Jan 9, 1989 - LleveDIllen,Jan DeBlock, Lutyart Van Lear,andWerner De Potter'. This is an .... After a 10-mm incubation in the dark at room temperature,.
CLIN. CHEM. 35/9, 1934-1938 (1989)
Enzyme-Linked Immunosorbent Assay for Chromogranin A LleveDIllen,Jan De Block, Lutyart Van Lear,and Werner De Potter’ This is an enzyme-linked immunosorbent assay (ELISA) for determining chromogranin A (CGA) with use of a monoclonal antibody. CGA was isolated from bovine chromaffin granules. The analytical ELISA procedure for bovine CGA was developed and optimized. Typical standard curves ranged from 500 pg to 500 ng of CGA. We then studied human plasma CGA-immunoreactivity as measured by this assay. The curve for dilutions of human plasma paralleled the standard curve for bovine CGA. The intra-assay coefficient of variation for determination of human plasma CGA was 4.56%, indicating that reliable determinations can be performed for human plasma. However, further study revealed the presence of two CGA-immunoreactive substances in human plasma, one of which corresponds to the native CGA. The nature of the second immunoreactive substance still remains unknown. Nevertheless the measured CGA concentrations (ranging from 0.19 to 0.35 mg/L) in plasma are comparable with previously reported values. Addftlonal Keyphrasea: monoclonal antibodies fes’val chromaffingranules
reference in-
Chromogranin A (CGA), Mr 70000, is a major soluble protein of the chromaffin granule, accounting for about 40% of its soluble protein content (1_4).2 CGA and its degradation fragments have apI of 4.2, and its physicochemical and immunological properties have been well characterized (1-4). CGA is released from the adrenal medulla together with catecholamines upon stimulation of the splanchnic nerve (1, 4) but the physiological role of CGA is still unknown. Some investigators suggest that it is a catecholamine-binding protein (5, 6); others propose a calciumbinding role (3). Deftos et al. (7) recently reported that CGA is not confined just to chromaffin cells of the adrenal medulla and sympathetic neurons but is also present in various neuroendocrine tissues. Thus the protein might be expected to also be secreted from these tissues; indeed, in the presence of some tumors of these tissues, the concentrations of CGA in blood have been shown to be increased (8). These findings throw some new light on the problem of the function of CGA, the elucidation of which will require improved methods for quantifying it. Thus far, semiquantitative immunological techniques have been used to determine CGA. O’Connor and Bernstein (9) developed sensitive radioimmunoassays for chromo-
Laboratory of Neuropharmarology, Department of Medicine, University of Antwerp, Umversiteitsplein 1, B-2610 Wilrijk, Bel-
gium. 1To whom correspondence should be addressed. 2Nonstandard abbreviations: CGA, chromogranin A; Bistris, [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane; FPLC, “Fast Protein Liquid Chromatography”; ELISA, enzyme-linked immunosorbent assay; S1)S-PAGE, sodium dodecylsu]fate-polyacrylamide gel electrophoresis; DH, dopamine-p-hydroxylase; and PBS, phosphate-buffered isotonic saline. Received January 9, 1989; acceptedJune 1, 1989. 1934
CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989
granins, but experienced difficulties with interspecies crosa-reactivities. Here we describe our development of an ELISA (enzyme-linked immunosorbent assay) for bovine
CGA with use of a monoclonal antibody. We investigated the cross-reactivity of this antibody to human, dog, and rat CGA, and detected immunoreactivity in human plasma as well.
Materials and Methods Apparatus The “Fast Protein Liquid Chromatography” (FPLC) system was from Pharmacia, Uppsala, Sweden. The Multiphor H Novablot system was from LKB, Bromma, Sweden. Reagents Sephacryl S-200 HR, concanavalin-A-Sepharose, Mono 5/5, and Protein G-Sepharose were from Pharmacia. ‘251-Labeled anti-mouse IgG was from Amersham International, Amersham, Buckinghamshire, U.K., and the imniunoblot assay kit was from Bio-Rad Labs, Richmond, CA.
Q HR
Procedures
Isolation
of CGA. We purified CGA from the chromaflIn of bovine adrenal medullas, as follows. The granules were isolated according to Smith and Winkler (2). We lysed them by resuspending in a fivefold dilution with distilled water and by repeatedly freezing and thawing this suspension. The granule lysate was in the supernate after centrifugation at 100000 x g for 1 h. Dopamine-f3-hydroxylase and some other glycoproteins were removed from the lysate of these granules by affinity chromatography on a column of concanavalin-A-Sepharose (7 cm x 1.4 cm2), with the elution buffer consisting of 0.2 mol of NaCl per liter of KH2PO4 (50 mmol/L, pH 6.5). From the unbound fraction of the concanavalin-A-Sepharose column, CGA was further purified by gel ifitration on a column of Sephacryl S-200 HR (90 cm x 2 cm2) at 4#{176}C, at a flow rate of 60 mL/h, the elution buffer being Bistris (20 mmol/L, pH 6). Chromogranin-containing fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sos-PAGE), pooled, and subsequently subjected to anionexchange chromatography on an FPLC Mono Q, HR 5/5 column at pH 6 (20 mmol/L Bistris) with a linear gradient from 0 to 0.7 mol/L NaC1 in 40 mm at a flow rate of 0.8 mLfmin. Again, the purity of these fractions was tested by granules
SDS-PAGE.
Production of monoclonal antibodies. Three Balb/c mice were immunized by intraperitoneal injection of granule lysate (100 pg of protein) mixed with an equal volume of complete Freund’s adjuvant. After three booster injections with the protein solution, the mice were killed and the spleen cells were fused with mouse myeloma cells. After several days we screened confluent hybridoma cells for antibody activity by using lmIlabeled goat anti-mouse IgO. We subcloned the positive clones and selected the F1 55/7 clone for use in the ELISA technique. The IgGs were isolated from the culture media or from ascites by passage through a column of Protein G. The antibodies were also
in immunoblot experiments. After two-dimensional gel electrophoresis (10), we blotted the proteins of the chromaflin granules onto nitrocellulose by using the Multiphor II Novablot system. For immunodetection of the proteins we used Bio-Rad’s immunoblot goat anti-mousehorseradish peroxidase conjugate assay kit (11, 12). Enzyme-linked immunosorbent assay. Before performing the ELISA, we biotinylated CGA by incubating 200 pL of a 1 g/L solution of biotin-aminocaproyl-N-hydroxysuccinimide in dimethylformamide with 1 mL of a 1 g/L solution of chromogranin A in phosphate-buffered saline (PBS; phosphate 100 mmol/L, NaC1 150 mmol/L, pH 7.4) for 4 h at room temperature. Then we dialyzed the mixture (10 mL) by elution over a Sephadex G-25 column (30cm x 2 2) vs PBS containing 0.2 g of thimerosal per liter, and stored the dialysate at 4#{176}C. For the assay itself we coated 96-well plates (Costar, Badhoevedorp, The Netherlands) with 1 pg of antibody in 100 L coating buffer (carbonate buffer, pH 9.6,0.05 moIIL) and incubated for 3-4 h at 37 #{176}C. After saturating the plates with coating buffer containing 10 g of bovine serum albumin per liter for 3-4 h at 37#{176}C, we washed them five times with PBS containing 1 mL of Tween 20 per liter. CGA standard or unknown and biotinylated CGA were added in 100 pL of PBS containing bovine serum albumin, 1 gIL, and Tween 20, 1 mLIL. The plates were incubated overnight (about 20 h) at 4#{176}C. After five washings with the PBS-Tween 20 solution, we added to each well 100 pL of 1000-fold-diluted streptavidin-peroxidase conjugate and incubated for 1 h at 37 #{176}C. We then washed the wells five times with the PBS-Tween 20 solution and added to each well 100 pL of o-phenylenediamine in pH 5 citrate/phosphate buffer (per liter, 0.4 g of o-phenylenediamine, 0.1 mol of citrate, 0.2 mol of phosphate, plus 1 g of urea peroxide). After a 10-mm incubation in the dark at room temperature, we added 50 L of 2 molJL H2S04 to stop the reaction and measured the absorbance of each well at 492 nm with a vertical-lightpath microplate reader in the dualwavelength mode (ifiter 1, at 492 nm; ifiter 2 at 690 nm; Titertek Multiskan MCC/ 340 MK H; Flow Laboratories, Helsinki, Finland). Calculations. The ELISA results were calculated by a logit-log transformation by means of an RIA data-reduction program from IBM. screened
Results Isolation of CGA After eliminating some glycoproteins of the bovine chromaffin granule lysate (such as DpH) by chromatography on concanavalin-A-Sepharose, we passed the unbound fraction through a column of Sephacryl S-200 HR. Figure 1A shows the elution pattern. CGA is eluted at a higher apparent Mr than would be expected from its known relative molecular mass of 70000, probably as a result of aggregation. The chromogranin-containing fractions (fractions 18-21, Figure 1B) were combined and further purified by anion-exchange chromatography. Figure 2A shows the elution pattern following a linear gradient of NaC1 from 0 to 0.7 mol per liter of elution buffer in 40 mm. We performed the ion-exchange chromatography at pH 6. Gel electrophoresis demonstrated the presence of a purified chromogranin peak in fractions 8-9 (Figure 2B). We dialyzed these fractions overnight vs distilled water to remove NaCl, then measured the protein concentration and stored the fractions at -20 #{176}C.
B
*220
Mr x103 9467 43-
3020-i
14-1 0
10
20
30
40
80 FCTIONNUM#{216}ER
Fig. 1. A. Elution pattern of bovine chromaffin granule lysate on a Sephacryl S-200 HR column, monitored at 280 nm Column dimensions: 90 cm x 2 cm2; elutionbuffer:20 mmoUL Bistris pH 6; flow rate, 0.8 mLimin; sample size, 10 mL Fractions collected at 6-mm intervals.TheCGA-containing fractionsare indicated B. SOS-PAGEof the pooled CGA-containlng fractions
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910
Fig. 2. A. Anion-exchange chromoatography of the CGA-containing fractions (Fig. 1, fractions 18-20) in an FPLC system We loaded 10 mL on the Mono 0 (HR5/5) column and eluted at a flow rateof 0.8 mLJmin (elution buffer; 20 mmol/L Bistris pH 6) and monitored the absorbance of 280 nm. When the absorption returned to baseline,westarted a linear gradient of NaCI, from 0 to 0.7 mol/L in 40 mm. Fractions were collected at I .5-mm Intervals. SDS-gel electrophoresis revealedthepresence of chrornogranin A in fractions 8 and 9. The CGA-containing fractionsare indicated B. SOS-PAGE of purified bovine CGA after ion-exchangechromatography (fractions 7-10, Fig. 2A)
CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989
1935
Identification of the Monoclonal Antibody
ELISA for CGA
We demonstrated the anti-chromogranmn A activity in the lysate of bovine chromaffin granules upon immunoblotting with the selected and Protein G-purifled F1 55/7 antibody (Figure 3). Only CGA and some degradation fragments with approximately the same p1, but no other proteins of the chromaffin granule, were visible on the nitrocellulose membrane after staining with peroxidase (EC 1.11.1.7).
We developed a competitive First of all, we determined the lated CGA, using dilutions of Figure 4A shows the dilution antigen. A 4000-fold dilution experiments. Standard curves
4;5
#{182}
5;5
Pt
94
for quantif’ing CGA. best dilution of the biotinya 100 mg/L stock solution. curve for the biotinylated
EUSA
was used throughout these ranging from 500 pg to 500 ng of CGA gave good results (Figure 4B). Correlation coefficients of 0.98 or more between the CGA concentrations and the absorption values were obtained after logitlog transformation. Prolonging the incubation time for the antigen and biotinylated antigen step did not improve the results.
:
We tested also the coating of the wells. In standard experiments, 1 pg of antibody was coated. We investigated the effect of 2 pg and 0.5 pg of coated antibody, but the most satisfactory results were obtained with the standard coating conditions (1 2g). We studied the cross-reactivity with the major possible contaminant of chromaflin granule lysate D(3H. Although the imniunoblot screening with the monoclonal antibody
30-
20-
14-.
Fig. 3. Two-dimensional gel electrophoresisof bovine chromaffin
granule lysate (10) and immunoblotting with anti-CGA Afterthe proteIns (100 tog)were blotted on nltrocellulose, thechromogranin A-anti-chromogranlnA (antibodies purifiedfromthe F1 5517 clone) complexes weremadevisible by using the Bio-RadImmunoblotassay kit
revealed no D/3H reactivity, the absence of cross-reactivity with DpH was confirmed with the ELISA procedure. We analyzed purified DH fractions in the ELISA in the same concentration range as for standard CGA. Figure 48 shows that only very high amounts of D(3H (500 ng) can compete, weakly, with biotinylated CGA for binding with the antibody. In fact, we could demonstrate no significant crossreactivity of D/3H. The detection of CGA immunoreactivity in human plasma with the newly developed ELISA motivated the study of the identification of this immunoreactivity in
A492
A 15-
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-
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LA9’l
#{149} plasma
CGA
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Fig. 4. A. Effect of dilution of a 100 mg/L stock solution of the biotinylated antigen, as measured with the ELISA Ag is the quantity (mg) of the biotinylated antigenpresent in 50 zL B. CGA standard curve (A, ranging from 10 pg to 500 ng) in ELISAperformed with a 4000-fold dilution of the biotinylated chromogranin Abscissa:absorbance percentageof the maximal absorptionat 492 nm. The correlationbetweenthe CGA concentrationsand theabsorbancewascalculated to be 0.99 afterlogit-logtransformation. Results obtainedwith the EUSAfor dopammne-p-hydroxylase(#{149}, DPI-I,100 pg to 500 ng) and humanplasma(U, dIlutions of 1/1, 1/2, 1/4, 1/8, 1/10, 1/20, 1/40, and 1/100)are also shown. [CGAIand(DPH] are the quantitiesof CGA and DpI-I, in picograms
ass
1936
CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989
human plasma. Initially, we clearly demonstrated a parallelism of human CGA with standard CGA (Figure 48), which served as a first measure of the ELISA accuracy.
Thereafter, we tried to determine the normal range of CGA concentrations in human plasma. Seventeen control plasma samples, obtained from a blood-transfusion center, showed CGA-concentrations ranging from 0.187 to 0.352 mg/L. For calculation of the intra-assay variation, we measured CGA concentrations in quintuplicate for four plasma samples. The mean CV was 4.56%. Because we were aware that parallelism as an indication of the EUSA8 accuracy has several shortcomings, we further identified the CGA immunoreactivity in human plasma. Purified bovine CGA and 10 mL of human plasma were gel-filtered on the Sephacryl S-200 HR column (see Methods). We measured the CGA content of the fractions with our ELISA, which revealed the presence of two immunoreactive peaks in human plasma (Figure 5). The first peak coincides with bovine CGA. However, a second and more important CGA-immunoreactive substance with a lower apparent Mr was also present in human plasma. At pH 6 this second CGA-immunoreactive fraction was not able to bind to an anion-exchange matrix, in contrast to standard bovine CGA and to the first CGA-immunoreactive fraction in human plasma. SDS-PAGE of this fraction in combination with immunoblot did not reveal the presence of CGA immunoreactivity, probably owing to the small concentrations of this protein together with high concentrations of other contaminants. Furthermore, in the peak fraction, only 2 ng/50 pL could be detected (Figure 5), which is below the detection limit of the immunoblot technique, about 10 ng. Similarly, immunoblotting of the first CGA-immunoreactive peak of the gel ifitration also failed to reveal immunoreactivity, also owing to the too-low concentrations.
0
0 U
10
20
30 FRACTIONNUMBER
Fig.5. ELISA of pooled fractions of purified CGA from bovine granule lysate (- - -) or human plasma (_) after gel filtration on Sephacryl S-200 HR Column, elution, and fractionation characteristics are the same asdescribedin Fig.I
Discussion We purified standard CGA from bovine chromaifin granule lysate, after concanavalin-A affinity chromatography, gel filtration, and anion-exchange chromatography (Figures 1,2, and 3). The fractions were monitored for purity by SDS-PAGE. Gel filtration was done at 4#{176}C, because we sometimes experienced breakdown of CGA when it was doneat roomtemperature. The most likely explanation for this phenomenonis that chromaflin granules also contain enzymes that can degrade chromogranins, such as acetylcholinesterase and trypsin-like enzymes (13). Perhaps small amounts of these enzymes are still present at this stage of the purification. Therefore, we are now testing proteolytic enzyme-inhibitors to block the breakdown of this protein. An alternative explanation might be that the initial chromaffin granule fraction contained lysosomal contaminants. In plasma, for example, it is known that the biological half-life of CGA is only 18 min (9). Purified standard CGA was also used for biotinylation. Again, this biotinylated CGA was not stable for several weeks at 4#{176}C. However, we wondered whether insufficient dialysis, resulting in the presence of unreacted biotin, caused this breakdown. We therefore performed dialysis on a Sephadex G-25 column to ensure complete elimination of unreacted biotin, which resulted in an improvement of the stability for several weeks. Our assay for CGA gave reliable results. The dilution curve of the biotinylated antigen showed an optimal absorption between a dilution of 1/1000 (5 pg) and 1/10 000 (0.5 pg) (Figure 4A). We initially worked with a dilution of 1/4000; however, this dilution was changed to 1/2000 when we noticed a decrease in the maximal absorption during the experiments, for reasons mentioned before. Also, standard curves prepared with standard chromogranin A ranging from 500 pg to 500 ng gave reliable results, proving the usefulness of the EUSA as an analytical procedure for standard bovine CGA. With the use of a monoclonal antibody in the ELISA, we expected cross-reactivity with other granular contaminants to be minor. However, when a common or very similar epitope is recognized, cross-reactivity can be appreciable. Therefore the cross-reactivity of the major possible chromaflin granule contaminant, D3H, was investigated. In the past, attempts to raise antisera to chromogranin A were not always successful. Small contaminations with the very immunogenic DpH sufficed to give predominantly anti-DI3H antiserum. In our approach, the monoclonal antibody for CGA reacted only with CGA and some of its degradation fragments (see immunoblot, Figure 3). In the EUSA only high concentrations of DflH (500 ng) begin to show cross-reactivity with the CGA-antibody (Figure 48). We initially developed this ELISA assay for research purposes. However, the observation that CGA could be detected in human plasma with this ELISA procedure was surprising and interesting. As species differences in the amino acid sequence of CGA become known, lack in crossreactivity of an antibody raised against bovine CGA with human CGA is possible. Indeed, O’Connor and Bernstein (9) found that, with their bovine CGA radioimmunoassay, no human CGA could be detected. These authors purified human CGA in an effort to develop an RIA for this antigen in human tissues and (or) plasma. In our ELISA assay, the monoclonal antibody directed against a single epitope could recognize the human CGA. Whenever a monoclonal antibody recognizes a common epitope on human CGA, higher CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989 1937
is to be expected in comparison with a polyclonal antiserum. Cross-reactivity of 100% can be reached when no small structural differences in the epitope region can interfere. Only comparison of standard curves prepared with use of isolated, purified human CGA with those for bovine CGA can answer this question unambiguously. The same problem arises for dog plasma CGA, because we could detect CGA immunoreactivity in dog plasma. However, in rat vas deferens and in rat brain homogenates (tissues tested in the course of current research projects), no cross-reactivity
CGA immunoreactivity was detectable. A first measure of the accuracy of CGA identification in human plasma was the observed parallelism with standard bovine CGA. We further identified CGA immunoreactivity in plasma, performing gel ifitration and ion-exchange chromatography of a plasma sample. Surprisingly, and in contrast with the previously mentioned arguments in favor of minor cross-reactivity, two CGA-immunoreactive substances were observed in human plasma. The first was completely co-eluted with bovine CGA; the second and larger peak was not. The immunoblot technique failed to demonstrate the presence of this CGA-immunoreactive substance. Identification of human granular CGA is necessary, to find out whether this second immunoreactive substance can be ascribed to species differences. Screening of bovine plasma samples for CGA immunoreactivity can reveal whether two CGA-immunoreactive peaks specifically are associated with plasma samples. The second immunoreactive substance may be a degradation product of the native CGA, which is not present in other tissues. Nevertheless, CGA concentrations in human plasma as measured by this ELISA procedure are in the same range as those found by Sobol et al. (8). In their investigation, control patients were in undisturbed recumbency for at least 20 mm. When they were in the standing position, plasma CGA increased. We had no control on this aspect of the blood sampling of our patients, probably explaining the
higher variation
of our results.
1938 CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989
This work was supported by the Queen Elisabeth Foundation of Belgium. We also thank P. Stroobants for her technicalassistance. References 1. Banks P, Hell K. The release of proteins from the stimulated adrenal medulla [Abstract]. Biochem J 1965;97:40C. 2. Smith AD, Winkler H. Purification and properties of an acidic
protein from chromaffin granules of bovine adrenal medulla. Biochem J 1967;103:482-92. 3. O’Connor DT, Frigon RP. Chromogranin A, the major catecholamine storage vesicle soluble protein. J Biol Chem 1984;259:323747. 4. Eiden EL, lacangelo A, Hsu CM, Hotchkiss AJ, Bader MF, Aunis D. Chromogranin A synthesis and secretion in chromaffin cells. J Neurochem 1987;49:65-74. 5. Da Prada M, Berneis KH, Pletscher A. Storage of catecholamines in adrenal medullary granules: formation aggregates with nucleotides. Life Sci 1971;10:639-46. 6. Helle KB, Reed RK, Pihl KE, Serck-Hanssen G. Osmotic properties of the chromogranins and relation to osmoticpressure in catecholaxnine storage granules. Acts Physiol Scand 1985;123:21-33. 7. Deftos U, Linnoila RI, Carney DN, et al. Demonstration of chromogranin A in human neuroendocrine cell lines by immunohistology and immunoassay. Cancer 1988;62:92-7.
8. Sobol RE, O’Connor DT, Addison J, Suchocki K, Royston, Deftos U. Elevated serum chromograrnn A concentrations in small-cell lung carcinoma. Intern Med 1986;105:698-700. 9. O’Connor D’F,Bernstein KN. Radioimmunoassay of chroniogranin A in plasma as a measure of exocytotic sympatho-adrenal activity in normal subjects and patients with pheochromocytoma. N Engi J Med 1984;311:764-70. 10. O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007-21. 11. Towbin H, Staehlin T, Gordon J. Electrophoretic transfer of proteinsfrom polyacrylanjidegels to nitrocellulose sheets: procedure and some applications. Proc Natl Aced Sci USA 1979;76:4350-.4. 12. Burnette WN. “Western blotting”: electrophoretic transfer of proteins from sodium dodecylsulphate-polyacrylainide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981;112:195-203. 13. Small DH, Ismael Z, Chubb 1W. Acetylcholinesterasehydrolyses chromogranin A to yield low molecular weight peptides. Neuroscience1986;19:289-95.
