Immunoradiometric assay of succinylated corticotropin: an improved method for quantification of ACTH

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	Abstract

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In this paper we describe the development and the evaluation of a new type of immunoassay for human corticotropin (ACTH). We succeeded, by using an original approach based upon immunization with ACTH derivatized with succinic anhydride, in raising monoclonal antibodies against this poorly immunogenic peptide. Three of the antibodies were selected to develop an immunoassay for ACTH. The assay requires the prior succinylation of the plasma samples for optimal sensitivity and specificity. This acylation treatment is fast, reproducible, and, in addition, improves the stability of the ACTH molecule in plasma, thus facilitating sample handling. The assay is performed in only 3 h with a detection limit of 0.7 ng/L. Analytical evaluation showed excellent specificity, reproducibility, and reliability. A comparison with two commonly used but time-consuming ACTH IRMAs was carried out by assaying several plasma samples in parallel and gave in both cases very good correlation.

	Introduction

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The determination of corticotropin (ACTH) in human plasma is of major importance in the exploration of hypothalamic–pituitary–adrenal disorders (1)(2)(3).¹ For that purpose, immunoassays have advantageously replaced earlier biological (4)(5) and radioreceptor assays (6). However, since the first RIA described by Felber in 1963 (7), much progress has been made yielding improved sensitivity, specificity, and convenience. In particular, the production of more efficient antisera has obviated the need for extraction of ACTH from plasma samples and thus led to a decrease of the duration of the assay (8)(9).

The quantification of ACTH is now routinely performed in clinical laboratories by IRMAs (10)(11)(12)(13), which overcome most limitations of earlier methods. The use of monoclonal antibodies (MAbs) (14)(15) endows these immunoassays with the required sensitivity of 2 ng/L and avoids cross-reaction with metabolites and related peptides. Nevertheless, as most anti-ACTH antibodies currently available have slow association kinetics and poor affinity constants, this sensitivity is achieved often after an incubation of 20 h.

The poor immunogenicity of ACTH has always been the major difficulty in obtaining effective antibodies suitable for an immunoassay. This characteristic may be attributed to the amino acid sequence homology between human ACTH and the endogenous ACTH of immunized animals and by the short in vivo half-life of the hormone (16).

Therefore, to produce MAbs for a fast and sensitive immunoassay, we immunized mice with chemically modified ACTH. The aim of the modification was to alter the structure of the immunizing peptide sufficiently to differentiate it from the endogenous hormone of the immunized mice and to increase its resistance to the proteolytic enzymes responsible for in vivo ACTH degradation.

The chemical modification was performed with succinic anhydride, an acylation reagent that reacts preferentially with the -amino groups of lysine residues and NH₂ termini of proteins. This reagent was selected for three main reasons: (a) The succinic anhydride is sufficiently small (molecular mass 100.1 Da) to ensure that a modified amino acid would not constitute an epitope by itself; (b) as previously demonstrated with other analytes (17)(18), the acylation of human plasma with succinic anhydride is complete, rapid, and reproducible; (c) the derivatization of plasma samples is an easy one-step procedure.

By immunizing mice with succinylated ACTH (sACTH) conjugated to carrier proteins, we were able to obtain 39 MAbs. After their characterization, three of them, MAb 148, MAb 299, and MAb 314, were selected to develop an improved assay of ACTH. MAb 148 and MAb 314 were coated onto tubes and MAb 299 was radiolabeled to be used as tracer. Because the affinity of these antibodies for the succinylated ACTH is higher than for the native form, the assay requires the succinylation of plasma samples before their assay.

We report here the experiments that led to a two-step assay with a total incubation time of only 3 h and a detection limit of 0.7 ng/L. Furthermore, we demonstrate that the succinylation of plasma samples increases dramatically the stability of ACTH, thus facilitating sample handling and storage.

	Materials and Methods

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reagents
Human ACTH (1–39), its fragments (1–10, 1–24, 11–24, 18–39), and melanotropin (-MSH) were obtained from Neosystem. Succinic anhydride, from Sigma Chemical Co., was dissolved at 40 g/L in dioxane and lyophilized for reconstitution with dimethyl sulfoxide (DMSO), just before use, to a final concentration of 240 g/L. The lyophilized acylation reagent may be stored for 1 year at 18–25 °C and 6 weeks at 4 °C after reconstitution with DMSO.

mabs
Selection.
Anti-sACTH antibodies were obtained by immunization of BALB/c mice with sACTH conjugated with different carrier proteins. Immunization of mice, characterization of MAbs, and their use in epitope mapping have been previously reported (19). For the IRMA, three antibodies were selected on the basis of the epitope map of ACTH and of their dissociation constants for the succinylated hormone, found to be in the 10^-11 mol/L range. MAb 299, directed against the 1–13 amino acid sequence of sACTH, was used as tracer. MAb 148 and MAb 314, specific for the 18–24 and 25–39 sACTH regions, respectively, were coated onto the tubes.

Iodination.
MAb 299 was labeled with I by the chloramine T method (20). After iodination, the antibody was diluted in the assay buffer (0.1 mol/L phosphate buffer, pH 7.2, 0.5 g/L casein, 500 mmol/L NaCl, 10 mmol/L NaN₃) to 3 x 10 cpm/L. The labeled antibody may be stored for 6 weeks at 4 °C.

Biotinylation and coating.
Purified MAb at 1 g/L in 20 mmol/L borate buffer pH 8.2, 150 mmol/L NaCl was derivatized with a 20-fold excess of N-hydroxysuccinimide biotin (Sigma) in DMSO. The chemical reaction was stopped after 20 min at 18–25 °C by addition of a 1 mol/L ammonium chloride solution to a final concentration of 50 mmol/L. The biotinylated antibody was separated from biotin excess by gel filtration on G25 (PD10; Pharmacia) with PBS. Antibody-coated tubes were then prepared by overnight incubation at 4 °C with 1 mL per tube of a mixture of biotinylated MAb 148 and MAb 314 at 0.5 mg/L each in 20 mmol/L borate buffer, pH 7 containing 1 g/L bovine serum albumin, 9 g/L NaCl, and 10 mmol/L NaN₃ in avidin-coated tubes (Immunotech). After overnight incubation at 4 °C, tubes were desiccated. Dry tubes may be stored at 4 °C without loss of activity for 1 year.

calibrators
A solution of ACTH at 1 g/L was prepared by adding 500 µg of synthetic, highly purified human ACTH (1–39) to 500 µL of human serum previously depleted of ACTH and treated by addition of 1 mmol/L EDTA, 10 g/L ciprofloxacin, and 10 mmol/L NaN₃. This solution was then acylated by adding 250 µL of an alkaline solution (0.4 mol/L KOH and 0.16 mol/L KH₂PO₄ pH 6.7) and 50 µL of the succinylating reagent. A range of sACTH calibrators from 0 to 1500 ng/L was prepared by diluting the concentrated solution of sACTH in the depleted human serum previously succinylated by the same procedure. sACTH calibrators may be stored for 1 year at -20 °C and for at least 6 weeks at 4 °C.

plasma samples
Blood samples from healthy subjects and patients under evaluation for pituitary–adrenal disorders or receiving corticoid therapy were drawn into siliconized EDTA tubes (Vacutainer Tubes, Becton Dickinson). The blood was placed on ice immediately after collection and centrifuged at 4 °C; the plasma was then stored at -20 °C until use.

assay procedure
Five hundred microliters of plasma samples, 250 µL of alkaline solution, and 50 µL of succinylating reagent were dispensed successively into plastic tubes. Tubes were then vortex-mixed and incubated for 5 min at 18–25 °C.

Three hundred microliters of succinylated calibrator or succinylated sample were added to MAb 148- and MAb 314-coated tubes and incubated for 1 h at 18–25 °C with constant shaking. The contents of the tubes were then aspirated and 100 µL (3 x 10 cpm) per tube of tracer were added (specific activity at 1.2 mol of I per mole of antibody). After 2 h of additional incubation under the same conditions as above, tubes were washed twice with 2 mL of wash solution (9 g/L NaCl and 0.5 mL/L Tween 20 in distilled water). The bound radioactivity was then measured.

assay evaluation
Trinitrobenzene sulfonic acid (TNBS) assay.
Quantification of the -amino groups of plasma proteins was carried out by a colorimetric method described by Sashidhar et al. (21). Briefly, 1 mL of plasma sample and glycine calibrator at 0 to 1000 µmol/L in PBS was incubated with 1 mL of 40 g/L NaHCO₃ pH 8.5 and 1 mL of 1 mg/L TNBS in distilled water. After 2 h at 42 °C, 1 mL of 100 g/L sodium dodecyl sulfate in distilled water and 0.5 mL of 1 mol/L HCl were added to the reaction mixture. Absorbance was then measured at 335 nm. The number of amino groups present in the sample was directly determined from the glycine calibration curve.

Interference.
Different concentrations of ACTH fragments 1–10, 1–24, 11–24, and 18–39, as well as -MSH, were added to plasma samples previously depleted of or containing a known concentration of ACTH. These supplemented samples were then assayed according to the assay procedure.

Precision and reproducibility.
The within-run imprecision of the assay was evaluated by assaying 10 replicates of three serum samples. To evaluate total (day-to-day) imprecision, three plasma samples were assayed on 10 days by three different operators with reagents from three different lots.

Recovery.
Three plasma samples were assayed before and after addition of 22, 32, or 147 ng/L ACTH. Recovery was calculated as the ratio of recovered to added ACTH concentrations (expressed as a percentage).

Parallelism.
Three samples with high concentrations of ACTH were succinylated and serially diluted in ACTH-depleted human serum. The ACTH concentration was determined for each dilution following the assay procedure. Parallelism was assessed by calculation of the ratio of assay results to expected results (x 100%).

Method comparison.
The sACTH immunoassay was compared with two commercialized ACTH IRMAs (ELSA-ACTH, CIS Bio international, and R-ACTH, Nichols Institute) by assaying in parallel normal and pathological plasma samples. The correlation coefficients of the linear regression were calculated by the linear least-squares method.

stability of native and succinylated samples
At 4 °C.
Fifteen human plasma samples were succinylated and then assayed twice, once immediately after their derivatization and once after 1 week of storage at 4 °C. The stability of sACTH was evaluated by the percentage decrease of the ACTH concentration after 1 week of storage at 4 °C.

At 37 °C.
Ten human plasma samples were supplemented with 50 ng/L ACTH. One-half of each sample was succinylated and the ACTH concentration of both native and succinylated parts of samples were measured after different times of storage at 37 °C. The native samples were succinylated immediately before the assay; the succinylated samples were directly assayed. The stability of ACTH in both native and succinylated samples was determined by following ACTH concentrations during storage.

	Results

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assay optimization
Selection of MAbs.
MAbs were selected according to their dissociation constants and their capacity to bind simultaneously sACTH (19). The best results were obtained with MAb 299 as tracer and MAb 314 or MAb 148 immobilized on tubes. Interestingly, when both antibodies, MAb 314 and MAb 148, were used as capture antibodies (both antibodies immobilized together on the tubes), we observed that the specific signal was 16-fold stronger than with MAb 148-coated tubes and 3.6-fold stronger than with MAb 314-coated tubes. Thus, the specific signal for the calibrator at 1000 ng/L ACTH, expressed by the ratio, in percent, between the radioactivity bound to the tube and the total radioactivity, was 1.7%, 7.5%, and 27% for the MAb 148-coated tubes, MAb 314-coated tubes, and MAb 148–MAb 314-coated tubes, respectively, whereas the background was constant whatever the type of tube. This dramatic increase led us to select the combination of the two MAbs as capture antibodies.

Optimization of the succinylation procedure.
Succinic anhydride reacts primarily with the -amino groups of lysine residues and the NH₂ terminus of plasma proteins.

To optimize the succinylation procedure, we evaluated therefore the optimal succinic anhydride concentration required to acylate a plasma sample and the minimum incubation time required. ACTH was serially diluted in human plasma to prepare solutions containing 0 to 1000 ng/L. Solutions were acylated with succinic anhydride at 60 to 480 g/L in DMSO and assayed 30 min later. Unlike the background, which was low whatever the conditions used, the maximum signal was reached with succinic anhydride at 240 g/L. With higher concentrations of the succinylating reagent, the specific binding decreased slightly (up to 25% for a concentration of 480 g/L).

The optimal duration for succinylation was then studied. ACTH solutions were treated with succinic anhydride at 240 g/L and left at room temperature. Aliquots were taken at intervals from 0 to 30 min for immediate assay. Because the signal obtained after 5 min of incubation before the immunoassay was not increased with longer incubation times, we concluded that succinylation was complete within 5 min.

Duration of immunological steps.
The kinetics of the two immunological steps, i.e., the binding of sACTH to the antibody-coated tubes and the binding of the labeled antibody to sACTH, were independently studied.

Thus, when we studied the kinetics of the first immunological step, the binding of sACTH to the capture antibodies, we observed that equilibrium was reached in about 1 h. For the second step, at least 20 h were necessary to reach equilibrium. The greatest sensitivity, however, was attained after only 2 h of incubation with the tracer. At this time, 70% of maximum binding of the radiolabeled antibody was observed. Over a longer period of time, the nonspecific binding increased more than specific binding, thus affecting the sensitivity of the assay.

Incubation volume for immunological steps.
The volumes for the immunological incubations were optimized by using the two-step, 1- 2-h protocol. To obtain the greatest sensitivity with the strongest signal, the volume for the first incubation was set for practical reasons at 300 µL. For the second incubation, different volumes of tracer were tested. For the same quantity of radioactivity (3 x 10 cpm/tube), the most concentrated tracer solution gave the strongest signal. Thus, for each calibrator, the specific signal was twice higher by using 100 µL instead of 300 µL of labeled antibody; the background remained unchanged.

assay performance
Reproducibility of the succinylation procedure.
The reproducibility of the derivatization procedure was evaluated by quantitative measurement of the amino groups of 10 plasma samples, before and after succinylation with the colorimetric TNBS assay. An average of 86.6% (CV 2.8%) of the amino groups were succinylated under the conditions defined above. These data demonstrate that, with the optimized derivatization procedure, the succinylation of plasma proteins is almost complete and highly reproducible.

Calibration curve.
The radioactivity bound to coated tubes was directly proportional to the amount of ACTH between 0 and 120 ng/L (Fig. 1 ), the range of the physiological concentrations. The detection limit, calculated as two SDs (95% confidence) from the mean binding value of 10 zero calibrators, was 0.7 ng/L.

View larger version (17K): [in this window] [in a new window]   Figure 1. Representative calibration curve. Inset: calibration curve at low ACTH concentration.

Specificity.
Potential cross-reactivity of the ACTH fragments 1–10, 1–24, 11–24, 18–39, and -MSH was studied by addition of known concentrations of these molecules to a human plasma sample previously ACTH-depleted. As shown in Table 1 , no appreciable cross-reaction was noted with ACTH 1–10, 18–39, 11–24, and -MSH. However, ACTH 1–24 cross-reacted slightly in the assay since we measured apparent ACTH concentrations equivalent to 9–10% of the ACTH 1–24 concentration studied, expressed in ng/L. This means that the reactivity of the assay with ACTH 1–24 is approximately 20 times lower than the reactivity with ACTH 1–39, if we take into account the respective molecular masses. The potential interference by the ACTH fragment 1–24 and -MSH was also studied by addition of known concentration of these molecules to a plasma sample containing ACTH at 263 ng/L. As shown in Table 1 , the addition of -MSH did not interfere significantly at concentrations of up to 1 µg/L, whereas the addition of 1 µg/L of ACTH 1–24 led to the same increase of signal as in the ACTH-depleted plasma sample.

Imprecision.
The calculated CVs ranged from 6.9% to 9.1% for the intraassay and from 6.2% to 9.6% for the interassay (Table 2 ).

Parallelism and recovery.
Parallelism was assessed by serially diluting in the zero calibrator three succinylated patient samples containing high concentrations of ACTH. Recovery was estimated by assaying three different plasma samples supplemented with different amounts of ACTH (31, 146, or 670 ng/L). The ratio of observed/expected ACTH concentrations (expressed as percentage) in studies of parallelism (dilution) and recovery was 100% to 116% for the diluted samples and from 100% to 111% for the supplemented samples (Tables 3 and 4, respectively).

Correlation.
Human plasma samples were assayed in parallel for ACTH with our assay and two commercial IRMAs (ELSA-ACTH, CIS Bio international and R-ACTH, Nichols Institute). The results of this study are shown in Fig. 2 .

View larger version (17K): [in this window] [in a new window]   Figure 2. Correlation between the ACTH IRMA of Immunotech (x) and two commercialized IRMAs (y): (A) For 60 sera, y = 0.87x + 3.1, r2 = 0.89 for the ELSA-ACTH; (B) for 52 sera, y = 0.74x - 0.18, r2 = 0.87 for the R-ACTH. Sy|x, confidence intervals for the slope and y-intercept of both curves are: (A) 5.547, 0.789–0.943 and 1.049–5.949; (B) 7.288, 0.661–0.821 and -3.161–2.794.

Normal and pathological values.
A preliminary study was performed on the plasma, drawn in the morning, of 28 apparently healthy adults and 7 patients with pituitary-dependent Cushing disease (Fig. 3 ). For the plasma of healthy individuals, the mean value was 20.2 ng/L with a range of 10 to 50 ng/L. For the pathological samples, the median value was 40 ng/L with a range from 18 to 70 ng/L.

View larger version (12K): [in this window] [in a new window]   Figure 3. Plasma ACTH values in 28 healthy subjects and 7 patients with Cushing syndrome.

Stability of ACTH samples.
The stability of ACTH in native and succinylated samples was evaluated in two studies. In the first one, plasma samples were stored at 37 °C in both native and succinylated form. At different times, aliquots of native samples were succinylated and immediately assayed while succinylated samples were directly assayed. As shown in Table 5 , native samples lost 11.1% and 15.6% after 1 and 2 h of storage at 37 °C, respectively, while succinylated samples were stable for at least 4 h, the average loss being 0.7%.

In the second study, 15 succinylated plasma samples were assayed immediately after chemical modification and after 1 week of storage at 4 °C. Under these conditions, the decrease of the ACTH concentration in succinylated samples was on average only 3.6%.

	Discussion

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The immunoassays of chemically modified antigens were first successfully used for haptens. The aim of the modification was to produce antibodies with high affinity suitable for sensitive immunoassays. The binding sites of antibodies raised against small molecules included most of the time also the chemical linker and, possibly, amino acid residues on the carrier protein used during immunization. These antibodies were therefore unable to recognize the hapten itself with high affinity. One way to maximize the affinity of the antibody is to convert the hapten in the sample into a derivative that mimics the immunogen by the chemical addition of a reagent identical to the spacer. For example, the derivatization by succinic anhydride of two haptens, c-AMP and serotonin, increased the antibody affinity by an average of 300-fold to 2600-fold for c-AMP, depending on the antiserum (22), and about 1000-fold for serotonin (18). For histamine, with its lower molecular mass (111.1 Da), succinylation was combined with glycinamidation and led to an affinity enhancement of 5 x 10 (23). This demonstrates that the immunoassays of chemically modified haptens in biological fluids are easy and reliable, provided that derivatization is complete and reproducible.

In the present experiments, we derivatized the antigen not to increase its size but to improve its immunogenic properties. Because ACTH is an evolutionarily conserved polypeptide with only three amino acid differences between the human and the murine hormone, classical immunization does not lead readily to a strong immune response. Consequently, it is difficult to obtain effective MAbs against this hormone. The chemical derivatization with succinic anhydride allowed the differentiation of the structure of the immunizing ACTH from the cognate hormone of injected mice. The efficacy of this approach was demonstrated by the ease with which we obtained high-titer immune responses and performed three fusions leading to 39 MAbs previously described (19). Three of these antibodies were selected to develop an immunoassay for ACTH, with two antibodies on the solid phase and the third as tracer. The optimization and analytical performance of the assay in which they are used are described in this paper.

It is noteworthy that the use of two MAbs on the solid phase rather than either one alone increased the specific signal. We hypothesize that this increase of signal resulted from an increase of avidity of the solid phase for the antigen due to the simultaneous binding of sACTH to both immobilized antibodies.

In addition to its rapidity and sensitivity, the assay is also highly specific, as neither ACTH fragments 1–10, 11–24, 18–39, nor -MSH interfere. We observed only weak cross-reactivity with the biologically active ACTH fragment 1–24, which is not clinically relevant. This cross-reactivity is due to the binding of the peptide to the tracer, MAb 299, specific for the 1–13 sequence of ACTH, and to the capture antibody MAb 148, which is specific for the 18–24 sequence of ACTH. Because ACTH 1–24 is recognized by only one antibody immobilized on the solid phase, it is not affected by the phenomenon of affinity enhancement observed with whole ACTH, resulting in a low binding of the ACTH fragment to the coated tubes and consequently in a low interference in the assay.

The reproducibility of the succinylation was confirmed by the colorimetric determination of the amino groups of 10 plasma samples before and after succinylation. Approximately 87% of amino groups were succinylated. These derivatized groups presumably correspond to the reactive amino groups in human plasma since we failed to improve succinylation whatever the conditions used. Because we previously demonstrated by HPLC analysis that acylation of ACTH was complete in plasma (19), the 13% remaining probably correspond to inaccessible amino groups of plasma proteins, perhaps for reasons of steric hindrance.

The demonstration that the stability of ACTH in plasma is increased by succinylation is of considerable practical importance, since it greatly improves the handling of ACTH samples. Contradictory results on the stability of the hormone in biologic fluids have been reported (24)(25); it is appropriate therefore to take much care with biological samples to avoid ACTH degradation, e.g., by keeping them on ice during manipulation. This instability also obliged manufacturers of kits to supply lyophilized ACTH calibrators and controls that after reconstitution must be stored frozen for limited periods of time. The stability of succinylated ACTH allows us now to supply ready-for-use liquid calibrators that can be stored for 6 weeks at 4 °C without any detectable loss of activity. After succinylation, plasma samples are also very stable and may be left at room temperature for several hours or at 4 °C for at least 1 week without any loss of ACTH.

To conclude, the assay that we developed makes possible the accurate measurement of ACTH concentrations in human plasma samples. Its detection limit is at least as good as currently commercialized IRMAs, but the assay requires only 3 h of incubation instead of 20 h for the other assays. Finally, the succinylation that stabilizes the samples should improve the clinical usefulness of ACTH immunoassays by avoiding underestimation of the concentration resulting from antigen degradation.

	Acknowledgments

 
We thank H. Rickenberg for comments and careful reading of the manuscript. We are grateful to Professor P. Jaquet for the gift of clinically identified samples.

	Footnotes

 
Immunotech, 130 ave. de Lattre de Tassigny, BP 177F, 13276 Marseille Cedex 9, France.

¹ Nonstandard abbreviations: ACTH, corticotropin; sACTH, succinylated ACTH; MAb, monoclonal antibody; -MSH, melanotropin; DMSO, dimethyl sulfoxide; and TNBS, trinitrobenzene sulfonic acid.

	References

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