Journal oflmmunological Methods, 9 (1976) 297--305 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
297
A RAPID, SENSITIVE AND SPECIFIC RADIOIM~UNOASSAY FOR HUMAN CHORIONIC GONADOTROPHIN
A. KARDANA and K.D. BAGSHAWE
Department of Medical Oncology, Charing Cross Hospital, Fulham, London, England (Received 10 July 1975, accepted 14 July 1975)
A specific, quantitative assay for human chorionic gonadotrophin (hCG) in plasma has been developed using an hCO-~ antiserum and an assay procedure lasting four hours. It employs a double antibody method and in a procedure prior to assay the two antibodies are pre-incubated in bulk and then stored in ampoules in lyophilised form ready for use. The assay procedure is a dis-equilibrium (sequential saturation) system in which the sample or unlabelled antigen is first incubated with the pre-incubated antisera followed by the addition of the labelled antigen and a further incubation. The antigen--antibody complex is separated from free antigen by vacuum filtration on glass fibre discs. The assay sensitivity is 100--200 picograms hCG/ml (0.68 mIU/ml) and is uninfluenced by normal concentrations of luteinising hormone in the sample.
INTRODUCTION The use o f r a d i o i m m u n o a s s a y has m a d e it possible t o d e t e c t h u m a n chorionic g o n a d o t r o p h i n (hCG) in c o n c e n t r a t i o n s o f a b o u t 1 ng/ml. However, r a d i o i m m u n o a s s a y s using antisera raised to w h o l e hCG also d e t e c t luteinising h o r m o n e (Wilde et al., 1967). The a m o u n t o f luteinising h o r m o n e (hLH) in b l o o d and urine changes with the e n d o c r i n e status o f the individual, the m e n s t r u a l c y c l e and to a lesser e x t e n t with the episodic secretion o f h L H t h r o u g h o u t the d a y (Wide et al., 1973 and Sami et al., 1973). F o r the d e t e c t i o n o f minimal hCG p r o d u c i n g t u m o u r s , and for the d e t e c t i o n o f early p r e g n a n c y , the sensitivity o f hCG assays is limited b y this cross-reaction with hLH. The f o u r h u m a n g l y c o p r o t e i n h o r m o n e s hCG, h L H , h F S H and h T S H contain dissimilar n o n - c o v a l e n t l y linked subunits ( S w a m i n a t h a n and Bahl, 1970; Morgan and Canfield, 1971; Pierce et al., 1971). T h e a - s u b u n i t is c o m m o n to all f o u r h o r m o n e s and specificity is c o n f e r r e d b y the ~-subunit. Assays o f the subunits have been described using i n c u b a t i o n times in excess o f 24 h (Vaitukaitis et al., 1972; Kosasa et al., 1 9 7 4 ; J o n e s et al., 1975). This p a p e r describes the d e v e l o p m e n t o f an assay w h i c h uses an a n t i b o d y raised t o the ~-subunit o f hCG, gives high sensitivity, requires o n l y a 4.h i n c u b a t i o n period and selectively measures hCG in t h e presence o f hLH.
298 MATERIALS AND METHODS Antigens Alpha and ~-subunits of hCG (Batch CR 117) were donated by the National Institute for Health, Bethesda, Maryland, U.S.A. Highly purified hCG (HP-hCG) was prepared from the urine of patients with trophoblastic tumours by the methods of Wilde and Bagshawe (1965). The Second International Reference preparation o f hCG (2nd IRP-hCG), provided by Dr. Derek Bangham, Institute of Biological Standards, Mill Hill, London, was used as the calibration standard for the laboratory standard. The h u man luteinising h o r m o n e (hLH) preparation (IRC-2) used was prepared from pituitary glands and was supplied by Dr. Ann Hartree, Cambridge, England. A n t isera Specific antisera to the #-subunit of hCG (hCG-~) were raised using a modification o f the m e t hod described by Vaitukaitis et al. (1971). Eighty t~g of hCG-~ in complete Freunds adjuvant were injected intradermally at 30--40 sites on the backs of New Zealand white rabbits. This was repeated after four weeks and the rabbits were bled four weeks later. Serial dilutions of hCG and hLH preparations were precipitated by a fixed dilution o f each antiserum and antiserum L17 was selected for its ability to give the greatest discrimination between the two antigens. Guinea-pig anti-rabbit-~,-globulin precipitating sera were obtained after intradermal injections of 1 mg rabbit-3,-globulin in complete Freunds adjuvant, followed by further 1 mg injections at 6-week and 9-week intervals. The guinea-pigs were bled within 10 to 14 days of the last immunisation. Iodination Iodination was per f or m ed by the chloramine-T m e t h o d of Greenwood, Hunter and Glover (1963). Carrier-free sodium [ , 2 s i ] i o d i d e (IMS-3) was obtained from the Radiochemical Centre, Amersham, England, and was used to label both hCG-~ and HP-hCG. Five mCi were used per 10 t~g antigen and specific activities for the labelled ligand were over 200 t~Ci/#g. Preparation o f the pre-incubated double antibody Using the met hod of Wilde et al. (1967), o p t i m u m titres for the rabbit anti-hCG-/3 and guinea pig anti-rabbit-3,-globulin sera were obtained. Following a procedure similar to t ha t described by Hales and Randle (1963) equal quantities of the two sera at their optimised dilutions were pre-incubated at 4~'C for a minimum of 3 days. Aliquots were lyophilised
299 and v a c u u m sealed in vials. T h e y were r e c o n s t i t u t e d in 0.04 M p h o s p h a t e buffer, p H 7.0 c o n t a i n i n g 0.1% bovine albumin, as required. T h e r a b b i t hCG-fi a n t i s e r u m was used at a final assay d i l u t i o n o f 1 : 7 2 , 0 0 0 and t h e guinea pig anti rabbit-y-globulin serum at a final dilution o f 1 : 900.
Assay procedure T h e assay was p e r f o r m e d on plasma samples c o n t a i n i n g 2.5 m g / m l E D T A as an anticoagulant. 50 /~1 o f the p r e - i n c u b a t e d 'antiserum was added to 100 pl o f the plasma sample to be assayed. If t h e plasma sample was to be assayed u n d i l u t e d , 100 pl o f 0.04 M p h o s p h a t e buffer, pH 7.0, c o n t a i n i n g 0.1% albumin, was added to the tube. When diluted plasma was used, 100 pl o f n o r m a l male E D T A plasma was added to the tube, so t h a t the reaction v o l u m e was c o n s t a n t and p r o t e i n c o n t e n t r e m a i n e d a p p r o x i m a t e l y equal. T h e a d d i t i o n o f n o r m a l plasma was necessary to avoid non-specific effects f r o m plasma p r o t e i n s observed with o t h e r assay systems (Burr et al., 1969; Van Hall et al., 1971; Vaitukaitis et al., 1972). 100 pl o f n o r m a l male plasma was also added to t u b e s c o n t a i n i n g r e f e r e n c e standards and the zero antigen controls. All the t u b e s were i n c u b a t e d at 37°C for 1.5 h, t h e n 50 pl o f labelled antigen was added to every tube. [ 12 5 I] hCG-~ was used at first b u t later [ ~2 s I] hCG was used with identical results. All tubes were subjected to a f u r t h e r i n c u b a t i o n at 37°C for a m i n i m u m o f 2 h. T h e h C C z - a n t i b o d y c o m p l e x was separated f r o m the free labelled hCG by v a c u u m filtration on either glass fibre ( W b a t m a n G F / F ) or cellulose acetate (Oxoid) discs. T h e discs were placed in p o l y s t y r e n e t u b e s and the radioactivit y o n the discs c o u n t e d in a Wallac m o d e l No. 8 0 , 0 0 0 7 - c o u n t e r , for 60 see. In r o u t i n e operations, the assay was p e r f o r m e d using an a u t o m a t e d analyser which p e r f o r m e d and calculated this and similar assays at a rate o f 300 reactions per h o u r (Bagshawe, 1975). Calculations were based on the logit t r a n s f o r m o f the response variant versus t h e log dose o f the antigen ( R o d b a r d et al., 1969; R o d b a r d and Lewald, 1970). A series o f c o n t r o l plasma samples, c o n t a i n i n g b e t w e e n 1 and 80 ng h C G / m l was run in replicate on the same assay and also in consecutive assays to serve as a basis for calculation o f the intra and interassay variation coefficients. RESULTS Specific activities o b t a i n e d for i o d i n a t i o n were over 200 pCi/pg for the whole hCG molecule. This c o r r e s p o n d s to the labelling o f 4 o f the 7 t y r o s i n e molecules. U n d e r the assay c o n d i t i o n s described antiserum (L17) b o u n d 15% o f the
300
"'~.
0
190
80 7O
X
50
B B 0 x 100
40
ii L,,. l
h 100
L 10
0 20
hCG
10 1000
n~ m l
Fig. 1. Inhibition curves shown by the hCG-~ subunit assay. • ----o, 2nd International reference preparation of hCG; i!. . . . . . ~, highly purified desialylated hCG; X. . . . . ×, hCG-~; • - - - - A , hCG
297
A RAPID, SENSITIVE AND SPECIFIC RADIOIM~UNOASSAY FOR HUMAN CHORIONIC GONADOTROPHIN
A. KARDANA and K.D. BAGSHAWE
Department of Medical Oncology, Charing Cross Hospital, Fulham, London, England (Received 10 July 1975, accepted 14 July 1975)
A specific, quantitative assay for human chorionic gonadotrophin (hCG) in plasma has been developed using an hCO-~ antiserum and an assay procedure lasting four hours. It employs a double antibody method and in a procedure prior to assay the two antibodies are pre-incubated in bulk and then stored in ampoules in lyophilised form ready for use. The assay procedure is a dis-equilibrium (sequential saturation) system in which the sample or unlabelled antigen is first incubated with the pre-incubated antisera followed by the addition of the labelled antigen and a further incubation. The antigen--antibody complex is separated from free antigen by vacuum filtration on glass fibre discs. The assay sensitivity is 100--200 picograms hCG/ml (0.68 mIU/ml) and is uninfluenced by normal concentrations of luteinising hormone in the sample.
INTRODUCTION The use o f r a d i o i m m u n o a s s a y has m a d e it possible t o d e t e c t h u m a n chorionic g o n a d o t r o p h i n (hCG) in c o n c e n t r a t i o n s o f a b o u t 1 ng/ml. However, r a d i o i m m u n o a s s a y s using antisera raised to w h o l e hCG also d e t e c t luteinising h o r m o n e (Wilde et al., 1967). The a m o u n t o f luteinising h o r m o n e (hLH) in b l o o d and urine changes with the e n d o c r i n e status o f the individual, the m e n s t r u a l c y c l e and to a lesser e x t e n t with the episodic secretion o f h L H t h r o u g h o u t the d a y (Wide et al., 1973 and Sami et al., 1973). F o r the d e t e c t i o n o f minimal hCG p r o d u c i n g t u m o u r s , and for the d e t e c t i o n o f early p r e g n a n c y , the sensitivity o f hCG assays is limited b y this cross-reaction with hLH. The f o u r h u m a n g l y c o p r o t e i n h o r m o n e s hCG, h L H , h F S H and h T S H contain dissimilar n o n - c o v a l e n t l y linked subunits ( S w a m i n a t h a n and Bahl, 1970; Morgan and Canfield, 1971; Pierce et al., 1971). T h e a - s u b u n i t is c o m m o n to all f o u r h o r m o n e s and specificity is c o n f e r r e d b y the ~-subunit. Assays o f the subunits have been described using i n c u b a t i o n times in excess o f 24 h (Vaitukaitis et al., 1972; Kosasa et al., 1 9 7 4 ; J o n e s et al., 1975). This p a p e r describes the d e v e l o p m e n t o f an assay w h i c h uses an a n t i b o d y raised t o the ~-subunit o f hCG, gives high sensitivity, requires o n l y a 4.h i n c u b a t i o n period and selectively measures hCG in t h e presence o f hLH.
298 MATERIALS AND METHODS Antigens Alpha and ~-subunits of hCG (Batch CR 117) were donated by the National Institute for Health, Bethesda, Maryland, U.S.A. Highly purified hCG (HP-hCG) was prepared from the urine of patients with trophoblastic tumours by the methods of Wilde and Bagshawe (1965). The Second International Reference preparation o f hCG (2nd IRP-hCG), provided by Dr. Derek Bangham, Institute of Biological Standards, Mill Hill, London, was used as the calibration standard for the laboratory standard. The h u man luteinising h o r m o n e (hLH) preparation (IRC-2) used was prepared from pituitary glands and was supplied by Dr. Ann Hartree, Cambridge, England. A n t isera Specific antisera to the #-subunit of hCG (hCG-~) were raised using a modification o f the m e t hod described by Vaitukaitis et al. (1971). Eighty t~g of hCG-~ in complete Freunds adjuvant were injected intradermally at 30--40 sites on the backs of New Zealand white rabbits. This was repeated after four weeks and the rabbits were bled four weeks later. Serial dilutions of hCG and hLH preparations were precipitated by a fixed dilution o f each antiserum and antiserum L17 was selected for its ability to give the greatest discrimination between the two antigens. Guinea-pig anti-rabbit-~,-globulin precipitating sera were obtained after intradermal injections of 1 mg rabbit-3,-globulin in complete Freunds adjuvant, followed by further 1 mg injections at 6-week and 9-week intervals. The guinea-pigs were bled within 10 to 14 days of the last immunisation. Iodination Iodination was per f or m ed by the chloramine-T m e t h o d of Greenwood, Hunter and Glover (1963). Carrier-free sodium [ , 2 s i ] i o d i d e (IMS-3) was obtained from the Radiochemical Centre, Amersham, England, and was used to label both hCG-~ and HP-hCG. Five mCi were used per 10 t~g antigen and specific activities for the labelled ligand were over 200 t~Ci/#g. Preparation o f the pre-incubated double antibody Using the met hod of Wilde et al. (1967), o p t i m u m titres for the rabbit anti-hCG-/3 and guinea pig anti-rabbit-3,-globulin sera were obtained. Following a procedure similar to t ha t described by Hales and Randle (1963) equal quantities of the two sera at their optimised dilutions were pre-incubated at 4~'C for a minimum of 3 days. Aliquots were lyophilised
299 and v a c u u m sealed in vials. T h e y were r e c o n s t i t u t e d in 0.04 M p h o s p h a t e buffer, p H 7.0 c o n t a i n i n g 0.1% bovine albumin, as required. T h e r a b b i t hCG-fi a n t i s e r u m was used at a final assay d i l u t i o n o f 1 : 7 2 , 0 0 0 and t h e guinea pig anti rabbit-y-globulin serum at a final dilution o f 1 : 900.
Assay procedure T h e assay was p e r f o r m e d on plasma samples c o n t a i n i n g 2.5 m g / m l E D T A as an anticoagulant. 50 /~1 o f the p r e - i n c u b a t e d 'antiserum was added to 100 pl o f the plasma sample to be assayed. If t h e plasma sample was to be assayed u n d i l u t e d , 100 pl o f 0.04 M p h o s p h a t e buffer, pH 7.0, c o n t a i n i n g 0.1% albumin, was added to the tube. When diluted plasma was used, 100 pl o f n o r m a l male E D T A plasma was added to the tube, so t h a t the reaction v o l u m e was c o n s t a n t and p r o t e i n c o n t e n t r e m a i n e d a p p r o x i m a t e l y equal. T h e a d d i t i o n o f n o r m a l plasma was necessary to avoid non-specific effects f r o m plasma p r o t e i n s observed with o t h e r assay systems (Burr et al., 1969; Van Hall et al., 1971; Vaitukaitis et al., 1972). 100 pl o f n o r m a l male plasma was also added to t u b e s c o n t a i n i n g r e f e r e n c e standards and the zero antigen controls. All the t u b e s were i n c u b a t e d at 37°C for 1.5 h, t h e n 50 pl o f labelled antigen was added to every tube. [ 12 5 I] hCG-~ was used at first b u t later [ ~2 s I] hCG was used with identical results. All tubes were subjected to a f u r t h e r i n c u b a t i o n at 37°C for a m i n i m u m o f 2 h. T h e h C C z - a n t i b o d y c o m p l e x was separated f r o m the free labelled hCG by v a c u u m filtration on either glass fibre ( W b a t m a n G F / F ) or cellulose acetate (Oxoid) discs. T h e discs were placed in p o l y s t y r e n e t u b e s and the radioactivit y o n the discs c o u n t e d in a Wallac m o d e l No. 8 0 , 0 0 0 7 - c o u n t e r , for 60 see. In r o u t i n e operations, the assay was p e r f o r m e d using an a u t o m a t e d analyser which p e r f o r m e d and calculated this and similar assays at a rate o f 300 reactions per h o u r (Bagshawe, 1975). Calculations were based on the logit t r a n s f o r m o f the response variant versus t h e log dose o f the antigen ( R o d b a r d et al., 1969; R o d b a r d and Lewald, 1970). A series o f c o n t r o l plasma samples, c o n t a i n i n g b e t w e e n 1 and 80 ng h C G / m l was run in replicate on the same assay and also in consecutive assays to serve as a basis for calculation o f the intra and interassay variation coefficients. RESULTS Specific activities o b t a i n e d for i o d i n a t i o n were over 200 pCi/pg for the whole hCG molecule. This c o r r e s p o n d s to the labelling o f 4 o f the 7 t y r o s i n e molecules. U n d e r the assay c o n d i t i o n s described antiserum (L17) b o u n d 15% o f the
300
"'~.
0
190
80 7O
X
50
B B 0 x 100
40
ii L,,. l
h 100
L 10
0 20
hCG
10 1000
n~ m l
Fig. 1. Inhibition curves shown by the hCG-~ subunit assay. • ----o, 2nd International reference preparation of hCG; i!. . . . . . ~, highly purified desialylated hCG; X. . . . . ×, hCG-~; • - - - - A , hCG
