Production of antibody to CAMP was confirmed by classical immunological techniques (ring test, precipitin test) (19) using a test antigen of O*‘monosuccinyl-CAMP-@lactoglobulin prepared in the same manner as the HSA conjugate. The final antibody solution for the assay proposed was obtained from the y-globulin fraction of this serum after 3x

486

MEYERHOFF

AND

RECHNITZ

ammonium sulfate fractionation (20) and as to the degree of conjugation can be made. final dialysis vs Tris-HCl-EDTA buffer. Typically, a conjugation between 2 and 7 This antibody solution contained between mol of nucleotide per mole of enzyme is 0.5 and 1.0 mg/ml anti-CAMP antibody as obtained based on a urease molecular determined by precipitin analysis (19) weight of 480,000 (15). using the p-lactoglobulin test antigen. The activity of the urease-cyclic nucleoUrease-cyclic nucleotide conjugates were tide conjugates was not significantly innot prepared by the carbodiimide method hibited by the presence of a lOO-fold exbecause of high loss of urease activity cess of anti-CAMP binding sites, with reduring the course of the reaction. Instead, spect to the concentration of enzyme-linked a modification of the mixed anhydride cyclic nucleotide (as determined by spectromethod used elsewhere to form steroidphotometric calculations and use of a 0.5 protein conjugates was used (21). Typically, mg/ml antibody solution). This suggests l-2 mg of the 02’-monosuccinyl derivatives that a homogeneous inhibition of ureaseof the cyclic nucleotide (free acid) was sus- cyclic nucleotide conjugate activity is not pended in 0.15 ml dioxane and 0.15 ml of possible and all eventual EIA systems indimethyl formamide with stirring at 4°C. volving these conjugates will be of the solidFive microliters of tributylamine, followed phase type. by 5 ~1 of isobutylchloroformate, were Procedure for determining urease acadded to the reaction mixture. The reac- tivity bound to antibody. It has been shown tion was run for 1 h at 4°C at which point the that the activity of enzymes can simply and mixed anhydride of the succinylated nucleoreadily be determined through the use of tide was formed. The approximately 0.3 ml potentiometric-type electrodes (23-26). In of organic solvent present was then re- earlier work, we used an ammonia gasmoved by rotoevaporation. Five milliliters sensing electrode to kinetically follow actiof cold urease (0.4 mg/ml) in 0.1 M NaHCO, vation of creatininase enzyme (23). It was at pH 9.4 was added to the residue material found that in a properly designed cell, and with constant stirring the conjugation activity measurements can be made in reaction proceeded for 2 h more at 4°C. volume as small as 0.8- 1.O ml, with stirring, by following the initial rate of potential Finally the urease conjugate was dialyzed extensively vs Tris-HCl-EDTA. change over a short period of time. In preliminary experiments for this work, a Contrary to other previously published assay system for urease was mixed anhydride procedures using other potentiometric proteins, it is an absolute requirement in designed to determine the lower limit of the case of urease that all organic solvent urease concentration one could measure be removed prior to final conjugation since accurately in a reasonably short assay time it was found that exposure of the urease to (4 min). This lower limit will ultimately determine detection limits for the EIA system even slight amounts of hydrophobic solvent renders it irreversibly inactive. Using the using urease. In these experiments, 1 ml of above method excellent final yields of urease buffer solution (prepared from a lot enzyme activity are obtained (approxicontaining 40,000 U/gram) was stirred in a thermostated cell at 25°C. An ammonia gasmately 80%). Conjugation of the nucleotides to urease sensing electrode was placed in the soluwas confirmed by uv spectra between 300 tion, and the potential was allowed to reach and 240 nm. Based on the change in the a steady-state value. Then 15 ~1 of 6 M urea was added and potential vs time curves urease spectra before and after conjugation and the known molar extinction coefficients recorded. A typical recording is shown in (22) of the cyclic nucleotides, an estimate Fig. 2. The “rate portion of curve” ex-

UREASE

flme

(min

CONJUGATES

IN ENZYME

)

FIG. 2. Typical potential (E) vs time recording obtained for assay of urease with an ammonia gassensing electrode. Curve shown is for addition of 15 ~1 of 6 M urea to 1 ml of 4 X lo-‘” M urease in 0.1 M Tri-HCI-I

x 1O-3

M

EDTA,

pH 7.5.

pressed in mV/min can be calculated by graphically extending the straight line portion of the curve. This rate can then be plotted vs urease concentration and a curve shown in Fig. 3 is obtained. At lower enzyme concentrations the correlation is linear and at higher levels, nonlinearity occurs due to the time response of the electrode ultimately becoming the rate limiting step. All EIA work was performed at the lower rate region and thus the rates seen in the EIA assays are directly proportional to urease activity. Furthermore, reproducible starting potentials within * 1 mV are achieved by dialyzing the electrode back to background ammonia levels in a large volume of buffer for a brief period of time (2-3 min) between each assay. This assay system offers a distinct advantage over the spectrophotometric method for assay of urease in an EIA system. It is most convenient for solid-phase EIAs to

IMMUNOASSAYS

487

measure the amount of label bound to the antibody, because as previously mentioned total purification of the label is therefore not necessary. Spectrophotometric assay of bound enzyme on solid-support particles is difficult due to turbidity problems. Electrode assay of bound enzyme circumvents this problem and allows for a truly kinetic assay of the enzyme. In this work we employed the insolubilized second-antibody approach to separate bound from free label (27). A j-ml quantity of goat anti-rabbit y-globulin serum from Calbiochem [containing 125 units, as one unit is defined in the Calbiochem Catalog (28)] was reconstituted and dialyzed vs 0.15 M NaCl. This antiserum was then polymerized into finely grained immunoreactive particles with ethyl chloroformate according to the method outlined by Avrameas (29). These particles can easily be pipetted, thus making them useful for quantitative immunoassays (29). From the initial 5-ml bottle of goat serum (125 units), the resulting particles after thorough washing were resuspended to a final volume of 15 ml with

FIG. 3. Typical rate of potential change vs urease concentration curve as determined in I ml of ureaseTris-HCI-EDTA, pH 7.5.

488

MEYERHOFF

working buffer. It was found that for both BSA and CAMP assays, 300 ~1 of this suspension was enough to remove all rabbit y-globulins from the assay solution. Urease activity on these particles can easily be quantitated by the above electrode method after centrifugation and washing steps. Final urease assays of washed particles is done in 1 ml of 0.1 M Tris-HCl-EDTA buffer, pH 7.5. Procedures for EIA experiments. For both BSA and CAMP systems, all assays were carried out in 3-ml conical centrifuge tubes. A given amount of rabbit antibody and enzyme conjugate and a standard amount of BSA or nucleotide were mixed for 1 h at 4°C (constant shaking action). Three hundred microliters of polymerized goat antirabbit y-globulin were added, followed by further incubation with mixing for 2 h more. The tubes were centrifuged at 1OOOgand the pellet washed three times with working buffer. Finally the pellet was resuspended in 1 ml buffer for urease assay. Calibration curves are expressed in percentage activity relative to a tube in which plain buffer was substituted for antigen or nucleotide aliquot. This tube’s pellet contains maximum activity as determined by the change in potential with time (mV/min) by the ammonia electrode method for urease determinations. This rate is designated as 100% activity and is indicative of the amount of urease label bound to the antibody in the absence of competing free antigen or hapten. Presence of standard CAMP or BSA in the respective assay tubes reduces the amount of urease label bound. Nonspecific binding of enzyme label to second-antibody particles was very low, usually <5% of maximum activity bound, as determined by a blank tube, in which all reagents were added except the first antibody and hapten or antigen standard. For BSA assay: Typically 40 ~1 of a 1: 100 dilution of rabbit anti-BSA (2.4 mg/ml) and 40 ~1 of a 1:lO dilution of urease-BSA conjugate were added to 1.5 ml of buffer-

AND

RECHNITZ

BSA solution. For CAMP assay: 30 ~1 of a 1:lO dilution of the (NH&SOI fraction of CAMP antiserum (0.5 mg/ml) and 30 ~1 of a 1: 10 dilution of enzyme-cyclic nucleotide conjugate were added to 1.O ml of buffer or buffer-nucleotide standard. RESULTS

AND DISCUSSION

In order to fully evaluate urease as a potential label for EIA in general, we felt it was necessary to demonstrate that urease could be covalently coupled to an antigen and still maintain sufficiently high activity for sensitive immunoassay. In addition, the effect of antibody binding to the labeled antigen should be tested to see if enzyme activity can be homogeneously inhibited by the antibody. BSA was chosen as a model protein antigen and coupled to urease as described in the experimental section. Homogeneous experiments using excess antibody to BSA indicated that no significant inhibition of the conjugate activity took place and that any useful EIA system employing ureaseprotein conjugates would have to be of the solid-phase type. Figure 4 shows the typical decrease in binding of a urease-BSA conjugate to anti-BSA antibody in the presence of increasing amounts of BSA. One hundred percent activity refers to the relative amount of urease activity present on the insolubilized second-antibody particles when no BSA is present. This 100% value was typically 14-15 mV/min using a potentiometric ammonia electrode as described above (activity assay time of 4 min). The limits of detection in this case (
UREASE CONJUGATES

IN ENZYME

IMMUNOASSAYS

489

80. > .z .? f

40.

8 20.

1

10 BSA

102 cont..

103

104

rig/ml

FIG. 4. Calibration curve obtained for BSA by monitoring amount of urease-BSA conjugate bound to anti-BSA antibody. Data obtained with tubes containing 1.5 ml BSA standard, 40 ~1 of 1:lOO anti-BSA serum, 40 ~1 of I:10 urease-BSA conjugate, and 300 ~1 of insolubilized goat anti-rabbit y-globulin suspension, ah prepared in 0.1 M Tris-HCI-EDTA, pH 7.5.

duction were monitored over a comparable alter the assay in any way. Ammonia produced from the urea present in the sample time period (either by rate or fixed-time method) an even more sensitive EIA sys- is adequately removed by the washing tem would result. procedures. Incubation times to reach equiThe optimum amounts of antibody, urease librium enzyme bound value levels were conjugate, and insolubilized second anti- surprisingly short, e.g., 1 h for rabbit antibody used in an assay must be determined from a series of titration experiments. For example, to obtain analytically reproducible antigen inhibition data, it is desirable to have a minimum amount of antibody bind a minimum amount of enzyme label to produce a sufficiently fast rate at 100% activity conditions. Figure 5 shows a typical series of titrations with rabbit anti-BSA using three different amounts of urease-BSA conjugate. These experiments were carried out by adding varying amounts of antiBSA antibody to tubes containing fixed amounts of urease-BSA conjugate in working buffer followed by the normal separation and assay steps. To obtain a sensitive assay system, it is important to work at the ug antibody antibody level at which saturation begins FIG. 5. Titration of various amounts of a 1: 10 diluto occur and also have this point correspond tion of urease-BSA conjugate with rabbit antibody to to a reasonably fast rate (12- 15 mV/min). BSA; 0, 25 ~1; & 80 yl; q , 200 ~1. Conditions as The effect of 10e3 M urea in the assay mixin Fig. 4, except that 1.5 ml buffer replaces standards ture was tested and as expected it did not in all tubes.

490

MEYERHOFF

AND RECHNITZ

body and conjugate, and 2 h following addition of the second antibody. Longer incubation times (up to 24 h) did not significantly change the results. The same general approach was used to develop an EIA procedure for CAMP. Cyclic AMP was conjugated to urease by the mixed anhydride procedure using the same 02’-monosuccinyl derivative previously employed to prepare the immunizing protein (HSA-CAMP). Preliminary experiments with this urease-CAMP conjugate and anti-CAMP antibodies (see Experimental) indicated that as in the case with the urease-BSA conjugate, no homogeneous inhibition of the conjugate could take place. This is probably a result of the small size of the substrate and the inability of antibody binding to sterically hinder urea from getting to the active site of the enzyme. Therefore, the double-antibody solid phase was also used for the CAMP assay. Figure 6 shows a typical calibration curve obtained for the inhibition of binding of a urease-CAMP conjugate to anti-CAMP antibody as determined by the ammonia electrode. One hundred percent of activity refers to blank tubes which had rates of

loo-

ll- 12 mV/min in the absence of CAMP. Selectivity of the assay over structurally similar cGMP is also shown in Fig. 6. It takes approximately 1000 times more cGMP than CAMP to get equal inhibition, indicating the high selectivity of the antibody. However, the relative insensitivity (> lo-’ M) toward CAMP presented a problem if one wanted to eventually use such a system in physiological samples where CAMP levels are quite low (10p8- lop6 M). Van Weeman and Schuurs (27) demonstrated that for EIA-hapten systems, the nature of the hapten linked to the enzyme can have a profound influence on sensitivity. They found that if the hapten of interest is linked to the enzyme in the same manner as the hapten was linked to the immunizing protein, a relatively insensitive assay may result because antibody production is elicited to the bridging group as well as the rest of the haptenic structure. Evidence of this type of antibody specificity in CAMP antibodies has been shown by RIA methods. Immunization with the 02’-monosuccinyl derivative gives rise to antibody production with strong recognition of the ester linkage at the 02’ position of the ribose

*

80. > I: 51 60.

8 40.

20.

I 10-B

10-6

10-7 ““cleotide

cont..

10-5 M

10-4

10-J

FIG. 6. Calibration curves obtained for CAMP and cGMP using a urease-CAMP conjugate and CAMP antibody. Data obtained in 0.1 M Tris-HCl-EDTA, pH 7.5, using 1.0 ml of nucleotide standard, 30 ~1 1:lO rabbit anti-CAMP antibody, 30 ~1 1:lO urease-CAMP conjugate, and 300 ~1 of second-antibody suspension.

UREASE CONJUGATES

IN ENZYME

IMMUNOASSAYS

491

FIG. 7. Calibration curves obtained from inhibition response to CAMP, cGMP, AMP, and GMP, using a urease-cGMP conjugate and CAMP antibody. Data obtained as in Fig. 6.

ring. Initial acetylation of CAMP samples (at the 02’ position) has brought forth increased sensitivity in the RIA method (30, 31) as a result of stronger affinity between antibody and the acetylated free CAMP. To increase sensitivity for EIA systems, Van Weeman and Schuurs showed that one could alter the site of hapten attachment to the enzyme, change the nature of the bridging group (i.e., succinyl to glutaryl), or use a hapten structurally similar to that to be measured. This last approach was pursued here in an attempt to improve the sensitivity of the CAMP assay. Figure 7 shows a typical calibration curve for CAMP when using a urease-cGMP conjugate with CAMP antibody in the electrode-based system. Comparison with Fig. 6 illustrates the dramatic improvement in sensitivity obtained. Inhibition of label binding begins to occur at less than lop9 M CAMP. The urease-cGMP conjugate is prepared with the O*‘-monosuccinyl derivative of cGMP and only differs from the initial urease-CAMP conjugate and immunogen by a guanine instead of adenosine moiety in the haptenic structure. This substitution effectively decreases the binding constant between the label and the CAMP antibody, thus allowing free CAMP to inhibit at lower

concentrations. Direct comparison of the two conjugates is possible because final activity and degree of nucleotide conjugation were very similar for both. Figure 7 also shows the selectivity of this assay system over cGMP, GMP, and AMP. As expected, in switching to a ureasecGMP conjugate, selectivity over cGMP itself is reduced, but it still takes 20 times more cGMP than CAMP to produce the same amount of inhibition. This again can be explained by the relative affinity of CAMP antibody for free cGMP vs cGMP conjugated to the enzyme. The succinyl group present in the enzyme conjugate causes greater affinity for enzyme-linked cGMP than for free cGMP. The system is highly selective over the corresponding noncyclic nucleotides AMP and GMP. This result agrees well with previous RIA systems, indicating the CAMP antibodies have strong recognition of the cyclic phosphate ring (18). Along this same line, a urease-cIMP conjugate was prepared and tested in the EIA system for CAMP. Figure 8 shows the resulting inhibition curves obtained for both cAMPand cGMP. Sensitivity for CAMP is not as good as when using the ureasecGMP conjugate, and selectivity over cGMP

492

MEYERHOFF

AND RECHNITZ

100

so.

,z :E

so.

5 o*

40.

20.

FIG. 8. Calibration curves obtained from inhibition response to CAMP and cGMP using a ureasecIMP conjugate and CAMP antibody. Data obtained as in Figs. 6 and 7.

also appears to be somewhere between that obtained with the other conjugates (approximately 80 times). These results seem appropriate in view of the fact that cIMP is more similar in structure to CAMP than cGMP. It is important to note here that cIMP was not tested for inhibition since it has not yet been found to exist in physiological fluids at detectable levels. cGMP itself is present in serum and urine but at levels 10 times lower than CAMP (31). Furthermore, the antiserum to CAMP used throughout this work was taken from a single rabbit and there was no attempt to produce higher quality antisera in other rabbits, which, if obtainable, could lead to even more sensitive and selective assays. Analytical precision of an EIA method is ultimately limited by how reproducibly one can assay enzyme concentration via activity determinations. Previous potentiometric activity determinations have resulted in excellent relative standard deviations of 10% or less (23,24,26). In the preliminary part of this work, analysis of urease by our potentiometric system yielded similar precision, with maximum standard deviations at low urease levels (i.e.,
in Table 1. These data represent the relative percentage activity values for three different CAMP concentrations when using a urease-cGMP conjugate (as in Fig. 6) over a 4-day period. It can be seen that excellent reproducibility is observed, indicating the good precision of the activity assay as well as the time stability of the reagents involved. In fact, all urease conjugates prepared in this work can be stored for at least 4 months without significant loss of immuno- or enzymatic activity. Moreover, working buffer conditions have been chosen such that, possible inhibitors of urease which may be present in real samples (e.g., heavy metals) would not be expected to create a problem with the assays [EDTA TABLE REPRODUCIBILITY

Cont. CAMP w 2.5 x 10-E 2.5 x 10-7 2.5 x 1O-6

OF

1

CAMP CALIBRATION

Activity (%) Day 1 Day 2 Day 3 Day 4 66.1 34.8 8.7

67.7 33.1 8.5

64.0 30.1 10.0

69.0 33.0 8.0

CURVES

Mean activity (%I 66.7 32.8 8.8

D Rates of 100% activity tubes were 11.8, 11.5, 10.8, and 11.5 mVlmin for Days l-4. Average, 11.5; relative SD, 3.7%.

UREASE CONJUGATES

IN ENZYME

preserves full activity of urease under physiological conditions (16)]. We have shown in this work that urease can be effectively employed as a label for EIA of both protein-type antigens and low molecular weight haptens through the use of an ammonia electrode to measure bound enzyme. Furthermore, the first EIA procedure for CAMP has been demonstrated using urease-cyclic nucleotide conjugates. In view of the great interest in measuring CAMP in physiological samples, the EIA system described here should provide the basis for developing an attractive alternative to traditional CAMP assay procedures.

We thank Drs. 0. Roholt, A. Grossberg, and D. Pressman of the Department of Immunology, Roswell Park Memorial Institute, Buffalo, New York, for generous assistance in this project. The support of grant GM25308 from the National Institutes of Health is gratefully acknowledged.

14.

15. 16. 17. 18.

20. 21. 22.

REFERENCES I. Schuurs, A. H. W. M., and Van Weeman, B. K. (1977) Clin. Chim. Acta 81, l-40. 2. Scharpe. S. L., Cooreman, W. M., Bloome, W. J., and Leekeman, G. M. (1976) C/in.

23.

WHO

Biochim.

Biophys.

Acfa

251, 427-434.

10. Rubenstein, K. F., Schneider, R. S., and Ullman, E. F. (1972)

Biochem.

Biophys.

37, 846-851. 1 I. Rowley. G. L., Rubenstein,

Res. Commun.

and Ullman, E. F. (1975) J. Biol. Chem. 250, 3759-3766. Bastiani, R. J., Phillips, R. C., Schneider, R. S., and Ullman, E. F. (1973) Amer. J. Med. Tech. 39,211-216. Sigma Chemical Company Catalog (1977) Sigma Chemical Co., St. Louis, MO. Boehringer-Mannheim Biochemical Catalog (1976- 1977). Boehringer-Mannheim Biochemicals, Indianapolis, Ind. Biochemica Information II (1975), Publication of Boehringer-Mannheim Biochemicals, lndianapolis, Ind. Papastathopoulos, D. S., and Rechnitz, G. A. (1975) Anal. Chim. Actu 79, 17-26. Avrameas, S. (1969) Immunochemistry 6, 43-52. Steiner, A. L., Kippis, D. M., Utiger, R., and Parker, C. (1969) Proc. Nat. Acad. Soi. USA 64, 367-373. Kabat, E. A., and Mayer, M. M. (1961) Experimental Immunochemistry, Chap. 2. Charles C Thomas, Springfield, Ill. Campbell, D. H., Garvey, J. S., Cremer. N. E., and Sussdorf, D. H. (1964) Immunology, pp. 118- 120, Benjamin, New York. Erlanger, B. F., Borek, F., Beiser, S. M., and Lieberman, S. (1957) J. Biol. Chem. 228, 713727. SchwardMann Radiochemical-Biochemical Catalog (1977). Becton. Dickinson, and Co., Orangeburg, N. Y. Meyerhoff, M., and Rechnitz, G. A. (1976) Anal. Chim.

25. 26.

53, 55-65.

6. Numazawa, M., Haryu, A., Kurosaka, K., and Nambera, T. (1977) FEBS Let?. 79, 3%-398. 7. Dray, F., Andrieu, J. M., and Renaud, F. (1975) Biochim. Biophys. Actu 403, 13 1- 137. 8. Engvall, E., and Perlman, P. (1972) J. fmmunol. 109, 129- 135. 9. Engvall, E., Jonsson, K.,and Perlman, P. (1971)

Acta

85, 277-285.

27.

28. 29

R. L., and Rechnitz. G. A. (1972) Anal. Chem. 44, 468-471. Cammann, K. (1977) Fresenius Z. Arm/. Chem. 257, l-9. D’Orazio, P., Meyerhoff, M. E.. and Rechnitz, G. A. (1978) Anal. Chem. 50, l531- 1534. Van Weeman, B. K., and Schuurs, A. H. W. M. (1976) in Immunoenzymatic Techniques (Feldman, G., Druet, P., Bignon, J., and Avrameas, S., eds.). pp. 125-133, American Elsevier, New York. Calbiochem Immunochemical Catalog (1978), Calbiochem-Behring Corp., La Jolla, Calif. Ternynck, T., and Avrameas, S. (1976) Stand. J. Immunol.

Suppl.

3, 29-35.

30 Harper, J. E., and Brooker, G. (1975) /. Cyclic Nucleotide

K. F., Haisjen, J.,

493

24. Llenado,

22, 733-738.

3. Wisdom, G. B. (1976) C/in. Chem. 22, 1243- 1255. 4. Landon, J. (1977) Nature 268, 483-484. 5. Voller, A., Bidwell, D. E., and Bartlett, A. (1976) Bull.

13.

19.

ACKNOWLEDGMENTS

Chem.

12.

IMMUNOASSAYS

Res.

1, 207-218.

31. Goldberg. M. L. (1977) Clin. Chem.

23, 576-580.