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Library of sequence-specific radioimmunoassays for human chromogranin A.

Human chromogranin A (CgA), a member of the granin family, is an acidic 439-amino acid protein with a widespread expression in endocrine cells and neurons (1-3). The role of CgA is unsettled, but chaperone functions in the cellular packaging of hormones and a role as precursor for biologically active peptides have been suggested (4-7). In addition, CgA seems to be a useful marker for most neuroendocrine tumors (8-18). Even silent neuroendocrine tumors without secretion of known hormones release CgA (19).

A major problem for the diagnostic use of CgA measurements is that CgA in a tissue- and tumor-specific manner is processed into a multitude of different fragments. To define the use of CgA as a marker for neuroendocrine tumors and to study the expression and processing of CgA in neuroendocrine tissue, we have now developed a library of sequence-specific RIAs for human CgA.

The aim of the present study was to describe the development and accuracy of the RIA library and to illustrate its potential. Therefore, we have measured CgA processing products in gastrointestinal tissues and neuroendocrine tumors as well as in plasma from healthy human subjects and from patients with carcinoid tumors or pituitary adenomas.

Materials and Methods

NOMENCLATURE

In the present study, the specificity of the assays (antisera) is described by indicating the N- or C-terminal amino acid of the hapten as recognized by the antiserum. Thus, "CgA(1[right arrow]) assay" indicates that the antiserum used in this assay binds an epitope that reaches from amino acid 1 of CgA, and spans a distance of 4-6 amino acid residues in the C-terminal direction.

PEPTIDES

The fragments of human CgA shown in Table 1 were custom synthesized by Cambridge Research Biochemicals (CRB) Ltd, Zeneca (Cheshire, UK) and Saxon Biochemicals GmbH (Hannover, Germany). N-terminal truncated fragments of hCgA(1-9)-Tyr and hCgA(340-348)-Tyr were obtained by controlled cleavage in an automated protein sequencer (20). The purity and content of the peptides were controlled in our laboratory by reversed-phase HPLC, amino acid analysis, and mass spectrometry analysis.

OTHER REAGENTS

[Na.sup.125] I (specific activity 14.5 Ci/mg, corresponding to 2.1 Ci/[micro]mol) was purchased from Amersham International. Trypsin (specific activity, 274 U/mg protein; treated with L-tosylamido-2-phenyl ethyl chloromethyl ketone) was from Worthington Diagnostic Systems Inc. Synthetic hCgA(250-301) amide was from Peninsula Laboratories Europe LTD.

ANTIBODIES

Two alternative coupling reagents were used for producing five different human CgA antigens (Fig. 1).

Coupling reagent I, maleimidobenzoic acid N-hydroxysuccinimide ester, was used in separate reactions to conjugate 8 mg each of CgA(1-9)-Tyr-Cys, CgA(116-124)-Tyr-Cys, CgA(340-348)-Tyr-Cys, and Cys-Tyr-CgA(432439) at the Cys residues to bovine serum albumin (20 mg). The coupled products were dissolved in 30 mL of distilled water. The antigen solution (2 mL) was mixed with 2.5 mL of a 9 g/L NaCl solution and then emulsified with an equal volume of complete Freund's adjuvant (the State Serum Institute, Denmark) and used for the first immunization. For the following booster injections, 1 mL of the antigen solution was mixed with 4 mL of a 9 g/L NaCl solution and with equal volumes of incomplete Freund's adjuvant. Four immunization series of each eight randomly bred Danish white rabbits were performed. Rabbits 94184-94191 received injections of CgA(1-9)-Tyr-Cys; rabbits 95045-95052 received injections of CgA(116-124)-Tyr-Cys; rabbits 95053-95060 received injections of CgA(340-348)-Tyr-Cys; and rabbits 95037-95044 received injections of Cys-Tyr-CgA(432-439). Each rabbit received subcutaneous injections at two sites in the lower back with 1 mL of mixture at 8-week intervals. Blood (20 mL) was bled from an ear vein 10 days after immunization, and the serum was stored at -20[degrees]C for evaluation.

[FIGURE 1 OMITTED]

Coupling reagent II, bis-diazotized tolidine, was used to C-terminally conjugate 6 mg of CgA(210-222)-Tyr to bovine serum albumin. The coupled product was then dissolved in 4.2 mL of 0.1 mol/L N[H.sub.4]HC[O.sub.3], pH 8.0. The antigen solution (1.4 mL) was emulsified with an equal volume of complete Freund's adjuvant and used for the first immunization. For the following five booster injections, 0.7 mL of the antigen solution was mixed with 0.7 mL of 0.1 mol/L N[H.sub.4]HC[O.sub.3], pH 8.0, and with equal volumes of incomplete Freund's adjuvant. Eight randomly bred Danish white rabbits (rabbits 1610-1613, 1626, 1628, 1629, and 1631) recieved subcutaneous injections at three sites in the lower back with 0.3 mL of mixture per rabbit at 8-week intervals. Blood (20 mL) was bled from an ear vein every second week, and the serum was stored at -20[degrees]C for evaluation.

CGA([left arrow] 301 AMIDE) ASSAY

Immunoreactive pancreastatin [CgA(273-301) amide] was measured by RIA using antiserum 871w-1, specific for the amidated COON terminus of pancreastatin, as detailed elsewhere (21). The antiserum reacted fully with the synthetic peptides hCgA(273-301) amide and hCgA(250-301) amide.

PREPARATION OF TRACERS

The tyrosine-extended fragments of human CgA-CgA(1-9)-Tyr (4.4 nmol), CgA(116-124)-Tyr (4.2 nmol), CgA(210-222)-Tyr (2.7 nmol), CgA(340-348)-Tyr (4.0 nmol), and Tyr-CgA(432-439) (4.5 nmol)--were monoiodinated using the chloramine-T method, as described previously (22), and purified on reversed-phase HPLC (Aquapore C-8 column, RP-300,220 X 4.6 mm, 7 [micro]m bead size). The following linear gradients were used: 10-30% ethanol (HPLC grade; Merck) for CgA(1-9)-Tyr and Tyr-CgA(432-439); 0-20% ethanol for CgA(116-124)-Tyr and CgA(340-348)-Tyr (Fig. 2A); or 10-30% acetonitrile (HPLC grade; Rathburn) for CgA(210-222)-Tyr, all in 1.0 mL/L trifluoroacetic acid (TFA; HPLC grade; Pierce) in water. Fractions (1 mL) were collected at a flow rate of 1.0 mL/min. To evaluate the chromatographic separation of labeled and nonlabeled peptides, 1 mL of the monoiodinated peak fraction was mixed with 10 pmol of the relevant synthetic tyrosine-extended chromogranin A fragment and reapplied to the HPLC column as described. Both the radioactivity of the labeled peptides and the immunoreactivity were measured (Fig. 2B). Using the assumption that the CgA assays measured iodinated and unlabeled fragments with identical affinity, we estimated the specific radioactivity of the tracers by self-displacement (23).

EXTRACTION OF TISSUES

Surgical specimens of healthy human neuroendocrine tissue (gastric antral mucosa, ileal mucosa, and pancreas; n = 6) and neuroendocrine tumors (foregut and midgut carcinoid, and pheochromocytoma; n = 6) were obtained at the Departments of Surgical Gastroenterology and Urology, Rigshospitalet, University of Copenhagen. The use of human tissue was approved by the local ethics committee, and informed consent was obtained from the patients (KF01-352/96 and KF01-290/97). Immediately after removal, the tissues were frozen in liquid nitrogen. The frozen tissue was weighed frozen within 2 h (wet weight) and subsequently stored at -80[degrees]C. Biopsies of the tissues were immersed in paraformaldehyde. Only pancreatic and gastrointestinal tissue shown to be histologically healthy as well as only nonnecrotic tumors with diagnoses verified by pathologists were included for additional studies. The tissue samples were extracted within 1 year. While frozen, the tissue was cut into pieces of a few milligrams and immersed in boiling distilled water (10 mL/g tissue) for 20 min, homogenized, and centrifuged at 10 000g for 30 min. The supernatant was subsequently stored at -20[degrees]C until analysis (boiling water extract). The pellet was reextracted by addition of 0.5 mol/L acetic acid, pH 1.5 (10 mL/g tissue), homogenized, left for 20 min at room temperature, and centrifuged as above; the supernatant was then stored at -20[degrees]C until analysis (acetic acid extract). The extracts were diluted in 20 mmol/L sodium barbital buffer, pH 8.4, containing 0.6 mmol/L merthiolate and 1.1 g/L bovine serum albumin. The extracts were analyzed in dilutions with the six CgA RIAs. The CgA peptide concentrations given below are the sums of the immunoreactivities measured in boiling water and acetic acid extracts.

[FIGURE 2 OMITTED]

The extractions described above were chosen after evaluation of six different extraction procedures. Frozen tissues (gastrointestinal and carcinoid) were cut separately into small pieces, divided into six portions, and extracted using the following methods: boiling water extraction (10 mL/g tissue), followed by reextraction of the pellet with either (a) 0.5 mol/L acetic acid, pH 1.5, or (b) 0.05 mol/L carbonate buffer, pH 10.0; (c) boiling water extraction (30 mL/g tissue); extraction with (d) Tris buffer (4[degrees]C, pH 8.2); (e) Tris buffer containing a mixture of protease inhibitors; (f) acidified ethanol extraction (700 mL/L ethanol containing 7.4 g/L HCl).

In all of the CgA assays, the highest peptide concentrations were measured in the boiling water and Tris buffer extracts. Reextraction with acetic acid added up to 15% of the total immunoreactivity, whereas <5% was obtained after reextraction with carbonate buffer. Acid ethanol extracted <50% of the immunoreactivity obtained using boiling water.

ENZYMATIC TREATMENT OF TISSUE EXTRACTS

For trypsin cleavage, extracts of human gastric antral mucosa, pancreas, carcinoid tumors, pheochromocytoma, and chromatographic fractions (carcinoid) were incubated with equal volumes of 0.2 g/L trypsin in 0.1 mol/L sodium phosphate buffer, pH 7.5, at room temperature for 30 min. The enzymatic reaction was terminated by boiling for 10 min. Parallel control experiments were performed by omitting trypsin from the sodium phosphate buffer. Enzyme-treated extracts were centrifuged at 3000g for 10 min at 4[degrees]C. Trypsin-treated antral and pheochromocytoma extracts and controls were assayed with the CgA(1 [right arrow]), CgA(116 [right arrow]), CgA(210 [right arrow]), CgA([left arrow] 301 amide), CgA(340 [right arrow]), and CgA([left arrow] 439) assays, whereas the extracts of pancreas and carcinoid tumors were analyzed only with the CgA(340 [right arrow]) assay. When the procedure described above was used, the C-terminal dipeptide [Arg.sup.438][Gly.sup.439] was cleaved from Tyr-CgA(432-439) and the C-terminal dipeptide Tyr-Cys was cleaved from CgA(1-9)-Tyr-Cys.

PLASMA SAMPLES

Consecutive EDTA plasma samples, drawn over periods of 8-10 months, were obtained after overnight fasting from three patients with midget carcinoid tumors. In addition, EDTA plasma samples were obtained after overnight fasting from an additional 10 patients with midget carcinoid tumors and from 9 patients with pituitary adenomas. To establish reference intervals, plasma samples were also obtained after overnight fasting from 28 healthy control subjects (14 men and 14 women; age range, 27-53 years). The samples were diluted in barbital buffer containing 12.5 mL/L Trasylol[R] (20 000 kIU/mL; Bayer) and analyzed at 4[degrees]C with the CgA(1 [right arrow]), CgA(210 [right arrow]), CgA([left arrow] 301 amide), and CgA(340 [right arrow]) assays. The use of human plasma was approved by the local ethics committee, and informed consent was obtained from the patients (KF01-352/96 and KF01-290/97).

RIA PROCEDURE FOR CGA(1 [right arrow]), CGA(116 [right arrow]), CGA(210 [right arrow]), CGA(340 [right arrow]), AND CGA([left arrow] 439) ASSAYS

Incubation was performed at 4[degrees]C using barbital buffer as diluent. The tyrosine-extended CgA fragments were used as calibrators. Tracer solution corresponding to 1000 counts/min was added to calibrator solutions, tissue extracts, chromatographic fractions, and antisera dilutions. After 3 days, the antibody bound and free tracer were separated by the addition of plasma-coated charcoal. The ratio of sample (calibrator, plasma, tissue extract, or chromatography fraction) volume relative to the total final volume varied from 0.06 to 0.18 for the five assays.

Known controls, buffer blanks, and extract blanks (without antiserum added) were included in the assays. The samples were assayed in duplicate. The reliability of the assays was evaluated with respect to detection limit, specificity, and precision.

CHROMATOGRAPHY

Gel chromatography of carcinoid tumor extracts was performed on a Superose 12 HR column; fractions were eluted with 0.02 mol/L phosphate buffer, pH 8.0, containing 0.25 mol/L NaCl. The void volume and total volume were indicated by Blue Dextran and [sup.22]NaCl, and the column was calibrated with synthetic hCgA(250-301 amide). The fractions were analyzed with the CgA(1 [right arrow]), CgA(116 [right arrow]), CgA(210 [right arrow]), CgA([left arrow] 301 amide), CgA(340 [right arrow]), and CgA([left arrow] 439) assays.

Results

RADIOIODINATION

Of the added [sup.125]I, 67-84% was incorporated in the tyrosine-extended fragments (Fig. 2A). For all CgA peptides, labeled and nonlabeled peptide separated completely (Fig. 2B). The nonspecific binding was <5%. The dilution curves of labeled and unlabeled antigen were parallel for the CgA(1 [right arrow]), CgA(116 [right arrow]), CgA(210 [right arrow]), CgA(340 [right arrow]), and CgA([left arrow] 439) assays. When estimated by self-displacement, four of the tracers had similar specific activities: 0.74 Ci/[micro]mol for CgA(116-124)-[sup.125] I-Tyr; 1.3 Ci/[micro]mol for CgA(210-222)-[sup.125] I-Tyr; 1.0 Ci/[micro]mol for CgA(340-348)-[sup.125]I-Tyr; and 0.95 Ci/[micro]mol for [sup.125]I -Tyr-CgA(432-439). CgA(1-9)-"I-Tyr apparently had a fivefold higher specific activity (5.9 Ci/[micro]mol), and because this exceeds the activity of the Na [sup.125]I used, it must be ascribed to a fivefold reduction in affinity of the antibody for the radioactive antigen in spite of the parallel dilution curves for labeled and unlabeled antigen. Thus, the specific activity of this tracer cannot be estimated by self-displacement.

[FIGURE 3 OMITTED]

EVALUATION OF ANTIBODIES

The estimated avidity of the antisera as expressed by the effective equilibrium constant ([K.sub.0.sub.eff]) (24,25) varied from 0.1 X [10.sub.12] to 1.0 X [10.sub.12] L/mol (Table 2). The index of heterogeneity, a, described by Sips (26), varied between 0.95 and 1.04 for the examined antisera, indicating that the ligand binding is highly homogeneous (27) and that each antiserum acts as a solution of monoclonal antibodies (28). The antiserum displaying the highest avidity and titer in each immunization series was selected for additional characterization and used for subsequent measurements. The detection limit was determined in two ways (Table 3). The specificity of the antisera was expressed as the ratio of median inhibitory dose ([ID.sub.50]) for the calibrator peptide vs the ID51 for the truncated or extended CgA fragments in tracer displacement (Table 4). Antibody dilution was adjusted to yield a [B.sub.0] of 35%. Removal of the two N-terminal amino acids, leucine and proline, of CgA(1-9)-Tyr and the N-terminal leucine of CgA(340-348)-Tyr substantially decreased the binding with antibodies 94188 and 95060 (Table 4). Consequently, the N-terminal amino acids constitute an essential part of the epitope for antisera 94188 and 95060, respectively. Correspondingly, removal of [Arg.sub.438][Gly.sub.439] minimized the binding with antiserum 95038, corroborating that the dipeptide is an essential part of the epitope for antiserum 95038 (Table 4). The ratio of the [ID.sub.50] of CgA(210-222)-Tyr to the [ID.sub.50] of CgA(208-216) for antibody 1612 was 0.007, suggesting that the antibody is dependent on the free NH2 terminus of the CgA(210-222)-Tyr sequence (Table 4). Removal of the C-terminal dipeptide Tyr-Cys from CgA(1-9)-Tyr-Cys by tryptic cleavage did not change the immunoreactivity measured with the CgA(1 [right arrow]) assay significantly: the mean concentration was 32 pmol/L before and 33 pmol/L after trypsin cleavage. The results indicate that the CgA(1 [right arrow]) assay measures the Tyr-Cys extended peptide [CgA(1-9)-Tyr-Cys] and the native peptide [(CgA(1-9)] with equimolar potency.

The computer homology program FASTA was used to search the SwissProt database for amino acid sequences resembling human CgA(1-9), CgA(116-124), CgA(340-348), or CgA(432-439). No relevant sequences other than the corresponding CgA sequences of other mammalian species were found.

Dilution curves for tissue extracts (antral mucosa and two carcinoid tumors) were parallel with the calibrator curves for the CgA(1 [right arrow]), CgA(116 [right arrow]), CgA(210 [right arrow]), CgA([left arrow] 301 amide), and CgA(340 [right arrow]) assays (Fig. 3). In the CgA([left arrow] 439) assay, none of the three extract dilution curves were parallel with the calibrator curve (Fig. 3).

SPECIFICITY OF ANTISERA EVALUATED BY TRYPTIC DIGESTION OF TISSUE EXTRACTS

Incubation of trypsin with tissue extracts cleaves CgA at numerous basic residues. Hence, trypsin treatment releases the CgA fragment [Leu.sup.340]-[Lys.sup.355] from further N-and C-terminally extended forms by cleavage after [Lys.sup.338][Arg.sup.339] and after [Lys.sup.355], and subsequent quantification by the CgA(340 [right arrow]) assay provides an estimate of the total CgA mRNA translation product, i.e., total CgA. The concentrations measured in the CgA(340 [right arrow]) assay by antibody 95060 were higher after tryptic cleavage than before (Table 5), and similar results were obtained with antibody 95058. As described above, [Leu.sup.340] was shown to constitute an essential part of the epitope for antibody 95060. In combination, these results showed that antibody 95060 is specific for the free NHZ terminus of CgA(340348). The ratio of CgA(340 [right arrow]) immunoreactivity to CgA(340 [right arrow]) immunoreactivity after trypsin provides an estimate of the extent of endogenous cleavage at the basic residues adjacent to the epitope.

Trypsin also cleaves CgA at [Lys.sub.9]. The immunoreactivity measured with the CgA(1 [right arrow]) assay, using antibody 94188, was not influenced by tryptic cleavage (Table 6), indicating that the antiserum binds CgA(1-9) and larger C-terminally extended molecular forms with equimolar potency. Similar results were obtained with antibody 94185. It is known that trypsin is less effective when acidic amino acid residues are adjacent to the basic residues; consequently, trypsin was not expected to cleave CgA at [Arg.sub.115] or [Arg.sup.209]. In agreement with this, trypsin had no effect on CgA(116 [right arrow]) or CgA(210 [right arrow]) immunoreactivity (Table 6). Because trypsin will cleave CgA at [Lys.sup.114], Lys123, and [Arg.sup.248][Lys.sup.249] the results also indicate that the CgA(116 [right arrow]) and CgA(210 [right arrow]) assays quantify small and C-terminally extended larger peptides with equimolar potency. Tryptic cleavage at [Arg.sup.300] substantially decreased CgA([left arrow] 301 amide) immunoreactivity (Table 6), and because cleavage at [Arg.sup.300] will remove [Gly.sup.301] amide, we concluded that the amidated residue constitutes an essential part of the epitope for the CgA([left arrow] 301 amide) assay. Similarly, tryptic cleavage eliminated CgA([left arrow] 439) immunoreactivity (Table 6), and because trypsin will cleave CgA at [Arg.sup.437], these data show that the dipeptide [Arg.sup.438] [Gly.sup.439] is necessary for measurement by the CgA([left arrow] 439) assay (Table 6).

SPECIFICITY OF ANTISERA EVALUATED BY CHROMATOGRAPHY OF A TUMOR EXTRACT

Gel chromatography of a carcinoid tumor extract monitored by the CgA assays showed that the CgA(340 [right arrow]) immunoreactivity eluted at a position [distribution constant ([K.sub.d]) of 0.69] corresponding to a lower molecular weight than that of synthetic CgA(250-301) amide ([K.sub.d] = 0.62; Fig. 4).

After tryptic cleavage of the chromatographic fractions, 25% of the CgA(340 [right arrow]) immunoreactivity eluted as described above. However, the majority of the CgA(340 [right arrow]) immunoreactivity after tryptic cleavage eluted in a broad peak (Kd = 0.10-0.55) that probably includes full-length CgA. The CgA(1 [right arrow]), CgA(116 [right arrow]), and CgA([left arrow] 439) immunoreactivity also eluted with [K.sub.d] values in the range of 0.10-0.55 (data not shown), suggesting that these assays also detect full-length CgA. The immunoreactivity measured by the CgA(210 [right arrow]) and CgA([left arrow] 301 amide) assays, which recognize the free NHZ terminus and the amidated COON terminus of the epitopes, respectively, eluted in well-defined peaks ([K.sub.d] = 0.40-0.42), but did not recognize full-length CgA (data not shown).

QUANTIFICATION OF CGA IN HUMAN TISSUE

In extracts of antral mucosa (Fig. 5) and pancreas, the concentrations measured by the different CgA RIAs varied from 5 to 48 pmol/g and 6 to 121 pmol/g, respectively. In both tissues, the highest concentrations were found using the antisera directed against the NHZ terminus of sequences adjacent to dibasic cleavage sites, i.e., the CgA(116 [right arrow]), CgA(210 [right arrow]), and CgA(340 [right arrow]) assays. Lower concentrations were measured using antisera specific for the N-terminal epitope of CgA and the pancreastatin antiserum. The CgA(340 [right arrow]) concentration measured after tryptic cleavage (i.e., total CgA) was 63 and 171 pmol/g in the antral mucosa and pancreas, respectively (Table 5).

In carcinoid and pheochromocytoma tumor extracts, the concentrations were approximately 100- to 1000-fold higher (0.50-34 nmol/g; Figs. 6 and 7). In the six tumors analyzed, the highest concentrations were measured with the antisera specific for the sequences in the mid portion of CgA, and low concentrations were measured with the CgA(1 [right arrow]) assay. The CgA(340 [right arrow]) concentration after tryptic cleavage was 5-38 nmol/g.

[FIGURE 4 OMITTED]

CHROMATOGRAPHY OF A MIDGUT CARCINOID TUMOR EXTRACT

The CgA(340 [right arrow]) immunoreactivity eluted in a narrow peak ([K.sub.d] = 0.69) after that of synthetic CgA(250-301) amide ([K.sub.d] = 0.62; Fig. 8). The CgA(1 [right arrow]) assay measured a heterogeneous peak comprising several molecular forms. The CgA(116 [right arrow]), CgA(210 [right arrow]), CgA([left arrow] 301 amide), and CgA([left arrow] 439) assays also measured heterogeneous peaks ([K.sub.d] = 0.40-0.60; results not shown).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

QUANTIFICATION OF CGA IN PLASMA FROM CARCINOID TUMOR AND PITUITARY ADENOMA PATIENTS

Assuming a gaussian distribution of the data, reference intervals including the central 95% of the results (mean [+ or -] 2 SD) were calculated for the four CgA assays: CgA(1 [right arrow]), 288-696 pmol/L; CgA(210 [right arrow]), 1-197 pmol/L; CgA([left arrow] 301 amide), 3-15 pmol/L; and CgA(340 [right arrow]), 0-53 pmol/L.

In plasma from the carcinoid tumor patients, the CgA(210 [right arrow]) and CgA(340 [right arrow]) assays consistently measured increased concentrations, whereas the concentrations measured with the CgA(1 [right arrow]) and CgA([left arrow] 301 amide) assays were within the reference interval or increased in only some of the samples (Tables 7 and 8). In the plasma from nine patients with pituitary adenomas (four growth hormone-producing and five silent), increased concentrations were measured by the CgA(340 [right arrow]) assay, whereas the concentrations measured with the other CgA assays were within the reference interval or increased in only a few samples (Table 9).

Discussion

The present study describes the development and evaluation of a library of five sequence-specific RIAs for human CgA. The sequences chosen for immunizations are distributed along the entire CgA sequence, adjacent to proteolytic cleavage sites. The antisera raised were of high avidity and homogeneity; when the antisera were used in combination with well-defined monoiodinated tracers, highly sensitive and specific assays were obtained.

Tyrosine-extended CgA peptides were used to radioiodinate the peptides using the chloramine-T method. The CgA(1 [right arrow]) assay was found to bind the CgA(1-9) fragment and the extended CgA(1-9)-Tyr-Cys fragment with equimolar potency. Hence, the tyrosyl extension did not affect immunoreactivity. This specific observation implies that the fivefold lower affinity of antiserum 94188 toward CgA(1-9) [sup.125]I-Tyr compared with CgA(1-9) must be attributed to the presence of iodine atoms on the tyrosine residues. Furthermore, it agrees with the study of Schechter (29), who showed that the combining sites on antibodies bind peptide epitopes corresponding to 4-6 amino acid residues.

The use of an immunoassay for exact quantitative measurements is based on the assumption of identical binding affinities of antibody toward calibrator and native antigen. If there are major differences in binding affinities, the concentration estimates will be erratic. Parallelism of dilution curves for native antigen and calibrator indicates, but does not prove, identity of binding affinities. Nonparallelism, however, demonstrates major differences and thus precludes quantitative measurements. Dilution curves of tissue extracts revealed nonidentity in the CgA([left arrow] 439) assay; consequently, this assay can give only semiquantitative estimates. For the other assays, the dilution curves appeared to be parallel. However, because even slight non-parallelism may conceal differences in affinity constants and, hence, errors in estimates of the concentrations of the unknown antigens, the numerical results must be interpreted with caution.

Our results show that the CgA(210 [right arrow])- and CgA(340 [right arrow])-specific antisera require a free NHZ terminus of the CgA(210-222) and CgA(340-348) sequences, respectively, for binding. The concentrations measured with these assays in tissue extracts therefore depend on the extent of endogenous cleavage at the dibasic amino acids adjacent to the epitopes. When the CgA(340 [right arrow]) assay is used after trypsin treatment of the extract, an estimate of total CgA immunoreactivity can be made. Whether the CgA(116 [right arrow]) assay is dependent of the free NHZ terminus is not known, but the chromatography results suggest that the assay detects full-length CgA.

[FIGURE 7 OMITTED]

In the extracts of antral mucosa and pancreatic tissue, concentrations between 35-43% and 26-51%, respectively, of the total CgA immunoreactivity [CgA(340 [right arrow]) after trypsin treatment] were measured with the antisera specific for the NHZ terminus of the CgA(210-222) and CgA(340-348) sequences, whereas only 8% of total CgA immunoreactivity was measured with the CgA(1 [right arrow]) assay. Furthermore, the dipeptide Leu'Pr02 was found to be an essential part of the epitope for the CgA(1 [right arrow]) assay. Therefore, the low concentrations measured with the CgA(1 [right arrow]) assay probably can be attributed to intracellular degradation of CgA, or perhaps to differentiated secretory pathways for CgA-derived peptides. When interpreting the measured pattern of immunoreactive CgA, one must recognize that the CgA(1 [right arrow]) and CgA(210 [right arrow]) assays quantify small tryptic peptides and larger peptides with equimolar potency and that the dilution curves of tissue extracts were parallel with the calibrator curves (except for the CgA([left arrow] 439) assay). However, the influence of molecular differences in the diluted antigens on the concentrations measured with the various CgA assays cannot be excluded.

[FIGURE 8 OMITTED]

The chosen extraction procedures (boiling water extraction followed by reextraction with acetic acid) have been used frequently for the extraction of CgA fragments. Accordingly, we found that boiling water (and cold Tris buffer) extraction gave the highest yield, which again is consistent with the hydrophilic nature of CgA, which enables it to remain soluble after boiling (30).

This study is the first to report concentrations measured by six different sequence-specific CgA assays in neuroendocrine tissues and plasma. In agreement with our results, pancreastatin concentrations in extracts of human antral mucosa and pancreas have been reported to range from 0.5 to 6.0 pmol/g tissue (31, 32), and CgA immunoreactivity of 5-10 pmol/g tissue has been found in human pancreas using antisera specific for two mid-sequences of hCgA(315-321) and hCgA(332-337) [the C-terminal region of WE-14 (33-35)]. Furthermore, CgA concentrations of 100-200 pmol/g tissue have been measured in human stomach and pancreas, using an antibody raised against CgA purified from pheochromocytoma (36). The CgA(1 [right arrow]) assay measured the human equivalents [CgA(1-76) and CgA(1-113)] to bovine vasostatins (37) as well as further C-terminally extended forms. Vasostatins have not been quantified in extracts of human neuroendocrine tissue. However, in cell lines derived from human lung cancers and from a medullary thyroid carcinoma, CgA concentrations have been measured with two CgA assays specific for the N- and C-terminal sequences as well as an RIA that recognizes full-length CgA (38). In agreement with our results, the lowest concentrations were also measured with the N-terminal RIA (38). Furthermore, Brandt et al. (38) demonstrated that the sequence-specific assays recognized mainly low-molecular weight forms, consistent with the previous observation that the terminal regions of CgA are important sites for processing.

Antisera against CgA have been used in immunohistochemical studies for establishing the diagnosis of neuroendocrine tumors [for reviews, see Refs. (2) and (8)]. Some studies have also used radioimmunochemical quantification of CgA in tumor tissue or in plasma as a marker for neuroendocrine tumors (9-12,17-19,35,39-44). In our study, six neuroendocrine tumors expressed 10-fold higher pancreastatin concentrations than those reported previously (31, 39). The CgA concentrations measured in carcinoid tumors with the CgA(315-321) and CgA(332-337) antisera (35) were in agreement with our results.

That tumor processing of prohormones varies considerably is known (45). Accordingly, the relative proportion of the CgA immunoreactivity measured with the six different CgA assays also varied in the six tumors examined in this study. However, one constant feature was the low concentrations measured with the CgA(1 [right arrow]) assay. In agreement with our results, various proportions of CgA(315-321) and CgA(332-337) immunoreactivity in neuroendocrine tumors as well as molecular heterogeneity of immunoreactive pancreastatin and immunoreactive CgA(315-321) have been reported (31, 35, 39). Cell-specific processing of CgA also has been demonstrated in human cell lines derived from lung cancers and medullary thyroid carcinomas using well-characterized sequence-specific RIAs, chromatography, and Western blot analysis (38, 46).

Consistent with the pattern of CgA immunoreactivity in the carcinoid tumors, the CgA concentrations in plasma from carcinoid tumor patients, as measured with the CgA(1 [right arrow]) and the CgA([left arrow] 301 amide) assays, were within the reference interval or increased in only some of the samples. In contrast, when the CgA(210 [right arrow]) and CgA(340 [right arrow]) assays were used, consistently increased concentrations were measured. Increased plasma concentrations of pancreastatin have been described previously in metastatic carcinoid tumors patients (39, 44). The CgA concentrations in plasma from the pituitary adenoma patients were above the upper reference limit when measured with the CgA(340 [right arrow]) assay. Accordingly, increased CgA concentrations have also been described in plasma from a fraction of patients with pituitary adenomas (16, 18,19).

Our results show that epitope specificity of CgA antisera may profoundly influence diagnostic sensitivity and hence, their usefulness. Furthermore, because tumor processing of proteins may vary considerably, the use of a library of sequence-specific CgA immunoassays, as presented in this study, should improve diagnosis and therapeutic control of neuroendocrine tumors.

This study was supported by grants from the Danish Medical Research Council, the Danish Biotechnology Program for Cellular Communication, and the Danish Cancer Union. We acknowledge the skillful technical assistance of Joan Christiansen and Alice von der Lieth. We thank Anders H. Johnsen for assistance with amino acid analysis, mass spectrometry, and truncation of peptides; Drs. Jens Gustafsen and Ulrich Knigge, Department of Surgical Gastroenterology, Rigshospitalet, University of Copenhagen, for providing the tissue; Dr. Simone Bjerregard Sneppen, Department of Endocrinology, Rigshospitalet, University of Copenhagen, for providing blood samples from pituitary adenoma patients; and Cathrine Orskov for the histological examination of the tissue.

References

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(33.) Conlon JM, Curry WJ, Shaw C, Johnston CF, Thim L, Buchanan KID. Isolation and primary structure of a novel chromogranin A-derived peptide, WE-14, from a mid-gut carcinoid tumor. FEBS Lett 1992; 301:319-21.

(34.) Hamberger B, Grimelius L. Isolation of peptides arising from the specific posttranslational processing of chromogranin A and chromogranin B from human pheochromocytoma tissue. Peptides 1992;13:639-44.

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(37.) Aardal S, Helle KB, Elsayed S, Reed RK, Serck-Hanssen G. Vasostatins, comprising the N-terminal domain of chromogranin A, suppress tension in isolated human blood vessel segments. J Neuroendocrinol 1993;5:405-12.

(38.) Brandt DW, Burton DW, Hogue-Angeletti R, Deftos U. Chromogranin A peptide-specific antisera and high-performance size exclusion chromatography demonstrate amino-terminal and car boxy-terminal fragments of the native molecule in human cell lines. Proc Soc Exp Biol Med 1994;205:316-20.

(39.) McGrath-Linden, Siobhan J, Johnston CF, O'Connor DT, Buchanan KID. Pancreastatin-like immunoreactivity in human carcinoid disease. Regul Pept 1991;33:55-77.

(40.) Janson ET, Holmberg L, Stridsberg M, Eriksson B, Theodorsson E, Wilander E, et al. Carcinoid tumors: analysis of prognostic factors and survival in 301 patients from a referral center. Ann Oncol 1997;8:685-90.

(41.) Grondal S, Eriksson B, Hamberger B, Theodorsson E. Plasma chromogranin A+B, neuropeptide Y and catecholamines in pheochromocytoma patients. J Intern Med 1991;229:453-6.

(42.) Schurmann G, Raeth U, Wiedenmann B, Buhr H, Herfarth C. Serum chromogranin A in the diagnosis and follow-up of neuroendocrine tumors of the gastroenteropancreatic tract. World J Surg 1992;16:697-702.

(43.) Nobels FIRE, Kwekkeboom DJ, Coopmans W, Schoenmakers CHH, Lindemans J, De Herder WW, et al. Chromogranin A as serum marker for neuroendocrine neoplasia: comparison with neuron specific enolase and the a-subunit of glycoprotein hormones. J Clin Endocrinol Metab 1997;82:2622-8.

(44.) Syversen U, Jacobsen MB, O'Connor DT, Ronning K, Waldum HL. Immunoassays for measurement of chromogranin A and pancreastatin-like immunoreactivity in humans: correspondence in patients with neuroendocrine neoplasia. Neuropeptides 1994;26: 201-6.

(45.) Rehfeld JF, Bardram L, Blanke S, Bundgaard JR, Friis-Hansen L, Hilsted L, et al. Peptide hormone processing in tumours: biogenetic and diagnostic implications. Tumor Biol 1993;14:174-83.

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TINE BORGLUM JENSEN, LINDA HILSTED, and JENS F. REHFELD *

University Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark.

Address correspondence to this author at: Department of Clinical Biochemistry (KB3013), Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen O, Denmark. Fax 45-35454640; e-mail rehfeld@rh.dk.

Received November 24, 1998; accepted February 3, 1999.
Table 1. Human CgA peptides.

CgA(1-9)-[Tyr (a)
 Leu-Pro-Val-Asn-Ser-Pro-Nie (c) -Asn-Lys-Tyr
CgA(1-9)-Tyr-CyS (a)
 Leu-Pro-Val-Asn-Ser-Pro-Nie (c) -Asn-Lys-Tyr-Cys
CgA(116-124)-Tyr (b)
 Glu-Asp-Ser-Lys-Glu-Ala-Glu-Lys-Ser-Tyr
CgA(116-124)-Tyr-Cys (b)
 Glu-Asp-Ser-Lys-Glu-Ala-Glu-Lys-Ser-Tyr-Cys
CgA(208-216) (b)
 Lys-Arg-Glu-Glu-Glu-Glu-Glu-Glu-Glu
CgA(210-222)-Tyr (b)
 Glu-Glu-Glu-Glu-Glu-Glu-Glu-Glu-Glu-Ala-Glu-Ala-Gly-Tyr
CgA(340-348)-Tyr (b)
 Leu-Glu-Gly-Gin-Glu-Glu-Glu-Glu-Asp-Tyr
CgA(340-348)-Tyr-Cys (b)
 Leu-Glu-Gly-Gin-Glu-Glu-Glu-Glu-Asp-Tyr-Cys
Tyr-CgA(432-439) (b)
 Tyr-Gin-Leu-Gin-Ala-Leu-Arg-Arg-Gly
Cys-Tyr-CgA(432-439) (b)
 Cys-Tyr-Gin-Leu-Gin-Ala-Leu-Arg-Arg-Gly

(a,b) The human CgA peptides were custom synthesized by (a)
Cambridge Research Biochemicals (CRB) Ltd, Zeneca (Cheshire, UK),
and (b) Saxon Biochemicals GmbH (Hannover, Germany).
(c) Residue 7. Met was substituted with Nle.

Table 2. Characteristics of antisera using monoiodinated
tyrosine-extended peptides as tracers.

Calibrator Antiserum/bleeding Titer, (a) x [10.sup.3]

CgA(1-9)-Tyr 94185/4 400
 94188/4 470
CgA(116-124)-Tyr 95045/4 10
 95050/4 5
CgA(210-222)-Tyr 1610/29 30
 1612/29 100
CgA(340-348)-Tyr 95058/4 50
 95060/4 60
Tyr-CgA(432-439) 95038/4 35
 95043/9 35

 [K.sup.0.sub.eff], (b)
 x [10.sup.12]
Calibrator L/mol [alpha] (c)

CgA(1-9)-Tyr 0.8 1.04
 0.9 0.95
CgA(116-124)-Tyr 0.1 0.98
 <0.1 NC (d)
CgA(210-222)-Tyr <0.1 NC
 0.8 1.00
CgA(340-348)-Tyr 0.9 1.04
 1.0 1.00
Tyr-CgA(432-439) 0.6 0.98
 0.5 0.97

(a) Antiserum dilution at which 35% of tracer is bound
at equilibrium.

(b) Effective equilibrium constant Keff according
to Ekins and Newman (24).

(c) Index of heterogeneity according to Sips (26).

(d) NC, [alpha] was not calculated for antisera
with [K.sup.0.sub.eff] <0.1 x [10.sup.12] L/mol.

Table 3. Detection limits was determined in two ways,
using monoiodinated tyrosine-extended peptides as tracers
and tyrosine-extended peptides as calibrators.

 Detection limit

 2[epsilon]/
 [K.sup.0.sub.- Mean - 2 SD, (c)
Antiserum no. Calibrator eff], (b) fmol/L fmol/L

94188 CgA(1-9)-Tyr 70 27
95045 CgA(116-124)-Tyr 100 177
1612 CgA(210-222)-Tyr 80 105
95060 CgA(340-348)-Tyr 40 110
95038 Tyr-CgA(432-439) 150 140

(a) All results are given in fmol/L incubation mixture.

(b) As suggested by Ekins and Newman (24).

(c) Ten replicates of "0"-calibrator were included in the assay.

Table 4. Specificity of antisera, expressed as the ratio of
ID,, of the peptide calibrator vs the [ID.sub.50] for the
truncated or extended CgA fragment in tracer displacement.

 Truncated/extended
Antiserum no. Calibrator peptide Specificity

94188 CgA(1-9)-Tyr CgA(2-9)-Tyr 0.0004
94188 CgA(1-9)-Tyr CgA(3-9)-Tyr 0.0001
1612 CgA(210-222)-Tyr CgA(208-216) 0.007
95060 CgA(340-348)-Tyr CgA(341-348)-Tyr 0.0004
95038 Tyr-CgA(432-439) Tyr-CgA(432-437) 0.001

Table 5. CgA(340 [right arrow]) immunoreactivity in extracts
before and after tryptic cleavage.

 CgA(340
 CgA(340 [right arrow])
 [right arrow]) IR (a,b)
 Tissue IR (a,b) after trypsin

Antrum mucosa
 I 31 70
 II 36 59
 III 18 57
 IV 22 64

Mean [+ or -] SE 27 [+ or -] 4 63 [+ or -] 3

Pancreas 87 171

Midgut carcinoid
 I 16.5 37.6
 II 15.4 34.9
 III 1.2 5.0
 IV 5.6 17.9

Foregut carcinoid
 I 0.60 4.6

Pheochromocytoma
 I 5.2 26.2

 CgA(340 [right arrow])IR/
 CgA(340 [right arrow])IR
 Tissue after trypsin

Antrum mucosa
 I 0.44
 II 0.61
 III 0.32
 IV 0.34

Mean [+ or -] SE 0.43 [+ or -] 0.07

Pancreas 0.51

Midgut carcinoid
 I 0.43
 II 0.44
 III 0.24
 IV 0.31

Foregut carcinoid
 I 0.13

Pheochromocytoma
 I 0.20

(a) IR, immunoreactivity.

(b) Immunoreactivity in pmol/g tissue for antrum mucosa
and pancreas, in nmol/g tissue for midgut carcinoid,
foregut carcinoid, and pheochromocytoma.

Table 6. CgA(1 [right arrow]), CgA(116 [right arrow]),
CgA(210 [right arrow]), CgA([left arrow] 301 amide),
CgA(340 [right arrow]), and CgA([left arrow] 439)
immunoreactivity in extracts before and after
tryptic cleavage.

 CgA(1 [right CgA(116 [right CgA(210 [right
 arrow]) IR (a) arrow]) IR arrow]) IR
 before and before and before and
 Tissue after trypsin after trypsin after trypsin

Antrum mucosa, 6 6 56 55 21 20
 pmol/g tissue
Pheochromocytoma, 0.89 0.90 26.0 25.9 2.06 2.07
 nmol/g tissue

 CgA([left CgA (340 [right CgA([left
 arrow]301) IR arrow]) IR arrow]439) IR
 before and before and before and
 Tissue after trypsin after trypsin after trypsin

Antrum mucosa, 5 <0.1 31 70 12 <0.1
 pmol/g tissue
Pheochromocytoma, 0.22 <0.001 5.20 26.20 1.07 <0.001
 nmol/g tissue

(a) IR, immunoreactivity.

Table 7. Concentrations measured with four CgA assays
in consecutive plasma samples from three midgut
carcinoid tumor patients.

 CgA(1 [right
 arrow])
 IR, (a) CgA(210 [right arrow])
 pmol/L IR, pmol/L

Patient 1
 Initial sample 487 990 (b)
 2 months later 503 1103 (b)
 4 months later 475 917 (b)
 8 months later 540 1087 (b)

Patient 2
 Initial sample 425 635 (b)
 4 months later 470 850 (b)
 7 months later 470 970 (b)
 10 months later 520 895 (b)

Patient 3
 Initial sample 625 1345 (b)
 3 months later 720 (b) 1470 (b)
 7 months later 565 1305 (b)
 10 months later 710 (b) 1495 (b)

 CgA([left
 arrow]301) CgA(340 [right arrow])
 IR, pmol/L IR, pmol/L

Patient 1
 Initial sample <10 240 (b)
 2 months later <10 270 (b)
 4 months later 80 (b) 305 (b)
 8 months later 130 (b) 345 (b)

Patient 2
 Initial sample <10 180 (b)
 4 months later <10 160 (b)
 7 months later <10 140 (b)
 10 months later <10 200 (b)

Patient 3
 Initial sample 370 (b) 310 (b)
 3 months later 420 (b) 330 (b)
 7 months later 285 (b) 220 (b)
 10 months later 523 (b) 290 (b)

(a) IR, immunoreactivity.

(b) Increased compared with the upper reference limit.

Table 8. Concentrations measured with four CPFA assays
in plasma samples from 10 midprut carcinoid tumor patients.

Carcinoid CgA(1 [right arrow]) CgA(210 [right arrow])
tumor IR, (a) pmol/L IR, pmol/L

Midgut 670 570
Midgut 1600 (b) 2300 (b)
Midgut 951 (b) 480 (b)
Midgut 465 270 (b)
Midgut 830 630
Midgut 1100 (b) 1800 (b)
Midgut 1965 (b) 1500
Midgut 470 840 (b)
Midgut 560 1155 (b)
Midgut 600 1650 (b)

Carcinoid CgA([left arrow] 301) CgA(340 [right arrow])
tumor IR, pmol/L IR, pmol/L

Midgut 225 (b) 280
Midgut 900 (b) 1185 (b)
Midgut 52 (b) 210 (b)
Midgut 60 (b) 160 (b)
Midgut 382 (b) 500
Midgut 850 980 (b)
Midgut 325 (b) 670
Midgut 305 (b) 725 (b)
Midgut 6 350 (b)
Midgut 510 (b) 240 (b)

(a) IR, immunoreactivity.

(b) Increased compared with the upper reference limit.

Table 9. Concentrations measured with three CgA assays
in plasma samples from nine patients with
pituitary adenomas.

 Pituitary CgA(1 [right arrow]) CgA([left arrow])
 adenoma IR, (a) pmol/L 301) IR, pmol/L

Nonsecretory 650 9
Nonsecretory 585 15
Nonsecretory 780 (b) 9
Nonsecretory 595 12
Nonsecretory 425 15
GH-producing 520 6
GH-producing 460 12
GH-producing 745 (b) 3
GH-producing 490 33 (b)

 Pituitary CgA(340 [right arrow])
 adenoma IR, pmol/L

Nonsecretory 170 (b)
Nonsecretory 226 (b)
Nonsecretory 52
Nonsecretory 307 (b)
Nonsecretory 203 (b)
GH-producing 72 (b)
GH-producing 227 (b)
GH-producing 120 (b)
GH-producing 126 (b)

(a) IR, immunoreactivity; GH, growth hormone.

(b) Increased compared with the upper reference limit.
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Title Annotation:Endocrinology and Metabolism
Author:Jensen, Tine Borglum; Hilsted, Linda; Rehfeld, Jens F.
Publication:Clinical Chemistry
Date:Apr 1, 1999
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