SiegfrIed Schwarz, Peter Berger, and Georg WIck. Knowing the epitope ...... Cancer Research Fund, by the Austrian Fonda zur Forderung der wissenschaftlichen.
CLIN. CHEM. 31/8, 1322-1328 (1985)
Epitope-Selective,Monoclonal-Antibody-Based Immunoradiometric Assaysof PredictableSpecificityfor DifferentialMeasurementof Choriogonadotropin and Its Subunits SiegfrIed Schwarz, Peter Berger, and Georg WIck Knowing the epitope specificities of our monoclonal antibodies (MCA) to human choriogonadotropin (hCG), we could design three classes of two-site immunoradiometric assays
much as 100% with lutropin, but more recent protocols in which the antigen used is either the /3-subunit of hCG (3) or its carboxy-terminal peptide extension (by which it differs from lutropin) yielded more specific immune responses (4). (IRMA): a combinationof two MCA recognizingtwo separate Still, the affinity-i.e., the assay sensitivity-of such antisea-epitopes (a-MCA) provides a system (i.e., an a-IRMA) that measures holo-hCG plus free a-subunitsplus follitropin,ra occasionally seems to be inversely related to the molecular size of the immunogen (4). Despite such technical lutropin, and thyrotropin, whereas a /3-IRMA, consistingof two improvements, “discordant” (5), “aberrant” (6), or even /3-MCA, quantifies holo-hCG plus free /3-subunits. The “inconsistent qualitative” (7-9) results of hCG measureamount of either of the two subunitscan be calculated by ments have been observed. Consequently, parallel applicasubtractingthe amountof holo-hCGdeterminedin parallelin tion of different kits based on different methodological a holo-hCG-IRMA.In the latter,one of the a- or f3-MCA may be principles has been advocated (9, 10), because such differeithercross-combinedor,preferably, pairedwith an MCA ently based immunoassays are subject to interference in specific for a conformationalepitope.These analyticalspecidifferent ways. These include the structural similarity of ficities, predicted from our previously established epitope hCG to lutropin (11), the heterogeneity in the carbohydrate map of hCG, could be experimentally verified. With these content of hCG (12, 13), the presence of free subunits (14) IRMAS we could demonstrate that in certain choriocarcinoma and other fragments (15, 16), the possible coexistence of celllinesthe earliest and quantitatively predominanttumor hCG with antibodies against hCG (17) or against rabbit marker is the free a-subunit. Similar results showing an immunoglobulin (18), variations in the content of serine unbalancedsecretionof hCG and itssubunitswere obtained proteases in plasma (19), and other variations in the sample for patients with related tumors. These findings challenge the matrix (reviewed in 2, 10,20). present diagnostic practice of relying solely on “p-hCG” Sophisticated maneuvers such as multiple absorptions or radioimmunoassaysand at the same time offer a novel chromatographic sample-purification steps are often reanalyticalstrategy. quired, to render polyclonal antisera completely specific. Moreover, antisera to isolated subunits of hCG are of very Addftlonal Keyphrases: analytical error unbalanced secretion limited availability. Thus, monoclonal antibodies (MCA) ofgonadotropinand itssubunits choriocarcinoma exploiting appear to be advantageous in several aspects: not only can epifopespecificitiesof monoclonalantibodies . ‘iwich”-type they be obtained in unlimited quantities, facilitating assay assay standardization, but also, as shown in this paper, they can be selected more rationally and combined with one another Human choriogonadotropin (hCG)1 is produced eutopicalso as to establish immunoassays that may have greater ly by cells of trophoblasticorigin (placenta, hydatidiform specificity than their constituent reagents alone. As reportmole, choriocarcinoma) and ectopicaily by various noned (21), even their affinities can be enhanced by such a sotrophoblastic tumors (ovary, uterus, pancreas), and perhaps called cooperative immunoassay. even by normal somatic cells (for a review see ref. 2). Exact We have produced several different purified IgG-MCA to diagnostic elucidation of any of these possibilities crucially hCG of defined primary specificities-i.e., a-, /3-, or c- (= depends on absolutely reliable measurement of hCG. Classiconformational) (22)-and affinities (23). We have assessed cal bio- and hemagglutination tests are either insufficiently their exact epitope specificities. In consequence, we were able to construct an epitope map of hCG (1). On this basis, sensitive or specific, or both, and therefore they have been we report here the application of five of 21 MCA against replaced by radio- and enzymoimlnunoassays. The validity of these latter depends on both the quality of the binding hCG as selected reagents used in three different two-site reagent and that of the material used as the standard. immunoradiometric assays (nua) that allow concurrent Antisera formerly were raised mainly by immunization measurement of both intact hCG (“holo-hCG”) and its free a with intact hCG; hence the antibodies could cross react by as and /3subunits in various standard preparations and clinical samples. .
.
Institute for General and Experimental Pathology, University of Innsbruck Medical School, Fntz-Pregl-Strape 3, A-6020 Innsbruck, Austria. Parts of this work were presented (Abstract no. 1753) at the 7th mt. Congr. of EndocrinoL, Quebec, Canada, July 1984. 1 Nonstandard abbreviations: hCG, human choriogonadotropin; MCA, monoclonal antibodies; Ig, immunoglobulin; IRMA, immufloradiometric assay; c-, conformational; IS, international standard; IRP, international reference preparation; and INN-hCG-nn, MCA prepared against hCG in Innsbruck; MRC, Medical Research Council; NIH, U.S. National Institutes of Health. Received February 11, 1985; accepted May 29, 1985. 1322
CLINICAL CHEMISTRY, Vol. 31, No. 8, 1985
MaterIals and Methods Two-Site IRMA As previously described in detail (1), the technique adopted for the following studies was a two-site nm in which hCG was complexed between two MCA that recognize two different and sterically distant epitopes. One of these MCA, which we called the “capture-MCA,” is coated onto 12 x 75 mm polystyrene tubes. The other, the “detection-MCA,” is iodinated with Namil by the Chloramine T method (24). The
yield of specific radioactivity is on the order of 1500 to 1800 Ci per millimole of IgG. Ten micrograms of IgG-MCA in 0.3 mL of bicarbonate buffer (10 mmoL’L, pH 9.6) is allowed to adsorb to the inner wall of the tubes, resulting in approximately 10 nmol of IgG remaining stably bound. We assessed this by a saturation experiment (1), using iodinated hCG, and by Scatchard plot evaluation with use of a previously published computer program (25). Tubes prepared in this way were incubated for 60 mm at 37#{176}C with various concentrations of standard hCG (0.2-2000 mt. units/L) in 0.2 mL of assay buffer (phosphate buffer, 50 inmol/L, pH 7.4, supplemented with 3.3 g of bovine serum albumin per liter), followed by two rinses with phosphate-buffered isotonic saline. We then added 200 000 cpm (corresponding to about 0.5 nmol of IgG) of the detection-MCA. We terminated the reaction after a further 90 mm at 37#{176}C by rinsing the tube three times with phosphate-buffered isotonic saline. The radioactivity bound to the tube was then counted.
Design of Three Types of hCG-IRMAS The basis for the design of MA5 for the selective and differential quantification of holo-hCG and its free subunits was our epitope map of hCG (1). It displays nine different epitopes. Figure 1 shows a simplified version. From this map, the predicted specificities of three types of ms, referred to as a-m, (3-nt, and holo-hCG-m, could be delineated, as schematically depicted in Figure 2. Accordingly, the combination of an a2-capture-MCA with a radiolabeled a3-detection-MCA recognizes hCG via its a-subunit. This a-nu should measure free a-subunits but also holehCG and, if present, all the other glycoprotein hormones (foflitropin, lutropin, and thyrotropin) as well, because they all possess the same a-chain as hCG. By analogy, a /3-ma, consisting of two /3-MCA, such as f31-capthre MCA and a j33-detection-MCA, complexes the (3facet of hCG, thus quantifying free (3-subunits and holo-hCG without interference by other glycoprotein hormones. Not even lutropin would interfere, because the /31-epitope has been shown to be expressed neither by human lutropin nor r_tope
map of hcG in MERCATOR’s projection
Fig. 1. Relief diagram of the immunological surface of molecule
the hCG
Thiswasrecentlyestablushed(1)byusinga2l xli matrixoftwo-sitebinding assays in which 21 different MCA to hCG were involved. This computergenerated grid-plotshows the spherical surface of hCG in Meioatorprojection as a plane from which its nine epitopes, tentatively assumed to resemble rotational elhpsoids, protrude. Three epitopes are located on the a-hemisphere (in the ‘north”),four epitopes on the /3-hemisphere (south”), and the two conformational epitopes that are solely found on intact hCG were consequently affocated to the equatorial area where the two subunits associate with each other
Fig. 2. Schematic representation of holo-hCG as analyte indifferent two-site IRMAS An open ua,e associated with a V-shaped antibody molecule denotes a capture-MCA,an asterisk a detection-MCA.For further explanation,see text
by lutropin from several other mammalian species. This contrasts with all other /3-epitopes (i.e., /32, /33, and /34), which apparently represent evolutionarily highly conserved structures. Holo-hCG alone should, in principle, be selectively quantified by simply cross-combining the aforementioned f3capture-MCA with the a3-detection-reagent. The other cross-combination-i.e., a2-capture-MCA with the /33-detection-MCA-would also react with lutropin. Quite obviously, the structural integrity of the hCG molecule is the prerequisite for such holo-hCG-mtas being operative. Thus, with only four different MCA, three different molecular species of hCG can be measured selectively by three different mrs. Use of an additional reagent, such as a cMCA, as the capture-MCA enables preselection of holo-hCG without interference from free subunits, because these are removed by washing before the detection step in a kind of affinity purification. Holo-hCG, complexed to such a solidphase c-MCA, can then be developed by adding the a3- or the /33-detection-MCA, the same as those being used in the respective a- or (3-mr&As, or by adding both of them for possible signal enhancement. In particular, for use in the ms described below, we selected MCA having the following code numbers: 1. INN-hCG-43 (an a2-MCA) as capture-element, combined with lssIlabeled INN-hCG-15 (an a3-MCA) in the a2. INN-hCG-32 (a th-MCA) with ‘I-labeled Li (a f33-MCA) in the /3-nuaA, and 3. INN-hCG-10 (a c1-MCA) with 1I-labeled Li (a /33-MCA) in the holo-hCG-nu. We also set up an inverse holo-hCG-mz to study the effects of excess amounts of free (3-subunits, consisting of 4. INN-hCG-22 (a /32-MCA) combined with ‘I-labeled INN-hCG-13 (another c1-MCA). Conforming to recently summarized criteria for radioimmunoassay standardization (26), we did each mtt in the same fashion: each assay included tubes with 0.2 to 2000 milli-int. units of the same holo-hCG standard (1st IRP of hCG for immunoassay, MRC 75/537), from which we con2 This INN-bLH-L1 MCA detected the same /33-epitope, present both on hCG and bovine lutropin, as INN-hCG-20, mNN-hCG-24, and INN-hCG-42. We used it solely because it was an exceptionally suitable detection-MCA.
CLINICALCHEMISTRY, Vol.31,No.8,1985 1323
structed calibration curves (counts/mm bound vs log dose). We used identical amounts of capture-MCA and detectionMCA (having equal specific radioactivities as indicated above) in each rnMA. We used the same pipet tip to deliver each sample and intermediate standard into the respective tubes for each IRMA. Eachof these samples was coded and, after being assayed, interpolated from the individual reference curves and expressed in terms of mt. units of hCG immunopotency equivalent to the 1st IRP. Consequently, subunit-standards, given in mass units (micrograms per liter) were also expressed as apparent mt. units of 1st IRP holo-hCG per liter.
Results Assay Stability, Precision, Sensitivity One of these mr.s, namely the inverse holo-hCG-nm (no. 4 above), was subjected to a long-term stability testing. Even though, as shown in Figure 3, the signal yield steadily declined with the time elapsed between preparation and actual use of the reagents (this decline being most prominent with the precoated tubes), the precision of the measurement of the control samples was equal in both systems (i.e., precoated and freshly coated tubes): the between-batch CVs were 12%, 8%, and 6%, respectively, for control samples containing 54, 180, and 650 mt. units of hCG per liter. In all other experiments described below we used only freshly prepared tubes and tracers that were not older than six weeks. The minimum detectable amount, defined by the concentration interpolated from the curve at the signal level three standard deviations above the zero signal, was between 0.2 and 1 mt. unitfL for all these ms, the actual value depending on the shelf life of the reagents used. For practical reasons, we designate here 1 mt. unit/L as the cutoff value, even when the calculated sensitivity was better.
Assay Accuracy and Specificity As shown in Table 1, we could account for 100% of the 1st of hCG, added as intermediate standard to buffer, in each of the three IRMA systems. Furthermore, both the aand the holo-hCG-ms accurately measure the 2nd IS of P
(. ...
d.O)
.b.,
I.-
I
*1 8
2#{149}
hCG(i8
IP)i.t
lUL
Fig.3. Precoated and freshly coated tubes compared with respect to stability On day 0, fresh wodong solutions of INN-hCG-22 and of freshly labeled llabeled INN-hCG-13 were prepared and stored at 4 ‘C. This same day, a set of tubes was coated with half of the INN-hCG-22solution, washed, dried and stored at 4 ‘C. At the time intervals indicated, we set up assays in parallel, using either the precoated tubes or the tubes coated on the day of assay. identical standard dilutions and control samples (freshly thawed, containing 15, 50, or 500 milli-int. units of hCG per milliliter, respectively) were measured in both sets of irus, with use of aliquots of the same tracer solution that was prepared on day 0. Note that during the first 20 days of observation freshly coated tubes yrekied equal or even slightly increasing binding signals
1324 CLINICAL CHEMISTRY, Vol. 31, No. 8, 1985
Table 1. Analytical Validation of the Three Epltope-Selective IRMAS Found Holo-hCG-
Substance assayed, and concn. 1st IRP-hCG (MRC 75/537): 200 mt.units/L
2nd IS-hCG: 200 mt. units/L
a-IRMA
1908 210 250 150 650
Pregnyl(Organon):250 mt.units/L a-Subunit(NIH):12.5Ag/L a-Subunit(NIH):50.0 g/L 150 milli-int. units1stIRP-hCG + 50 g a-subunitlL 800 250 milli-int. units1stIRP-hCG + 400 g a-subunitlL >2000 250 milli-int. units 1st IRP-hCG + 400 g (3-subunit/L 240
IRMA
200 210
p-IRMA
195
250
800 1500
