40

incubation. Whenever too high concentrations of tritiatedoestradiol are used Scatchard analysis of serum samples willshow the absence of specific binder.We describe below our technique which demonstrates high-

affinity oestradiol binders in serum when they are present inmeasurable amounts. Scatchard analyses of patients with andwithout high-affinity serum binder are shown in the figure.

Materials and Methods

Blood is collected in a 15 ml ’Vacutainer’ and allowed to clot for30 min at room temperature. The blood is then centrifuged and freshserum is separated. Serum is immediately transferred to an ice bath.Only blood from patients who are not on androgens, oestrogens, oranti-osstrogens will be of value for this assay.To 0.4 ml of 0-9% saline plus 0.3 ml of serum to be tested, add 0. 1

ml of the appropriate concentration of 2,4,6,7-tritiated oestradiol-175(New England Nuclear, specific activity of 80-100 Ci/mmol). TritiatedOEStradiol-17p was 97% pure on paper chromatography. The radioac-tive oestradiol is diluted in saline to the appropriate concentration forScatchard analysis and a working solution is made up weekly froma refrigerated stock solution. The assay concentrations of radioactiveoEstradiot-17 are (in pg in every 0-1 ml of solution): 7, 15, 30, 60,100, 200, 300, 500, and 1000. After addition of the tritiated oestra-diol-17p to the saline and serum mixture, the solutions are allowedto incubate for 2 h at 4°C without shaking.

Dextran-coated charcoal is prepared as follows: 2.5 g of charcoal(’Norit A’; PFA and Stiehl Laboratories, Waukegan, Illinois) is addedto 100 ml of solution containing 0.25 g of dextran (Sigma; clinicalgrade, average molecular weight 83 300) /dl of buffer. Buffer prepara-tion : 5.38 g NaH2PO. H2O; 16.35 g NaHPO - 7HO; 9.0 g NaCI;1.0 g sodium azide; 1.0 g gamma-globulin (Cohn fraction n); distilledwater to 1 litre. 0.5. ml of dextran-coated charcoal mixture is addedto serum/saline/oestradiol mixture, shaken for 40 s, and incubated inthe refrigerator at 40C for 1 h. The mixture is then centrifuged for 10min at 400 revolutions/min at 40C to remove the dextran-coated char-coal. All supernatant is then separated and prepared for liquid scintil-lation counting.

The scintillation fluid used is 4 g PPO+0.5 g of POPOP+250 mlTriton X-100+750 ml toluene. All the supernatant is added to 12 mlof scintillation fluid. The material is then counted in a Beckman LS150 liquid scintillation counter at room temperature. The quench cor-rection is made, and a Scatchard plot constructed by plottingbound/unbound steroid against bound. The supernatant control andpatient assays are done in duplicate.We hope that this information will resolve some of the diffi-

culties in demonstrating high-affinity oestradiol-17p binder inserum. If the techniques used for tissue are identically appliedto serum most investigators will fail to find high-affinity restra-diol binders because the concentration of oestradiol used mustbe matched to the concentration of high-affinity binder. Evenin tissue it is important not to use excess oestradiol since thiswill fail to demonstrate the vertical (specific) binding portionof the Scatchard analysis and would show only the horizontal(non-specific) binding portion.’ 1

Whittier-Montebello Cancer Research InstituteMontebello, California 90640, U.S.A.

GLEN TISMAN

SHOW-JEN GRACE WU

ENZYME IMMUNOASSAY IN RAPID PROGRESS

SIR,-You predict (Aug. 21, p. 406) that enzyme im-

munoassays will overtake radioimmunoassays within a fewyears. Arguments against ’this view appeared in the Sept. 11issue. One argument was that there is only radioimmunoassay(R.I.A.) for the analysis of femtomole (10-" mol) amounts ofsteroids, hormones, and so on to aid patient diagnosis andtreatment and that enzyme immunoassay (E.I.A.) would be un-likely to replace immunoassay for many years, if at all, sincethe scale of research expansion and application is much greaterwith R.I.A. than with E.I.A. We anticipate that E.I.A. will be assensitive as or more sensitive than R.I.A.We have developed a novel procedure for the conjugation of

the Fab’ fragments of rabbit antibody with p-D-galactosidaseE.C. 3. 2. 1. 23) from Escherichia coli using N, N’-o-pheny-

1. Hahnel, R., Twaddle, E. Cancer Res. 1973, 33, 559.

.1enedimaleimide.1 The Fab’ fragments which contain sulphyd-ryl groups are treated with excess dimaleimide, unreacteddimaleimide is removed, and the fragments containing malei-mide residues are coupled to j3-D-galactosidase which containssulphydryl groups. We have shown that 30 attomoles (1amol=10-18 mol) of 2, 4-dinitrophenyl human IgG, or ornith-ine 8-aminotransferase E.C. 2. 6. 1. 13) from rat liver are mea-surable by the sandwich procedure with the antibody/p-D-ga-lactosidase complex and rabbit antibody bound on silicone’ as

Sandwich immunoassay of ornithine -aminotransferase fromrat liver.

Assay done as described previously2 except that solid phase was 1.5mm cube silicon pieces.

solid phase. Furthermore, careful assay makes possible to

determine even 1 amol of ornithine 8-aminotransferase (figure).When one of us started studies on E.I.A. 4t years ago, he

never imagined measuring antigens at attomole levels. Later,we selected &bgr;-n-galactosidase for labelling antibodies andantigens to perform a highly sensitive immunoassay since p-D-galactosidase itself can be detected at single molecule levels.3We can detect attomole amounts of antigens. Developments inE.I.A. have been rapid; future development will be much moreso.

K. KATOY. HAMAGUCHIS. OKAWAE. ISHIKAWA

Department of Biochemistry,Medical College of Miyazaki,Kiyotake, Miyazaki 889-16, Japan

Department of Enzyme Chemistry,Institute for Enzyme Research, TokushimUniversity School of Medicine,Kuramoto, Tokushima 770

K. KOBAYASHIN. KATUNUMA

1. Kato, K., Fukui, H., Hamaguchi, Y., Ishikawa, E. J. Immun. 1976, 116,1554.

2. Hamaguchi, Y., Kato, K., Ishikawa, E., Kobayashi, K. Katunuma, N.F.E.B.S. Lett. 1976, 69, 11.

3. Rotman, B. Proc. natn. Acad Sci. U S.A. 1961, 47, 1981.