JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 7 195-201 (1992)
Cross-reactivity of a Commercial Chemiluminescence lmmunoassay for Human Chorionic Gonadotropin with t h e Free P-Subunit P. Luppa,''
G.Spott1,2 6.
D. Neumeier' and K. M a n n 2
Institute of Clinical Chemistry,' and Department of Internal Medicine 11,* University Hospital Grosshadern, D-8000 Munich 70, Germany
An enhanced chemiluminescence immunoassay for the determination of serum human chorionic gonadotropin (HCG) in specimens f r o m oncology patients has been assessed with respect t o i t s cross-reactivity with the free HCG @-subunit(HCG-$). The assay, standardized against t h e First International Reference Preparation 751537. had a crossreactivity with t h e free $-subunit o f 6 2 5 % (molar basis). Therefore this assay achieves high sensitivity for the detection of either intact HCG or free HCG-fl in serum of patients with seminomatous o r nonseminomatous testicular cancers. Results of both assays, t h e in-house immunoradiometric assay ( HCG-@) and the Amerlite H C G - 6 0 assay, showed a close correlation (R = 0.854-0.960) when serum samples f r o m tumour patients were analyzed. Moreover, t h e content o f free $-subunit determined in a specific HCG-$ assay, could be quantitatively measured in t h e enhanced chemiluminescence immunoassay. Thus, this assay is suitable for oncology use, b u t also highlights t h e limitations of measuring HCG in serum samples.
+
Keywords: Amerlite; HCG ; testicular cancer
I NTR 0 DU CTlO N
Human chorionic gonadotropin (HCG), a glycoprotein composed of two dissimilar subunits (a and P), is mainly produced by normal and neoplastic trophoblasts. In choriocarcinoma and testicular germ cell tumours, HCG and alphafetoprotein are well-established tumour markers. Saller et al. (1990) recently reported on the reliability of measuring intact HCG and the free /?-subunit of HCG (HCG-P) in sera from patients suffering from tro-
phoblastic tumours. The secretion pattern of intact HCG or the free subunits (a and /I) is variable in testicular cancer. Many investigations (Beastall et al., 1991; Mann, 1990) demonstrate that both the intact HCG and the P-subunit are sensitive markers for clinical use. It is important to notice that in about 20% of pure seminomas a selective elevation of the HCG-8 subunit can be observed (Mann and Siddle, 1988). For clinical practice a single assay, sensitive for both intact and fl-chain HCG, is most reliable in diagnosis and follow-up of
* Author for correspondence. 0884-3996/92/030195-07$08.50 0 1992 by John Wiley & Sons, Ltd.
Received 22 November 1991 Revised 3 February 1992
196
testicular tumours. It is thus important to use HCG assays which have a high P-subunit cross-reactivity in order to achieve high sensitivities in patients with testicular cancer, secreting either HCG or 8-HCG. We have compared the specificity of the Amerlite HCG-60 enhanced immunochemiluminometric assay (ICLMA) to an HCG (+ HCG-8) immunoradiometric assay (IRMA) (Saller et al., 1990) and studied its application in oncology.
P. LUPPA ET AL
this assay have been described previously (Saller et al., 1990). Serum specimens were collected from 87 patients with testicular cancer, who were admitted to our hospital for cytostatic treatment. Tumours were classified histologically as teratomas, seminomas or combination tumours of both, according to the WHO staging procedures. RESULTS
MATERIAL A N D METHODS
The ICLMA Amerlite HCG-60 (Amersham International plc, UK) consists of two monoclonal antibodies against HCG (+ HCG-8) and is standardized against the First International Reference Preparation (1.IRP 75/537). The performance data have been described previously (Bonini et al., 1990; Luppa et al., 1988). HCG standard 1.IRP 75/537 and HCG-p standard 1.IRP 75/551 as preparations CR 121 and CR 1238 were supplied by the Hormone Distribution Program of the NIDDK, USA. Cross-reactivity of the Amerlite HCG-60 assay was evaluated according to Saller et al. (1990), using the standard preparations CR 121 and CR 123p with known contents of HCG or HCG-8. Serial dilutions of standards of intact or &subunit HCG in HCG-free serum matrix were prepared. The mean values of measured top light intensities (percentage TLI, derived from the light emissions by comparing the measured photoelectron signals from samples to those of standards in percentages) from three different duplicate determinations were plotted against the standard HCG or HCG-/3 concentrations in ng/ml. The percentage of crossreaction was determined from the parallel section of both curves by the ratio (percentage TLI!,,,,, .,-,/percentage TLIHCG+)x 100. The conversion of results to a molar basis assumes a molecular weight of 37,500 Da for the intact HCG and 22,000 Da for the &subunit (Beastall et al., 1991). For method comparison we used the well-estabIished HCG (+HCG-P) IRMA (IRMA 1) (Mann and Siddle, 1988; Mann, 1990). The detection limit of this assay was 1.6 mIU/ml (Mann and Siddle, 1988), whereas the Amerlite assay has a detection limit of 1.25 mIU/ml (Luppa et al., 1988). Free HCG p-chain was measured using a second IRMA method (IRMA 2), and performance data of
The evaluation and the clinical trial of the HCG-60 ICLMA was performed to provide data specifically for the use of this assay in oncology. Fig. 1 shows that the measurement of increasing concentrations of the HCG-8 standard CR 1238 in the Amerlite assay (results expressed as a percentage of the light intensity of the top standard) yields high signals. The mean cross-reactivity with the 8subunit, calculated within the parallel section of the two curves in the range of 0.5 and 2.2 ng/ml (0.025 and 0.1 nmol/l) HCG-P, is 625% (Table 1 and Fig. 1 ). Supplementary additions of intact HCG standard IRP 75/537 and HCG-8 subunit standard IRP 75/551 to the zero standard were performed and the percentage TLI were plotted against the HCG concentrations. As shown in Fig. 2(a), the values for the assay standards supplied with the ICLMA kit are similar to standards prepared using the 1.IRP standard. However, analysis of standards prepared using the HCG-P standard indicates the high cross-reactivity of the ICLMA with the 8subunit. This is illustrated in Fig. 2(b), the insert shows that even low concentrations of HCG-p give measurable HCG ( HCG-8) values. In the Amerlite HCG-60 assay 1 ng intact HCG CR 121 corresponds to 9.3 mIU 1.IRP 75/573 (9.3 IU/pg; mean value from three different duplicate determinations). The specific HCG-8 IRMA 2 (Saller et ul., 1990) also used in this study, determines 1 ng HCG-8 CR 1238 to be equivalent to 1.6 mIU 1.IRP 75,551 (1.6 IU/pg). To check the specificity of the Amerlite assay, recovery experiments were performed by adding increasing amounts of HCG-,8 standard CR 1238 to different concentrations of intact HCG standard CR 121 and vice versa. The recovery rates were calculated from the resulting HCG ( + HCG-[j) values and varied between 84% and 105% (Table 2). This proves the equivalent ability of measuring both intact HCG and HCG-8 molecules.
+
CROSS-REACTIVITY OF A COMMERCIAL CHEMILUMINESCENCE IMMUNOASSAY TO HCG
197
% Top light intensity
50
r" 8 intact HCG
50
tree 13-HCG
-A-
40
40
30
30
20
20
10
10
0
0 0.1
10
1
HCG CR 121 and HCG CR 1238 [ng/mll Figure 1. Cross-reactivitiesof CR 121 and CR 123/3 in the ICLMA
Table 1. Cross-reactivities of the ICLMA HCG determination with added free HCG-fl CR 1238 HCG-b CR 1238
Signal
HCG CR 121
(ns/ml)
(% TLI)
(ng/ml)
Cross-reactivity (mol/mol; X)
0.51 0.75 1.21 1.30 1.98 2.20
4
6.00 7.87 12.37 13.33 19.87 25.1 2
705 625 610 590 560 660
6 7.5 12 15.5 18
The concentrations of HCG (+HCG-/?) in sera of 87 testicular cancer patients, measured by use of the ICLMA, revealed no significant differences from those obtained by the reference IRMA 1 (Saller et al., 1990), which has a cross-reactivity of 720%. The linear regression coefficients for HCG (+HCG-p) values in the ranges of 0-500 and 0-6000 mIU/ml are shown in Table 3, and the data,
presented in Fig. 3, reflect the close correlation between the two assay methods. In acidition, measuring the concentrations of the free /%subunit in the sera using the specific IRMA 2 method, similar correlations of the P-HCG to the respective HCG ( + HCG-8) values were observed for both assays. The determinations performed with either the ICLMA or the IRMA 1 (Saller et al.,
P. LUPPA ET AL
200
TOPtqht intensity
I%]
160
100
50
0 0.01
TTrrrriT-i-rrrmr
01
1
10 I00 HCG and HCG-E added lna/mll
-& & U y aimdards
*IRP
1000
761561 dllullon
Figure 2. Top light intensities (TLI), measured with the Arnerlite HCG-60 (ICLMA) assay for different HCG standards. (a) Addition of increasing amounts of intact HCG standard IRP 7 5 / 5 3 7 to zero assay standard. (b) Addition of increasing amounts of HCG-8 standard IRP 7 5 / 5 5 1 to the zero assay standard (inserr: lower HCG range up to 5 ng/ml)
Table 2. Recovery experiments t o demonstrate assay specificity f o r t h e ICLMA. (Increasing amounts o f HCG-fi standard CR 1238 were added t o different stock concentrations of intact HCG standard CR 121 and vice versa. The respective standard solutions were admixed 1 :I and recoveries were calculated by comparing t h e measured HCG ( + HCG-fi) values with those o f equivalent 1 :1 dilutions of t h e respective HCG standards with t h e zero assay standard) Variation of intact HCG
Variation of HCG-P --______
HCG intact
(ns/W 1.6 1.6 1.6 1.6 3.1 3.1 3.1 3.1 25.0 25.0 25.0
--__
HCG-8 (ng/mJ)
Recovery
0.4 1.6 6.2 25.0 0.8 1.6 12.5 25.0 6.2 12.5 25.0
95 92 90 90 96 96 105 94 99 86 98
(%I
HCG-8 (ng/ml)
HCG intact (ns/ml)
Recovery (%)
6.2 6.2 6.2 12.5 12.5 12.5 25.0 25.0 25.0
3.1 12.5 25.0 3.1 12.5 25.0 1.6 3.1 25.0
84 97 99 105 97 86 90 94 98
CROSS-REACTIVITY OF A COMMERCIAL CHEMILUMINESCENCE IMMUNOASSAY TO HCG ~
~
~~
Table 3. Method comparison data for HCG ( + HCG-p) using the ICLMA and IRMA 1. (The linear regression was calculated according t o Passing and Bablok(1983);R = linear regression coefficient) Number
Range
(N)
(mlU/ml)
87 61
CMOOO 0-500
(Y = a
R
0.9605 0.8543
+ bx)
Y = 29.875 y = 18.985
+ 0.807x + 0.958~
1990) are shown in Fig. 4 (coefficients of correlation, 0.760 vs 0.722). It must be stated that four sera with measurable free HCG-fl concentrations were below the detection limit of the ICLMA of 1.25 mIU/ml HCG ( HCG-B), whereas with the HCG +HCG-P) IRMA 1 all HCG-p-positive sera were above the detection limit of the assay.
+
199
DISCUSSION
The cross-reactivity of the enhanced chemiluminescence assay Amerlite HCG-60 with the HCG-fi subunit is higher (625%) than any of the 11 commercial assays tested by Saller et al. (1990). The recommendation was made, that the cross-reactivity of any HCG ( + HCG-8) assay should be similar to that of the in-house IRMA 1 (720%) to be reliable for diagnosis and follow-up of testicular tumours. In our opinion, a minimum of crossreactivity has still to be defined by additional clinical studies. The high cross-reactivity of the ICLMA reflects the excellent specificity of this excess reagent immunoassay. The antibodies used in the assay recognize identical epitopes on intact HCG and its 8subunit. Table 2 shows that quantitative recoveries of measured HCG ( + HCG-8) concentrations for intact and free &chain HCG standards can be achieved after admixture of the respective analyte and cross-reactant.
hCG (+ HCG-B) ICLMA [rnlU/rnl] 5000
500
50
5
0.5 0.5
5
50
500
5000
HCG (+ HCG-8) IRMA 1 IrnlU/rnll Figure 3. Comparison of concentrations of HCG ( + HCG-8) in sera of testicular cancer patients measured using the ICLMA and IRMA 1
200
P. LUPPA ET AL
The method comparison study performed with clinical specimens revealed a good correlation between the two assay methods. Remarkably, serum samples with elevated concentrations of the free psubunit had high values of measured HCG ( HCG-p) concentrations using the ICLMA, even if the fraction of intact HCG (data now shown) is small. However, the sensitivity of the ICLMA in the determination of HCG ( HCG-p) was limited in the lower range, since four of the 87 sera with HCG ( + HCG-/)) values of 3-5 mIU/ml in the reference IRMA 1 (Saller et al., 1990) were below the detection limit of the ICLMA assay (1.25 mIU/ml) (Fig. 3). These sera had detectable HCG-p concentrations as shown in Fig. 4. The spread of results in the lower range of 0-5 mIU/ml (Fig. 3) reflects both non-specific interferences in the assays and unidentified sample interferences such as jl-core fragments. However, it must be noted that a decision on the existence of a HCG-producing neoplasm is difficult to make in this low range of HCG concentrations. The close correlation of the HCG (+ HCG-j?) values to the free HCG-P concentrations, determined using the ICLMA, highlights the reliability of this method for the diagnosis and follow-up of trophoblastic tumours. Nonetheless, the divergent
+
+
results (IRMA 1 and ICLMA), shown in Fig. 4, indicate that a precise determination of cross-reactivity of HCG ( HCG-p) assays to the free p-chain is limited by putative contaminations of 8-core fragments (Wehmann and Nisula, 1989) either in standard preparations (Wehmann et al., 1988) or in individual serum specimens, e.g., some sera showed no linear relationship between the measurements of intact HCG, HCG (+ HCG-b) and HCG-fl. An additional limitation is that the HCG-60 assay may have false results due to a possible high-dose hook effect, since two-site immunoenzymometric assays are more sensitive to very high analyte concentrations than radioimmunoassays. During the evaluation we observed this effect in three different serum specimens (data not shown). In summary, these results support the use of the Amerlite HCG-60 assay for use in oncology. The variability of cross-reactivities of different HCG ( + HCG-fl) assays (Saller et al., 1990) to the free pchain, and the difficulties in defining cross-reactivities caused by contaminants in the WHO standard preparations described above, lead to the recommendation of using a single reliable HCG determination method in the follow-up of individual tumour patients.
+
,oo
1
0.010i 5
Q
;
I
6
60
HCG (+ HCG-6)- 1
600
IRMA 2 ImlU/mll -.
l
L..,...-r-m2
I-
R-13
6000
ImlU/ml
0.6
6
50
HCG (+ HCG-6)
600
5000
lmlU/mll
Figure 4. Comparison of HCG ( + H C G - p ) values in sera of affected patients-determined using (a) the ICLMA or (b) the IRMA 1 method-with the free HCG-B values, determined using the specific IRMA 2. Horizontallines: lower detection limit of the HCG-8 assay. vertical lines; lower detection limits of the respective HCG (+HCG-b) assays
CROSS-REACTIVITY OF A COMMERCIAL CHEMILUMINESCENCE IMMUNOASSAY TO HCG
Acknowledgement The skilful technical assistance of Mrs U. Langmandel and Mrs R. Kubbe-Spitzner is gratefully acknowledged.
R EFER ENCES Beastall, G. H., Cook, B., Rustin, G . J. S. and Jennings, J. (1991). A review of the role of established tumour markers. Ann. Clin. Biochem., 28, 5 - 18. Bonini, P. A,, Banfi, G. and Murone, M. (1990). Enhanced chemiluminescence in the measurement of proteins and haptens: evaluation of choriogonadotropin (hCG) and free thyroxin. J . Biolumin. Chemilumin., 5, 193 195. Luppa, P., Neumeier, D. and Knedel, M. (1988). Evaluation of a new chemiluminescence immunoassay for the determination of human chorionic gonadotropin in serum. J. Clin. Chem. Clin. Biochem., 26, 705-713. ~
201
Mann, K. (1990). Tumor markers in testicular cancer. Urologe A , 29, 77-86. Mann, K. and Siddle, K. (1988). Evidence for free beta-subunit secretion in so-called human chorionic gonadotropin-positive seminoma. Cancer, 6 2 , 2378-2382. Passing, H. and Bablok, W. (1983). A new biometrical procedure for testing the equality of measurements from two different analytical methods. J. Clin. Chem. Clin. Biochem., 21, 709- 720. Saller, B., Clara, R., Spottl, G., Siddle, K. and Mann, K. (1990). Testicular cancer secretes intact human choriogonadotropin (hCG) and its free beta-subunit: evidence that hCG (+ hCGbeta) assays are the most reliable in diagnosis and follow-up. Clin. Chem., 36, 234- 239. Wehrnann, R. E. and Nisula, B. C. (1989). Characterization of a discrete degradation product of the human chorionic gonadotropin 8-subunit in humans. J . Clin.Endocrinol. Metab., 51, 101 -105.
Wehmann, R. E., Blithe, D. L., Akar, A. H. and Nisula, B. C. (1988). j-Core fragments are contaminants of the World Health Organization reference preparations of human choriogonadotropin and its a-subunit. J . Endocrinol., 117, 147- 152.
Cross-reactivity of a Commercial Chemiluminescence lmmunoassay for Human Chorionic Gonadotropin with t h e Free P-Subunit P. Luppa,''
G.Spott1,2 6.
D. Neumeier' and K. M a n n 2
Institute of Clinical Chemistry,' and Department of Internal Medicine 11,* University Hospital Grosshadern, D-8000 Munich 70, Germany
An enhanced chemiluminescence immunoassay for the determination of serum human chorionic gonadotropin (HCG) in specimens f r o m oncology patients has been assessed with respect t o i t s cross-reactivity with the free HCG @-subunit(HCG-$). The assay, standardized against t h e First International Reference Preparation 751537. had a crossreactivity with t h e free $-subunit o f 6 2 5 % (molar basis). Therefore this assay achieves high sensitivity for the detection of either intact HCG or free HCG-fl in serum of patients with seminomatous o r nonseminomatous testicular cancers. Results of both assays, t h e in-house immunoradiometric assay ( HCG-@) and the Amerlite H C G - 6 0 assay, showed a close correlation (R = 0.854-0.960) when serum samples f r o m tumour patients were analyzed. Moreover, t h e content o f free $-subunit determined in a specific HCG-$ assay, could be quantitatively measured in t h e enhanced chemiluminescence immunoassay. Thus, this assay is suitable for oncology use, b u t also highlights t h e limitations of measuring HCG in serum samples.
+
Keywords: Amerlite; HCG ; testicular cancer
I NTR 0 DU CTlO N
Human chorionic gonadotropin (HCG), a glycoprotein composed of two dissimilar subunits (a and P), is mainly produced by normal and neoplastic trophoblasts. In choriocarcinoma and testicular germ cell tumours, HCG and alphafetoprotein are well-established tumour markers. Saller et al. (1990) recently reported on the reliability of measuring intact HCG and the free /?-subunit of HCG (HCG-P) in sera from patients suffering from tro-
phoblastic tumours. The secretion pattern of intact HCG or the free subunits (a and /I) is variable in testicular cancer. Many investigations (Beastall et al., 1991; Mann, 1990) demonstrate that both the intact HCG and the P-subunit are sensitive markers for clinical use. It is important to notice that in about 20% of pure seminomas a selective elevation of the HCG-8 subunit can be observed (Mann and Siddle, 1988). For clinical practice a single assay, sensitive for both intact and fl-chain HCG, is most reliable in diagnosis and follow-up of
* Author for correspondence. 0884-3996/92/030195-07$08.50 0 1992 by John Wiley & Sons, Ltd.
Received 22 November 1991 Revised 3 February 1992
196
testicular tumours. It is thus important to use HCG assays which have a high P-subunit cross-reactivity in order to achieve high sensitivities in patients with testicular cancer, secreting either HCG or 8-HCG. We have compared the specificity of the Amerlite HCG-60 enhanced immunochemiluminometric assay (ICLMA) to an HCG (+ HCG-8) immunoradiometric assay (IRMA) (Saller et al., 1990) and studied its application in oncology.
P. LUPPA ET AL
this assay have been described previously (Saller et al., 1990). Serum specimens were collected from 87 patients with testicular cancer, who were admitted to our hospital for cytostatic treatment. Tumours were classified histologically as teratomas, seminomas or combination tumours of both, according to the WHO staging procedures. RESULTS
MATERIAL A N D METHODS
The ICLMA Amerlite HCG-60 (Amersham International plc, UK) consists of two monoclonal antibodies against HCG (+ HCG-8) and is standardized against the First International Reference Preparation (1.IRP 75/537). The performance data have been described previously (Bonini et al., 1990; Luppa et al., 1988). HCG standard 1.IRP 75/537 and HCG-p standard 1.IRP 75/551 as preparations CR 121 and CR 1238 were supplied by the Hormone Distribution Program of the NIDDK, USA. Cross-reactivity of the Amerlite HCG-60 assay was evaluated according to Saller et al. (1990), using the standard preparations CR 121 and CR 123p with known contents of HCG or HCG-8. Serial dilutions of standards of intact or &subunit HCG in HCG-free serum matrix were prepared. The mean values of measured top light intensities (percentage TLI, derived from the light emissions by comparing the measured photoelectron signals from samples to those of standards in percentages) from three different duplicate determinations were plotted against the standard HCG or HCG-/3 concentrations in ng/ml. The percentage of crossreaction was determined from the parallel section of both curves by the ratio (percentage TLI!,,,,, .,-,/percentage TLIHCG+)x 100. The conversion of results to a molar basis assumes a molecular weight of 37,500 Da for the intact HCG and 22,000 Da for the &subunit (Beastall et al., 1991). For method comparison we used the well-estabIished HCG (+HCG-P) IRMA (IRMA 1) (Mann and Siddle, 1988; Mann, 1990). The detection limit of this assay was 1.6 mIU/ml (Mann and Siddle, 1988), whereas the Amerlite assay has a detection limit of 1.25 mIU/ml (Luppa et al., 1988). Free HCG p-chain was measured using a second IRMA method (IRMA 2), and performance data of
The evaluation and the clinical trial of the HCG-60 ICLMA was performed to provide data specifically for the use of this assay in oncology. Fig. 1 shows that the measurement of increasing concentrations of the HCG-8 standard CR 1238 in the Amerlite assay (results expressed as a percentage of the light intensity of the top standard) yields high signals. The mean cross-reactivity with the 8subunit, calculated within the parallel section of the two curves in the range of 0.5 and 2.2 ng/ml (0.025 and 0.1 nmol/l) HCG-P, is 625% (Table 1 and Fig. 1 ). Supplementary additions of intact HCG standard IRP 75/537 and HCG-8 subunit standard IRP 75/551 to the zero standard were performed and the percentage TLI were plotted against the HCG concentrations. As shown in Fig. 2(a), the values for the assay standards supplied with the ICLMA kit are similar to standards prepared using the 1.IRP standard. However, analysis of standards prepared using the HCG-P standard indicates the high cross-reactivity of the ICLMA with the 8subunit. This is illustrated in Fig. 2(b), the insert shows that even low concentrations of HCG-p give measurable HCG ( HCG-8) values. In the Amerlite HCG-60 assay 1 ng intact HCG CR 121 corresponds to 9.3 mIU 1.IRP 75/573 (9.3 IU/pg; mean value from three different duplicate determinations). The specific HCG-8 IRMA 2 (Saller et ul., 1990) also used in this study, determines 1 ng HCG-8 CR 1238 to be equivalent to 1.6 mIU 1.IRP 75,551 (1.6 IU/pg). To check the specificity of the Amerlite assay, recovery experiments were performed by adding increasing amounts of HCG-,8 standard CR 1238 to different concentrations of intact HCG standard CR 121 and vice versa. The recovery rates were calculated from the resulting HCG ( + HCG-[j) values and varied between 84% and 105% (Table 2). This proves the equivalent ability of measuring both intact HCG and HCG-8 molecules.
+
CROSS-REACTIVITY OF A COMMERCIAL CHEMILUMINESCENCE IMMUNOASSAY TO HCG
197
% Top light intensity
50
r" 8 intact HCG
50
tree 13-HCG
-A-
40
40
30
30
20
20
10
10
0
0 0.1
10
1
HCG CR 121 and HCG CR 1238 [ng/mll Figure 1. Cross-reactivitiesof CR 121 and CR 123/3 in the ICLMA
Table 1. Cross-reactivities of the ICLMA HCG determination with added free HCG-fl CR 1238 HCG-b CR 1238
Signal
HCG CR 121
(ns/ml)
(% TLI)
(ng/ml)
Cross-reactivity (mol/mol; X)
0.51 0.75 1.21 1.30 1.98 2.20
4
6.00 7.87 12.37 13.33 19.87 25.1 2
705 625 610 590 560 660
6 7.5 12 15.5 18
The concentrations of HCG (+HCG-/?) in sera of 87 testicular cancer patients, measured by use of the ICLMA, revealed no significant differences from those obtained by the reference IRMA 1 (Saller et al., 1990), which has a cross-reactivity of 720%. The linear regression coefficients for HCG (+HCG-p) values in the ranges of 0-500 and 0-6000 mIU/ml are shown in Table 3, and the data,
presented in Fig. 3, reflect the close correlation between the two assay methods. In acidition, measuring the concentrations of the free /%subunit in the sera using the specific IRMA 2 method, similar correlations of the P-HCG to the respective HCG ( + HCG-8) values were observed for both assays. The determinations performed with either the ICLMA or the IRMA 1 (Saller et al.,
P. LUPPA ET AL
200
TOPtqht intensity
I%]
160
100
50
0 0.01
TTrrrriT-i-rrrmr
01
1
10 I00 HCG and HCG-E added lna/mll
-& & U y aimdards
*IRP
1000
761561 dllullon
Figure 2. Top light intensities (TLI), measured with the Arnerlite HCG-60 (ICLMA) assay for different HCG standards. (a) Addition of increasing amounts of intact HCG standard IRP 7 5 / 5 3 7 to zero assay standard. (b) Addition of increasing amounts of HCG-8 standard IRP 7 5 / 5 5 1 to the zero assay standard (inserr: lower HCG range up to 5 ng/ml)
Table 2. Recovery experiments t o demonstrate assay specificity f o r t h e ICLMA. (Increasing amounts o f HCG-fi standard CR 1238 were added t o different stock concentrations of intact HCG standard CR 121 and vice versa. The respective standard solutions were admixed 1 :I and recoveries were calculated by comparing t h e measured HCG ( + HCG-fi) values with those o f equivalent 1 :1 dilutions of t h e respective HCG standards with t h e zero assay standard) Variation of intact HCG
Variation of HCG-P --______
HCG intact
(ns/W 1.6 1.6 1.6 1.6 3.1 3.1 3.1 3.1 25.0 25.0 25.0
--__
HCG-8 (ng/mJ)
Recovery
0.4 1.6 6.2 25.0 0.8 1.6 12.5 25.0 6.2 12.5 25.0
95 92 90 90 96 96 105 94 99 86 98
(%I
HCG-8 (ng/ml)
HCG intact (ns/ml)
Recovery (%)
6.2 6.2 6.2 12.5 12.5 12.5 25.0 25.0 25.0
3.1 12.5 25.0 3.1 12.5 25.0 1.6 3.1 25.0
84 97 99 105 97 86 90 94 98
CROSS-REACTIVITY OF A COMMERCIAL CHEMILUMINESCENCE IMMUNOASSAY TO HCG ~
~
~~
Table 3. Method comparison data for HCG ( + HCG-p) using the ICLMA and IRMA 1. (The linear regression was calculated according t o Passing and Bablok(1983);R = linear regression coefficient) Number
Range
(N)
(mlU/ml)
87 61
CMOOO 0-500
(Y = a
R
0.9605 0.8543
+ bx)
Y = 29.875 y = 18.985
+ 0.807x + 0.958~
1990) are shown in Fig. 4 (coefficients of correlation, 0.760 vs 0.722). It must be stated that four sera with measurable free HCG-fl concentrations were below the detection limit of the ICLMA of 1.25 mIU/ml HCG ( HCG-B), whereas with the HCG +HCG-P) IRMA 1 all HCG-p-positive sera were above the detection limit of the assay.
+
199
DISCUSSION
The cross-reactivity of the enhanced chemiluminescence assay Amerlite HCG-60 with the HCG-fi subunit is higher (625%) than any of the 11 commercial assays tested by Saller et al. (1990). The recommendation was made, that the cross-reactivity of any HCG ( + HCG-8) assay should be similar to that of the in-house IRMA 1 (720%) to be reliable for diagnosis and follow-up of testicular tumours. In our opinion, a minimum of crossreactivity has still to be defined by additional clinical studies. The high cross-reactivity of the ICLMA reflects the excellent specificity of this excess reagent immunoassay. The antibodies used in the assay recognize identical epitopes on intact HCG and its 8subunit. Table 2 shows that quantitative recoveries of measured HCG ( + HCG-8) concentrations for intact and free &chain HCG standards can be achieved after admixture of the respective analyte and cross-reactant.
hCG (+ HCG-B) ICLMA [rnlU/rnl] 5000
500
50
5
0.5 0.5
5
50
500
5000
HCG (+ HCG-8) IRMA 1 IrnlU/rnll Figure 3. Comparison of concentrations of HCG ( + HCG-8) in sera of testicular cancer patients measured using the ICLMA and IRMA 1
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P. LUPPA ET AL
The method comparison study performed with clinical specimens revealed a good correlation between the two assay methods. Remarkably, serum samples with elevated concentrations of the free psubunit had high values of measured HCG ( HCG-p) concentrations using the ICLMA, even if the fraction of intact HCG (data now shown) is small. However, the sensitivity of the ICLMA in the determination of HCG ( HCG-p) was limited in the lower range, since four of the 87 sera with HCG ( + HCG-/)) values of 3-5 mIU/ml in the reference IRMA 1 (Saller et al., 1990) were below the detection limit of the ICLMA assay (1.25 mIU/ml) (Fig. 3). These sera had detectable HCG-p concentrations as shown in Fig. 4. The spread of results in the lower range of 0-5 mIU/ml (Fig. 3) reflects both non-specific interferences in the assays and unidentified sample interferences such as jl-core fragments. However, it must be noted that a decision on the existence of a HCG-producing neoplasm is difficult to make in this low range of HCG concentrations. The close correlation of the HCG (+ HCG-j?) values to the free HCG-P concentrations, determined using the ICLMA, highlights the reliability of this method for the diagnosis and follow-up of trophoblastic tumours. Nonetheless, the divergent
+
+
results (IRMA 1 and ICLMA), shown in Fig. 4, indicate that a precise determination of cross-reactivity of HCG ( HCG-p) assays to the free p-chain is limited by putative contaminations of 8-core fragments (Wehmann and Nisula, 1989) either in standard preparations (Wehmann et al., 1988) or in individual serum specimens, e.g., some sera showed no linear relationship between the measurements of intact HCG, HCG (+ HCG-b) and HCG-fl. An additional limitation is that the HCG-60 assay may have false results due to a possible high-dose hook effect, since two-site immunoenzymometric assays are more sensitive to very high analyte concentrations than radioimmunoassays. During the evaluation we observed this effect in three different serum specimens (data not shown). In summary, these results support the use of the Amerlite HCG-60 assay for use in oncology. The variability of cross-reactivities of different HCG ( + HCG-fl) assays (Saller et al., 1990) to the free pchain, and the difficulties in defining cross-reactivities caused by contaminants in the WHO standard preparations described above, lead to the recommendation of using a single reliable HCG determination method in the follow-up of individual tumour patients.
+
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1
0.010i 5
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;
I
6
60
HCG (+ HCG-6)- 1
600
IRMA 2 ImlU/mll -.
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6000
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0.6
6
50
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600
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Figure 4. Comparison of HCG ( + H C G - p ) values in sera of affected patients-determined using (a) the ICLMA or (b) the IRMA 1 method-with the free HCG-B values, determined using the specific IRMA 2. Horizontallines: lower detection limit of the HCG-8 assay. vertical lines; lower detection limits of the respective HCG (+HCG-b) assays
CROSS-REACTIVITY OF A COMMERCIAL CHEMILUMINESCENCE IMMUNOASSAY TO HCG
Acknowledgement The skilful technical assistance of Mrs U. Langmandel and Mrs R. Kubbe-Spitzner is gratefully acknowledged.
R EFER ENCES Beastall, G. H., Cook, B., Rustin, G . J. S. and Jennings, J. (1991). A review of the role of established tumour markers. Ann. Clin. Biochem., 28, 5 - 18. Bonini, P. A,, Banfi, G. and Murone, M. (1990). Enhanced chemiluminescence in the measurement of proteins and haptens: evaluation of choriogonadotropin (hCG) and free thyroxin. J . Biolumin. Chemilumin., 5, 193 195. Luppa, P., Neumeier, D. and Knedel, M. (1988). Evaluation of a new chemiluminescence immunoassay for the determination of human chorionic gonadotropin in serum. J. Clin. Chem. Clin. Biochem., 26, 705-713. ~
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Mann, K. (1990). Tumor markers in testicular cancer. Urologe A , 29, 77-86. Mann, K. and Siddle, K. (1988). Evidence for free beta-subunit secretion in so-called human chorionic gonadotropin-positive seminoma. Cancer, 6 2 , 2378-2382. Passing, H. and Bablok, W. (1983). A new biometrical procedure for testing the equality of measurements from two different analytical methods. J. Clin. Chem. Clin. Biochem., 21, 709- 720. Saller, B., Clara, R., Spottl, G., Siddle, K. and Mann, K. (1990). Testicular cancer secretes intact human choriogonadotropin (hCG) and its free beta-subunit: evidence that hCG (+ hCGbeta) assays are the most reliable in diagnosis and follow-up. Clin. Chem., 36, 234- 239. Wehrnann, R. E. and Nisula, B. C. (1989). Characterization of a discrete degradation product of the human chorionic gonadotropin 8-subunit in humans. J . Clin.Endocrinol. Metab., 51, 101 -105.
Wehmann, R. E., Blithe, D. L., Akar, A. H. and Nisula, B. C. (1988). j-Core fragments are contaminants of the World Health Organization reference preparations of human choriogonadotropin and its a-subunit. J . Endocrinol., 117, 147- 152.
