11. Radioimmunoassay II 169

The antibody dilution was chosen so that the standard curve gave results between 50 and 1650nmo1.1-1 (0 .18- 5.17 pmol/tube), to allow all patient sera to be assayed at one dilution. The intra-assay precision is seen in Table 1.

The recovery of added cortisol to serum was 95.7 _+ 7.2 % (2 _+ s, n = 15). Different dilutions of a serum sample gave the same results after corrected for dilution. Cross reaction with other endogenous steroids was acceptable, only 11- deoxycor- tisol and l l-deoxycorticosterone (~30%) and cortisone (~85 %) being above 2.5 %. The normal range (9.00 a.m.) is between 152 and 593 nmol. 1-1 (n = 30). Correlation of this assay with the routine 3H-assay [1] was good (r = 0.877 and for the regression equation y = a + b x : a = 1.057, b = 0.971).

C o n c l u s i o n s . A fully automatable method for the cortisol RIA has been developed which requires no prior extraction using an antibody in an assay-system at pH 4.0. The use of 125I- labelled tracer has abolished the need for scintillation fluid and its attendant quench and radioactive waste problems. The capacity of this method is very high because of the

automation. The first clinical results have shown that this method is reliable, reproducible and gives comparable values with those from the 3H-assay. The fact that no centrifugation is needed, that 3 samples are counted simultaneously and that the capacity is over 1000 tubes per working day makes this method attractive to a routine laboratory with high through- put of serum cortisol samples. From the theoretical stand- point, this method is adaptable to almost any commercially available automatic or semi-automatic assay system.

References

1. Horn, K., Henner, J., Mfiller, O. A., Scriba, P. C. : Z. Klin. Chem. Klin. Biochem. 13, 173 (1975)

2. Marschner, I., Erhardt, F., Henner, J., Scriba, P. C.: Z. Klin. Chem. Klin. Biochem. 13, 481 (1975)

3. Marschner, I., Erhardt, F. W., Scriba, P. C.: Radio- imlnunoassay and related procedures in medicine, Vol. 1, p. 111. Proceedings, Symposium Istanbul, Internat. Atomic Energy Agency, Vienna 1974

11.8.

Fresenius Z. Anal. Chem. 290, 169-170 (1978) - �9 by Springer-Verlag 1978

Reversed Sequential Reaction in a Double Antibody Solid Phase Immunoradioassay (IRMA): A Novel Approach to Rapid Thyrotropin Assay

Paul Carter

The Radioassay Office, Department of Pathology, The Wilmington Medical Center, Wilmington, DE, 19899, U.S.A.,

Umgekehrte Reihenfolge beim doppelten Antik6rper-Festphasen-Radioimmunoassay (IRMA): Eine neuartige Methode zur schnellen Thyrotropinbestimmung

Key words: Best. yon Thyrotropin; Radioimmunoassay; umgek. Reihenfolge

High on the list of priorities in contemporary clinical judgement is thyrotropin (TSH) assay, which now ranks eighth place among laboratories offering radioimmunoassay (RIA) services as revealed in a recent poll [4]. However when a survey sponsored by the Deutsche Gesellschaft ffir Endokrinologie [5] was conducted among 32 laboratories, interlaboratory precision was as high as 128 % with a recovery rate ranging from 5 0 - 926 %. In the future clinical chemists must resolve to correct this unfortunate situation which exists collectively in the western world. In this communication we address ourselves to that task by evaluation and documen- tation of a novel method for TSH assay which proves to be rapid, precise and sufficiently accurate for clinical interpre- tation that it can be implemented confidently in the routine hospital laboratory.

In essence a solid phase immunoradiometric assay (IRMA) is performed as follows: 200 gl of human serum is pipetted into a 12 x 75 mm test tube followed by 200 gl of radioiodinated TSH antibody (Corning Medical, Medfield, MA. 02052). After incubation at 37 ~ C for 120 _+ 5 rain, 500 gl of second antibody immobilized covalently on glass particles is added followed by a second incubation for 30min. Radioactivity in the bound phase which is separated by centrifugation at 1600 x g is counted after the supernatant has been decanted.

Standardization was documented by a modification of the single parameter variation technique [1] in which an in- ternational reference preparation (World Health Organization, National Institute for Biological Standards and Control, Holly Hill, London) was innoculated in TSH free serum [2] and substituted for the manufacturer's standard based in defibrinated porcine plasma. A third standard based in equine serum for which no TSH cross reactivity was observed (Abbott, N., Chicago, Ill., 60004) was also used. Normalized curves based on the ratio of % bound at 40 gIU/ml were virtually identical by all three methods.

Accuracy was verified by comparison with a commercially available reference technique (Beckman, Fullerton, CA, 92634) where 125I-labeled human TSH in sequential satu- ration was performed followed by solid phase separation with a second antibody covalently bound to cellulose. Least mean square regression yielded the equation : y = 0.92x + 1.85 and a correlation coefficient 0.975. A second comparison on the identical 26 serum specimens ranging in value from about 5 - 55 gIU/ml TSH was also performed in a homologous non- equilibrium sequential saturation assay with rabbit antiserum TSH using a labeled antigen (Biorad, Richmond, CA. 94804) in which resin bead double antibody separation was em- ployed. Regression analysis revealed an estimating equation y = 1.52x 0.53 and a correlation 0.900 where x is the reference value. Generally values assayed by the proposed

170 Fresenius Z. Anal. Chem., Band 290 (1978)

Table 1. Effect of structurally related endogenous hormones innoculated in normal human serum, 5.5 ' IU/ml TSH, on thyrotropin assay

HCG (IU/ml) 10 50 100 TSH found (gIU/ml) 14.7 10.5 5.7

LH (IU/I) 10 50 100 TSH found (glU/ml) 7.3 6.5 7.3

FSH (IU/1) 10 50 100 TSH found (gIU/ml) 5.7 7.2 6.0

method were higher than comparative values by the second reference technique but in no instance did they lead to clinical misinterpretation. Mean recovery determined by addition of human based TSH [2] at three concentrations to pre-assayed specimens was 102 % in 10 experiments with a range of 91 - 111%. Samples containing elevated TSH concentration di- luted with hormone free serum and assayed as described for 3 specimens yielded mean recovery of 102 % with a range 9 6 - 106%.

Antibody specificity was determined by innoculation of normal human serum with human choriogonadotropin (HCG), human follitropin (FSH) and lutropin (LH), over a variety of concentration as illustrated in Table 1. At values four fold greater than expected in healthy pre-menopause patients, values did, not in any case exceed the upper limit of the observed ranged in a healthy population. Nevertheless in samples up to about 60IU/I HCG some erroneous hy- pothyroid values are encountered; hence results assayed during early pregnancy should be interpreted with caution.

Intra-assay variance was observed in the normal range and at about mid point of the calibration curve on replicate specimens of pooled serum for five successive assays to yield a value of 5.2 +_ 0.3, C.V. = 5.0~, 31.5 + 1.4, C.V. = 4.5~. Interassay variance at the same levels yielded values of 5.2 + 0.7, C.V. = 12.7% and 31.5 + 2.2, C.V. = 7.0%. Sensitivity, which we define as the smallest amount of antigen that can be distinguished from no antigen was determined from precision at low antigen concentration and was esti- mated to be about 0.8 gIU/ml. Non-specific binding was determined by modification of a method from this laboratory [3] in which the difference between apparent and corrected counts in the bound phase is expressed as a percentage of the total counts in the system and was found to vary between 2.5 % at 1.5 gIU/ml to 2.3 % at 60 gIU/ml. These values are comparable to non-specific binding obtained in commercially available RIA kits (0 .2-5 .6 %) where solid phase antibody coated tubes are employed. Frequency distribution of TSH in a healthy population was obtained from assayed specimens by way of donors to the Wilmington Medical Center Blood Bank. For 38 fasting normal adults morning values ranged from 1.6 to 9.5 gIU/ml with a mean of 5.0 _+ 2.0 gIU/ml.

References

1. Carter, P.: X. International Congress on Clinical Chemistry, Mexico 1978

2. Carter, P.: Clin. Chem. 23, 1122 (1977) 3. Carter, P.: Clin. Biochem. 10, P. 6 (1977) 4. Editorial: Lab, World 27, 26 (1976) 5. Marschner, I., Erhardt, F. W., Scriba, P. C. : J. Clin. Chem.

Clin. Biochem. 14, 345 (1976)

11.9

Fresenius Z. Anal. Chem. 290, 170-171 (1978) - �9 by Springer-Verlag 1978

Rapid and Sensitive Radioimmunoassays for the C-Terminal Region of Human Parathyrin

W. G. Wood, I. Marschner, and P. C. Scriba

Laboratorien f/Jr Klinische Chemie und Endokrinologie, Medizinische Klinik Innenstadt der Universit/it Mfinchen, Ziemssenstrage 1, D-8000 Mtinchen 2

Schnelle und empfindliche Radioimmunoassays fdr die C-terminale Region des mensehlichen Parathyrins

Key words: Analyse von Parathyrin; Radioimmunoassay.

A major problem with the development of sensitive assays for human parathyrin (hPTH) has been the long incubation times needed [1]. By optimising assay conditions, studying reaction kinetics and introduction of the technique described by Wide et al. [3], assays for the C-regional peptides of hPTH (C- hPTH), have been developed which give results withm 30 h of receipt of blood samples, in comparison with the original assay using the same components which required several days [2]. Such rapid assays are of great use both in the routine assay of C-hPTH and in cases where clinicians and surgeons require

rapid results for patients with suspected adenoma or hyper- plastic parathyroid glands in order to plan parathyroidec- tomy or future treatment.

The two antibodies used have been raised in sheep against a mixture of bovine (bPTH) and porcine (pPTH) and are coded S-469, which is purely C-hPTH specific and S-478 which also has determinants for the N-region of hPTH. The results of the study are presented here.

a) Original Assay-S-469 50L. The labelling details have been published in full elsewhere [2]. 50pl of serum and 200~tl of antiserum S-469 [initial dilution (i.d.) 1:20000] were prein- cubated for 4 days at 4 ~ C. 100/al of tracer (70 Bq) were added and a further incubation of 3 days at 4 ~ C followed. Bound and free hormone was separated with 12% polyethylene glycol and the precipitate counted after centrifugation for 5 min.

b) Short Assay-S-469 50Q. By shortening the preincubation to 18 h, increasing the antiserum concentration to an i.d. 1:14000, addition of 330Bq tracer and introduction of a second incubation time of 6 h the counting time could be reducted to 2 min. Results were obtained which were 3 times as high as those in the S-469 50 L assay. The reason for this was determined by kinetic studies which confirmed the totally different binding characteristics of the bPTH used as stan- dard and tracer, and the C-hPTH present in serum. Despite