0021-972X/79/4904-0557$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 49, No. 4 Printed in U.S.A.
Immunoradiometric Assay for Serum Thyroglobulin: Semiquantitative Measurement of Thyroglobulin in Antithyroglobulin-Positive Sera* MONIKA F. BAYER AND JOSEPH P. KRISS Departments of Radiology and Medicine, Division of Nuclear Medicine, Stanford University School of Medicine, Stanford, California 94305
ABSTRACT. All RIAs for human serum thyroglobulin (Tg) described hitherto are based on the same principle; namely, competitive binding between cold and labeled antigen, followed by separation of bound from free antigen by precipitation of the former by a second antibody. These RIAs for Tg are accurate only when applied to sera free of detectable anti-Tg because the presence of the latter can result in either falsely elevated or falsely depressed values. This report describes a solid phase, sandwich-type, immunoradiometric assay (IRA) for serum Tg. Tg in the sample or standard is first bound to plastic cups coated with rabbit anti-Tg and then measured by the binding of rabbit [l25I]anti-Tg. The sensitivity of this assay (detection limit, 2.5 ng Tg/ml serum) and its reproducibility, as indicated by intraassay coefficients of variation (CV20 „„, 11.3%; CV,,r, „„, 4.1%; CV310 ng, 7.0%) and inter-
A
assay coefficients of variation (CV2o „„, 14.0%; CV,ir, ,,„, 7.2%; CV310 ng, 7.1%), are comparable to or better than those previously described. Normal values are
FIG. 6. Effect of anti-Tg on Tg measurements. Analysis of solutions containing varying concentrations of purified Tg (standard preparation) and varying concentrations of anti-Tg (anti-Tg-IgG from a patient with Hashimoto's thyroiditis) at equilibrium. Correlation of Tgfoumi vs. Tg,heor. in the presence of anti-Tg concentrations of 30 (•—•), 60 (A—A), 125 (•—•), 250 (D—•), 500 (A—A); 2,000 (•—•), and 200,000 (O-Z,) U/ml.
were added to various patients' sera (all with high antiTg concentrations) and the mixtures were analyzed. In contrast to the previous results, there was no correlation between the amount of Tg recovered and the anti-Tg concentrations, viz. when the sera were ranked in order of increasing anti-Tg concentrations, measured by RIA, the recoveries of Tg did not decline in the same order (Table 2). Thus, it is impossible to extrapolate Tgtheor. from Tgfound and the anti-Tg concentration when the sera contain abnormal concentrations of both Tg and anti-Tg. However, it was observed that in each serum with a high anti-Tg concentration, the percent recoveries for 100 and 500 ng added Tg/ml differed from each other by less than 10% (Table 2). From these data, we conclude that the percent recovery for a limited concentration range of Tg can be regarded as constant, and the values found for Tg can be satisfactorily corrected for on the basis of recovery studies. For such sera, the approach we suggest involves three Tg measurements; namely, 1) the neat serum, 2) serum plus 100 ng Tg/ml, and 3) serum plus 200 ng Tg/ml, preferably all measured in the same assay run with corrections for TgfoUnd made accordingly: Tgtheor. = Tgfound in neat sample x 100/average % recovery. This procedure was tested by adding known quantities of Tg (50 or 100 ng) to normal serum and performing recovery studies before and after the addition of varying amounts
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 13:50 For personal use only. No other uses without permission. . All rights reserved.
JCE&M • 1979 Vol49 • No4
BAYER AND KRISS
562
of anti-Tg (50 or 500 U). It was found that the recovery of Tg in normal serum (before the addition of anti-Tg; anti-Tg, 1000
510 445 365 315 160 85
4
5 6 7
(ng/ml)
Initial +100 +500 value (ng Tg/ml) 1 7 10 2 2
Vol. 49, No. 4 Printed in U.S.A.
Immunoradiometric Assay for Serum Thyroglobulin: Semiquantitative Measurement of Thyroglobulin in Antithyroglobulin-Positive Sera* MONIKA F. BAYER AND JOSEPH P. KRISS Departments of Radiology and Medicine, Division of Nuclear Medicine, Stanford University School of Medicine, Stanford, California 94305
ABSTRACT. All RIAs for human serum thyroglobulin (Tg) described hitherto are based on the same principle; namely, competitive binding between cold and labeled antigen, followed by separation of bound from free antigen by precipitation of the former by a second antibody. These RIAs for Tg are accurate only when applied to sera free of detectable anti-Tg because the presence of the latter can result in either falsely elevated or falsely depressed values. This report describes a solid phase, sandwich-type, immunoradiometric assay (IRA) for serum Tg. Tg in the sample or standard is first bound to plastic cups coated with rabbit anti-Tg and then measured by the binding of rabbit [l25I]anti-Tg. The sensitivity of this assay (detection limit, 2.5 ng Tg/ml serum) and its reproducibility, as indicated by intraassay coefficients of variation (CV20 „„, 11.3%; CV,,r, „„, 4.1%; CV310 ng, 7.0%) and inter-
A
assay coefficients of variation (CV2o „„, 14.0%; CV,ir, ,,„, 7.2%; CV310 ng, 7.1%), are comparable to or better than those previously described. Normal values are
FIG. 6. Effect of anti-Tg on Tg measurements. Analysis of solutions containing varying concentrations of purified Tg (standard preparation) and varying concentrations of anti-Tg (anti-Tg-IgG from a patient with Hashimoto's thyroiditis) at equilibrium. Correlation of Tgfoumi vs. Tg,heor. in the presence of anti-Tg concentrations of 30 (•—•), 60 (A—A), 125 (•—•), 250 (D—•), 500 (A—A); 2,000 (•—•), and 200,000 (O-Z,) U/ml.
were added to various patients' sera (all with high antiTg concentrations) and the mixtures were analyzed. In contrast to the previous results, there was no correlation between the amount of Tg recovered and the anti-Tg concentrations, viz. when the sera were ranked in order of increasing anti-Tg concentrations, measured by RIA, the recoveries of Tg did not decline in the same order (Table 2). Thus, it is impossible to extrapolate Tgtheor. from Tgfound and the anti-Tg concentration when the sera contain abnormal concentrations of both Tg and anti-Tg. However, it was observed that in each serum with a high anti-Tg concentration, the percent recoveries for 100 and 500 ng added Tg/ml differed from each other by less than 10% (Table 2). From these data, we conclude that the percent recovery for a limited concentration range of Tg can be regarded as constant, and the values found for Tg can be satisfactorily corrected for on the basis of recovery studies. For such sera, the approach we suggest involves three Tg measurements; namely, 1) the neat serum, 2) serum plus 100 ng Tg/ml, and 3) serum plus 200 ng Tg/ml, preferably all measured in the same assay run with corrections for TgfoUnd made accordingly: Tgtheor. = Tgfound in neat sample x 100/average % recovery. This procedure was tested by adding known quantities of Tg (50 or 100 ng) to normal serum and performing recovery studies before and after the addition of varying amounts
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 13:50 For personal use only. No other uses without permission. . All rights reserved.
JCE&M • 1979 Vol49 • No4
BAYER AND KRISS
562
of anti-Tg (50 or 500 U). It was found that the recovery of Tg in normal serum (before the addition of anti-Tg; anti-Tg, 1000
510 445 365 315 160 85
4
5 6 7
(ng/ml)
Initial +100 +500 value (ng Tg/ml) 1 7 10 2 2
