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SUMMARY.~-D-galactosidase from Escherichia coli and horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human a-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The non-specific binding of Fab'-peroxidase conjugates to IgG-coated polystyrene halls was less than that of Fah'-[1-D-galactosidase conjugates. and the affinity-purified Fab'-peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding f1-D-galactosidase conjugates. However. a large quantity of Fah'-peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than thẽ -[)-galactosidase assay. Other advantages and disadvantages of the two enzymes are discussed.We developed a method for conjugating Fab' with~-D-ga\actosidase from Escherichia coli using thiol groups in the hinge of Fab' by the reaction between thiol and maleimide groups (the hinge methods};': 2 This enabled us to measure 1 amol of ornithine o-aminotransferase from rat liver. I 2 amol of human IgE, 0·05 amol of human ferritin and 2 amol of human thyroidstimulating hormone." We also developed a method for preparing a monomeric conjugate of Fab' and horseradish peroxidase using thiol groups in the hinge of Fab' by the reaction between thiol and maleimide groups" 5 or by the thiol-disulphide exchange reaction (the hinge method). The Fah-peroxidase conjugate thus prepared was shown to be superior to those prepared using amino groups of Fab' by the glutaraldehyde and periodate methods (the non-hinge methods) in both sandwich enzyme immunoassay for rnacromolecules": 5 and immunohistochemical staining of tissue sections. Furthermore. the use of affinity-purified antihuman growth hormone Fah'-peroxidase conjugate prepared by the hinge method enabled us to measure 3 amol of human growth hormone." Thus, both enzymes appear to provide a sandwich enzyme immunoassay at an 310 attomole level, but have not been critically evaluated.This paper describes a critical evaluation of -D-galactosidase from Escherichia coli and horseradish peroxidase as labels of Fab ' by the sandwich enzyme immunoassay technique. Materials and methods BUFFERThe most frequently used buffer was (l-Il! mol/l sodium phosphate buffer. pH 7·{), containing 0·\ molll NaCI and I gil bovine serum albumin (fraction V. Armour Pharmaceutical Co.. Kankakee, Illinois), and was abbreviated as buffer A. Calculation of the amount of enzymesThe amount of~-[)-galactosidase in mol was

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A h i g h l y s e n s i t i v e sandwich enzyme immunoassay (EIA) f o r human growth hormone (hGH) w a s developed. hGH t o be assayed31 Copyrlght 0 1983 by Marcel Dekker, Inc. 0003-27 19/83/1601-0031$3.50/0 32 HASHIDA ET AL.was i n c u b a t e d w i t h a n anti-hGH IgG-coated p o l y s t y r e n e b a l l , and t h e n t h e p o l y s t y r e n e b a l l a f t e r washing was i n c u b a t e d w i t h a n t i -hCH Fab ' -p e r o x i d a s e c o n j u g a t e . P e r o x i d a s e a c t i v i t y bound t o t h e p o l y s t y r e n e b a l l was c o r r e l a t e d t o t h e amount of hGH t o be a s s a y e d . P o l y s t y r e n e b a l l s were c o a t e d by p h y s i c a l a d s o r p t i o n w i t h r a b b i t anti-hGH LgG which w a s n c t a f f i n i t y -p u r i f i e d . R a b b i t anti-hGH Fab' which had been a f f i n i t y -p u r i f i e d w a s c o a j ug a t e d w i t h h o r s e r a d i s h p e r o x i d a s e u s i n g N-succinimidyl 4-(N-maleimidomethy1)cyclohexane-1-carboxylate. The s e n s i t i v i t y was 0.06 pg of hGH p e r t u b e o r 6 pg/ml of serum when 0.01 ml of serum s a m p l e s was u s e d . No s i g n i f i c a n t i n t e r f e r e n c e was o b s e r v e d i n t h e p r e s e n c e of 8 n g / t u b e of h u m n p r o l a c t i n , 8 UU/tube of human t h y r o i d -s t i m u l a t i n g hormone, 50 mU/tube of human l u t e i n i z i n g hormone, 10 mU/tube of human f o l l i c l e -s t i m u l a t i n g hormone, o r 5 U / t u b e of human c h o r i o n i c g o n a d o t r o p i n . The r e c o v e r i e s of hGH added t o human sera were 96.2-121 %. The c o e f f i c i e n t s of w i t h i n -a s s a y and between-assay v a r i a t i o n s were 3.6-10.0 % and 5.6-9.8 %, r e s p e c t i v e l y . The r e g r e s s i o n e q u a t i o n and c o e f f i c i e n t f o r c o r r e l a t i o n t o r a d i oinmunoassay (RIA) were Y(EIA)=l.04X(RIA)-O.60 and 0.98 (n=137), r e s p e c t i v e l y . P r e p a r a t i o n of hGH-Free hTSH and hPRL The l y o p h i l i z e d p r e p a r a t i o n of hTSH (160 VU) or hPRL (100 ng) o b t a i n e d w a s d i s s o l v e d i n 0.01 M s o d i u m p h o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 0 . 1 % b o v i n e serum a l b u m i n (0.5 ml) and p a s s e d t h r o u g h a n anti-hGH IgG-Sepharose 4 B column (0.5 x 1.0 c m f o r hTSH and 0.5 x 2.0 cm f o r hPRL) a t a f l o w r a t e of 1.0 m l / h u s i n g the same b u f f e r . The amounts of hTSH and hPRL i n the e f f l u e n t were d e t e r m i n e d by enzyme immunoassay and radioimmunoassay, r e s p e c t i v el y , as d e s c r i b e d below, and t h e r e c o v e r i e s of hTSH and hPRL i n t h e e f f l u e n t were 80 X a n d 85 %, r e s p e c t i v e l y . Radioimmunoassay f o r hGH A c o m p e t i t i v e radioimmunoassay w i t h d o u b l e a n t i b o d y was performed by u s i n g HGH R I A K I T (Dainabot R a d i o i s o t o p e L a b . , Tokyo) as 1 d e s c r i b e d p r e v i o u s l y . Enzyme Immunoassay f o r hTSH hTSH w a s d e t e r m i n e d by a sandwic...

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