60 tubes was -3.5 h with the use of a semi-automated luminometer. ... undertaken in clinical laboratories as part of ... of Clinical Biochemistry,. Addenbrooke's ...
CLIN. CHEM. 34/10, 2087-2090 (1988)
A Laboratory and Clinical Evaluationof an ImmunochemiluminometricAssay of Thyrotropinin Serum Debbie
J. Berty,PenelopeM.S. Clark,andChrIstopherP. Price
We evaluated an immunochemiluminometncassay for human thyrotropin. A chemiluminescent acridinium ester is used as a label, with magnetic-particleseparation.The lower limit of detection of the assay (mean + 3 SD of the zero standard) was 0.07 milli-int.unitlL, with a working range of 0.5 to >60.0 milli-int.units/L.Assay accuracy was good as judged from analyticalrecovery,analysisof external qualityassessment samples, and comparison with an enzymeamplified immunoassay. There were no significantinterferences or cross-reactivities. Twenty-four samples assayed showed aggregationof the magnetic particles.On re-assay, four of these samples showed a significantincrease in the measured TSH by the luminescence assay. Assay time for 60 tubes was -3.5 h with the use of a semi-automated luminometer. The reference interval, determinedfrom data on 144 healthy euthyroid subjects, was 0.3-4.0 milli-int.
units/L. Sixteen of 19 thyrotoxic patients showed clearly suppressed concentrations of thyrotropin in serum.
Materials and Methods Reagents “Magic Lite TSH Assay” kits were supplied by CibaCorning J)iagnostics, Halstead, Essex, U.K. Each TSH kit contains sufficient reagent for 100 tubes
and includes the following: 10 mL of TSH Lite reagent (labeled antibody), 50 mL of TSH antibody solid phase, 2 mL ofNH low calibrator, and 2 mL of TSH high calibrator. The calibrators are calibrated against the second International Preparation
(1RP) 80/558.
Two reagents are supplied for use with the luminometer: 225 mL of 5 g/L hydrogen peroxide solution in 0.1 molfL nitric acid; and 225 mL of 0.25 mol/L sodium hydroxide Department of Clinical Biochemistry, Addenbrooke’s Hills Road, Cambridge, CB2 2QR, U.K. Received October 26, 1987; accepted June 6, 1988.
lutropin (12 360 hit. units/mg) and follitropin (2925 mt. units/mg) were kindly donated by Dr. S. Lynch, The Birmingham and Midland Hospital for Women, Birmingham, U.K. Human choriogonadotropin was from Paines and Byrne Ltd., Greenford, U.K. Growth hormone was obtained from Dr. J. Seth, Edinburgh, and prolactin standard (coded as 83 PEL) was supplied by Prof. S. Jeffcoate, Chelsea Hospital for Women. The International Reference Preparation for TSH (IRP 80/558) was from the National Institute of Biological Standards and Controls, London, U.K. ‘‘Fri-Level” ligand controls, levels A, B, and C (Gilford Systems, Irvine, CA) were supplied by Ciba-Corning Diagnostics. “Lyphochek” Immunoassay Control Serum Levels I, H, and UI were purchased from Bio-Rad Labs., Watford, Herts., U.K. In addition, we prepared three pools of serum to give a range of TSH concentrations. Equipment
Measurement of human thyrotropin (TSH) is widely undertaken in clinical laboratories as part of thyroid-function testing and has traditionally been done by radioimmunoassay (RIA) (1). With development of immunometric assays and the application of monoclonal antibodies, several sensitive kits are now available for measuring TSH, based on radiometric (2), chemiluminometric (3), fluorometric (4), and enzyme-amplified (5) immunoassays. A principal advantage of these kits over conventional RIA is their potential to discriminate between subnormal and normal concentrations of TSH in serum. We report here our evaluation of a TSH assay in which a chemilumninescent label is used to detect bound analyte. The assay involves a 2-h incubation of sample with a monoclonal anti-TSH antibody that has been labeled with an acridinium ester. After incubation with a second monoclonal antibody, covalently bound to magnetic particles, the unbound antibody is washed away and the amount of analyte bound is quantified luminometrically.
Reference
containing surfactant. Preparations of human
Hospital,
All of the following equipment was supplied
by Ciba-
Corning
Diagnostics. The magnetic separation unit consists of a rack that holds as many as 60 12-mm x 75-mm test tubes and a base that contains the magnets. A luminometer is required for quantifying the results. We used the “Magic Lumninometer,” a small, semi-automated
bench-top unit comprising two reagent-injection pumps, a measurement chamber, and a photomultiplier tube. A microprocessor unit stores test variables and the calibration curve. Results are given as relative light units (RLU) and as microprocessor-calculated TSH concentrations. Procedures Magic Life assay. The assay protocol recommended by the was followed throughout. All samples, calibrators, and controls were assayed in duplicate. A calibration curve is assigned to each batch of assay kits and stored in the luminometer. Therefore, only a two-point calibration is required with each analytical batch (0 and 13.4 milli-int. units/L). Total assay time for 60 assay tubes is -3.5 h. Enzyme-amplified immunoassay. We compared the results of the assay with those of an enzyme-amplified immunoassay (“EuA”; Novo BioLabs, Cambridge, U.K.) (5,6), in which two monoclonal antibodies to TSH are used, one labeled with alkaline phosphatase (EC 3.1.3.1), the other bound to a microtiter plate as solid phase. After incubation with the sample, the activity of the bound enzyme is measured by an enzyme-amplification step. The assay is calibrated against IRP 80/558. We obtained between-batch CVs of 9.8, 5.1, 7.1, and 7.7% at TSH concentrations of 0.27, 1.12, 3.79, and 9.73 milli-int. units/L, respectively. The lower limit of detection of the assay (mean plus 3 SD of the zero signal) is 0.04 milli-int. unit/L. A full evaluation of this manufacturer
assay
carried
out in this laboratory
was reported
elsewhere
(5). CLINICALCHEMISTRY, Vol. 34, No. 10, 1988
2087
Other assays. We measured total thyroxin in serum with an in-house RIA (7); free triiodothyronine and free thyroxin were measured with the Amerlex-M RIA (Amersham International plc, Cardifl U.K.).
Results Assay Tolerance Figure 1 illustrates the effects of varying the duration of the first and second incubation steps. With a first incubation of 2 h, the reaction has not gone to completion. However, prolonging the second incubation gives little increase in luminescence. We assessed the washing procedure by washing either zero, one, two, or three times after the second incubation. When no washing step was included, results for low TSH concentrations were high. More than two washing steps caused the RLU value to be lower because of loss of magnetic particles. There appeared to be little difference in results for between one and two washes. The final chemiluminescent reading was made immediately after the assay was completed, 30, 60, and 180 mm later, and overnight. We recalibrated the luminometer for each set of readings. The results showed no substantial loss of luminescent signal with time. Assay Performance Lower limit of detection. Analysis of 20 replicates of the zero standard supplied with the kit and calculating the mean plus 3 SD of the zero signal, we determined the lower limit of detection as 0.07 milli-int. unitfL. Precision. Within-batch precision was assessed by replicate analysis (n = 20) of each of the three human serum pools; between-batch precision was assessed with qualitycontrol material and human serum pools in 21 analytical N
480
L,ph.ch.k
460
III
440
batches (Table 1). Assay of quality-control materials at the beginning and end of each analytical batch evidenced no assay drift. A precision proffle, obtained with duplicate analysis (8), showed a working range for the assay (CV 60.0 milli-int. units/L. Dose-response curve. Five patients’ samples containing high NH concentrations (23-71 milli-int. unitsfL) were serially diluted with zero calibrator and assayed. The responses for two samples were linearly related to the dilution. The remaining three samples also showed a linear response on dilution, but the value for the undiluted sample was lower than expected in each case. Adding IRP 80/558 to zero calibrator to give 70,100, and 200 milli-int. units/Land assaying revealed no high-dose “hook” effect. Anolytical recovery. Recovery of TSH IRP 80/558 added to a serum pool was 97% at 1 milli-int. unitfL, 95% at 10 miiihit. unitsfL, 112% at 50 milli-int. units/L, and 98% at 100 mill-mt. unitWL. Recovery estimated from the analysis of External Quality-Assessment Scheme samples (9) ranged from 68 to 123%. Comparison with enzyme-amplified immunoassay. Results by the Magic Lite assay were compared with those obtained by the Au assay for 111 samples from inpatients suspected of having thyroid disease and from patients attending a thyroid clinic. If necessary, samples with a high NH concentration were assayed after dilution. Figure 2 shows all the results. Least squares regression (10) of only those results 25 milliint. units/L from the calculation because the few results obtained at this concentration and the additional sample dilution required for the AELIA assay might bias the data. Twenty-four samples, assayed in several different batches, showed aggregation of the magnetic particles in the immunochemiluminometric assay. Although the original results for these samples agreed with those of the enzymeamplified immunoassay, on re-assay four samples showed a significant increase in the measured TSH by the lumines-
420 400
380
380
360
360
F
340 0
S
S
320
280
Nigh
C.Iib..tn.,
5
i
240
260
240 220
200
L7000.,h.k
II
200
-
180
180
‘C N-
160
160
,,
140 120
*____
-
-
*
Nigh
C
Lyph..,h.k
-
#{163}20
“-‘C’
-
#{163}00
80
NJ
60
I 30
80
I. 60
90
120 ISO
1,,b.tien
C.1ibrt.,r
140
100
180
210
Tl,n
Cm..,)
240
60 0/U
0
10
20
30
40
50
Tin,.,
(n,in)
60
70
80
90
100
Fig. 1. Effect of the first incubation time (left) and the second incubation time (nght) on the Magic Ute TSH assay 0/N,
Ill
280
220
S
Lypheh.k
.
N
300
260
g
340
overnight
2088 CLINICALCHEMISTRY, Vol. 34, No. 10, 1988
II
cence assay. The cause of the aggregation was not identified and requires further investigation. External quality-assessment. Samples sent to the laboratory as part of the U.K. External Quality-Assessment Scheme for Thyroid-Related Hormones were also analyzed by the Magic Lite assay. All but one sample showed good agreement with the “all-laboratory trimmed mean” (11,12): immunochemiluminometric assay result = 2.54 milli-int. units/L, AELIA result = 5.09 milli-int. units/L, all-laboratory trimmed mean = 4.3 milli-int. unitslL. Interferences. Comparison of results from the Magic Lite assay with those from the AELIA assay for samples from patients with renal disease (n = 10), liver disease (n = 11), and myeloma (n =9) showed no deviation from the Deming regression described above. In addition, there was no significant interference from hemoglobin (10 g/L) or bilirubin (300 zmol/L). The monoclonal pair did not show any detectable crossreaction with lutropin (up to 123 int. units/L), follitropin (up to 146 int. units/L), human choriogonadotropin (up to 10000 jut. unitsfL), somatotropin (70 milli-int. units/L), or prolactin (up to 2800 int. units/L). Clinical Studies
To determine a normal reference interval, we analyzed sera from 144 healthy euthyroid subjects. Of these, six were excluded because of a markedly increased value for serum TSH (>6 milli-int. units/L), and one was excluded because
TSH was undetectable in serum (200 nmol/L (reference interval 65-145 nmoJJL) or with a value for free thyroxin >58 pmol/L, and eight subjects with total thyroxin >160 nmol/L-had a serum TSH concentration of 25 milli-int. units/L) for the AELIA method and to 0
20
40
60
80
100
120
140
LeO
180
AEUA UN (.111 I.LesIIWL)
Fig. 4. Comparisonof results by Magic Ute TSH assay with those obtained by an enzyme amplified immunoassay for 15 hypothyroid patients
luminescent assay (
