Clin. Biochem. 8, 133-141 (1975)
A RAPID RADIOIMMUNOASSAY METHOD FOR PLASMA LUTEINIZING HORMONE K. GUPTA, H. BUTTERFIELD, W. H. C. WALKER and E. V. YOUNGLAI Depart~nen~s of Nuclear Medicine, Pathology and Obstet~'ics and Gynecology, MeMaster University Medical Centre, Hamilton, Ontario, LSS 1~J9 (Accepted October ~0, 1975)
CDBIA, 8 (2) 133-141 (1975 Clin. Bioche~. Gupta, K., Butterfield, H., Walker, W. H. C. and YoungLai, E. V. Departmvnts of Nuclear Medicine, Pathology and Obstetrics and Gynecology, McMaster University Medical Centre, Hamilton, Ontario LSS $J9
A RAPID RADIOIMMUN.OASSAY METHOD FOR PLASMA LUTEINIZING HORMONE 1. A rapid double antibody radioimmunoassay method for the determination of plasma luteinizing hormone (hLH) is described. 2. The method involves preincubation of the anti-LH serum with the precipitating second antibody and incubation of this mixture with unknowns and standards at room temperature overnight. The incubation tubes are then centrifuged, supernatants decanted and the precipitates counted. 3. A total of two working days is required to complete the assay and report the results. 4. This method is more rapid than the conventional ones currently used for the estimation of LH in blood.
SEVERAL RADIOIMMUNOASSAY METHODS for measuring gonadotrophins h a v e b e e n d e s c r i b e d 1"s. A l t h o u g h t h e p r i n c i p l e o f t h e m e t h o d s is t h e same, some laboratories use precipitating second antibody to separate free h o r m o n e f r o m b o u n d , w h i l e o t h e r s h a v e u s e d talc6 a n d alcoholT. Rad~oi m m u n o a s s a y s e m p l o y i n g i m m u n o s o r b e n t s s h a v e also f o u n d s o m e a p p l i c a tion. All t h e s e m e t h o d s h a v e one o r m o r e d i s a d v a n t a g e s f o r clinical p u r poses, t h e m a j o r o n e b e i n g t h e l e n g t h y t u r n r o u n d t i m e . P r e s e n t a s s a y s r e q u i r e f i v e to seven d a y s of i n c u b a t i o n . H a l e s a n d R a n d l e 9 f o u n d t h a t p r e - p r e c i p i t a t e d i n s u l i n a n t i s e r u m w a s still c a p a b l e of r e a c t i n g w i t h a n t i g e n . I n t h i s c o m m u n i c a t i o n , t h e s a m e a p p r o a c h h a s b e e n a p p l i e d to t h e a s s a y o f h u m a n l u t e i n i n z i n g h o r m o n e ( h L H ) . C o n d i t i o n s h a v e been selected to r e d u c e t h e i n c u b a t i o n t i m e of t h e a s s a y to a m i n i m u m . Satisf a c t o r y p e r f o r m a n c e is o b t a i n e d w i t h a t u r n r o u n d t i m e of less t h a n 2 days. Correspondence: Dr. E. V. YoungLai, Department of Obstetrics and Gynecology, McMaster University Medical Centre, Hamilton, Ontario LSS 4J9.
134
G U P T A et al
12000
a Z
8000
0 en IZ 0 0
4000
0 ....
I 0.5
I 1
I 5
I 10
I 100
LH n g / t u b e
Fig. 1 - - Mean ± 1 S.D. values for 6 d i f f e r e n t standard curves using the rapid h L H procedure. MATERIALS AND METHODS
L E R 907, h u m a n p i t u i t a r y gonadotrophin s t a n d a r d and a n t i - h L H were supplied by the Hormone Distribution P r o g r a m of the National Institutes of Arthritis and M e t a b o l i c Disease ( N I A M D ) , Bethesda, Maryland, U.S.A. (1 ng L E R 907 -- 0.23 MIU hLH). Goat Anti Rabbit G a m m a Globulin was obtained f r o m Antibodies Incorporated, Davis, California 9'5616. N o r m a l rabbit serum was bought f r o m Grand Island Biological Company. 11~ H i g h Specific Activity (NEZ-0334) was obtained f r o m New England Nuclear, Massachusetts 01862. Bovine Serum Albumin Fraction V was purchased f r o m Sigma Chemical Company. Quality Controls were p r e p a r e d in the l a b o r a t o r y by pooling h u m a n plasma.
Reagents PBS
--
0.15 M.NaCI in 0.01 M. phosphate buffer, p H 7.8 containing 0.1% sodium
azide -diluted with 2% normal rabbit serum in PBS containing 0.5 M. E D T A to a working dilution of 1:12,000 and final dilution in the a s s a y tubes of 1:30,000.
Anti-hLH
hLH-I
hLH
1~ ~ h L H ( L E R 960) was iodinated with 11'~ according to a modification of Greenwood et al (10) and diluted with 2% bovine serum albumin in PBS. standards
~
p r e p a r e d in 1% bovine serum albumin in PBS.
RIA
METHOD
FOR
hLH
135
TABLE 1 PROTOCOL FOR LUTEINIZING HORMONE ASSAY
Tube No.
Specification
1,2,3
Background
4,56
Zero tube
, ....
Volume
LH Antbody Precipitate
NRS - + EDTA
(200~1)
Total Volume
200~1
400 BI
200~1
200~1
200~i
100~1
Standards in duplicate (0.5, 1.0, 2.0, 5.0 10.0, 20.0 50.0, & 100 nanograms)
& 200~1
Quality Controls
200~1
200~1
Unknowns
lOOv.l*
200~1
IOOBI
Buffer PBS
hLH-II~5 (lO,O00cpm) ~"
100~1 100~1
"
.~
100~1
L~
200~I
~
i00~i
"
•..22 f.,J
23-29 29, 30
•Q
I00~.1
~4
IOOBI
"
or
to end
200~.1
200~1
"
~.,
lOOp.1 lOOp.1
Incubated for 20 to 24 hours Added 500 ~1 of PBS *The volume may be varied as long as the total volume of unknown or standard + PBS is 200 ~1. + NRS-EDTA: 0.5M EDTA, 2% normal rabbit serum in PBS.
Method An optimum ratio of working dilution anti-hLH to goat anti rabbit gamma globulin (second antibody) was preineubated for 24 hours at 4 °. With the reagents employed this corresponded to 39 ml anti-hLH (1:12,000) and 1 ml second antibody. The assay protocol is shown in Table 1. After incubation at room temperature for 20 hours and addition of 0.5 ml PBS the tubes were centrifuged for 25 rain at 2500 rpm at 4 °. The supernatants were decanted and the deposits counted in a well type Nuclear Chicago gamma counter. Standard curves were plotted as counts bound versus concentration of LH (Fig. 1). The method was compared with the 5 day method of Faiman and Ryan ~ (Standard method) which has been in regular use in our laboratory for the past 3 years. RESULTS
1. Second antibody. T h e e f f e c t s of v a r y i n g a m o u n t s of second a n t i b o d y in t h e p r e i n c u b a tion mixture were studied, using anti hLH at an initial dilution of 1 i n 12,000 a n d 1 i n 24,000. A t b o t h a n t i b o d y d i l u t i o n s , a n i n c r e a s e
G U P T A et al
136 2260~)-
A2 °'-..... ° 2000
A1o ~
a Z
A3 •
1500 °
o
o
O ea m IZ O
o
1000
.:j
5~
0
.... 0
I 0.8
! 1
l 5
, ! 10
I 50
I 100
LH ng/tube
Fig. 2 - - S t a n d a r d curves obtained with varying anti-hLH and varying second antibody concentrations.
anti hLH dilution anti hLH/tube (~l)_
A1
A2
A3
A4
B1
B2
B3
12000 190
12000 195
12000 196
12000 198
24000 192
24000 198
10
5
4
2
8
24000 196 ~ 4
second antibody/tube (~l)
2
All other assay conditions constant as in the protocol.
in counts bound occurred as second antibody was increased up to an o p t i m u m beyond which further increase of second antibody reduced binding. The results over the whole range of the standard curve are shown in Fig. 2 and the effect on the binding in the zero standard tube is seen in more detail in Fig. 3.
RIA
MF_~HOD
FOR
137
hLH
30
25 D Z 0 ¢B I.zw 0 e-
20
15
10
5
! 4
I 5
I 6
! 7
SECOND ANTIBODY
I 8
I 9
~l/tube
F i g . 3 - - Percentage bound in zero standard tube using a n t i - h L H 1:30,000 with v a r y i n g amounts of second antibody.
4
A ~ x
•
11 0
I 50
I 100
I 150
I 200
T I M E IN H O U R S
Fig. 4 m Rate of progression of binding in r a p i d a s s a y using procedure of protocol a t 25 ° ( . ) and a t 5 ° ( X ) .
138
GUPTA
el, aZ
2. Temperature and time. The rate of progression of binding was measured using a series of zero-standard tubes in which the reaction was terminated after increasing time intervals. The increase in percent counts bound was followed at 5 °, 25 °, and 37 °, until equilibrium was reached. The equilibrium position was unaffected by temperature from 5 ° to 25 ° (Fig. 4). Results at 37 ° were unsatisfactory, with a marked and variable decrease in binding on prolonged incubation which may be due to instability of the antibody at elevated temperature. The forward reaction rate was temperature dependent, being almost doubled by increasing the temperature from 5 ° to 25 ° . Under the conditions selected for the assay, the reaction reaches 85 % completion in the zero-standard tube.
3. Sensitivity, precision and specificity. Sensitivity, defined as the least amount distinguishable from zero was 0.25 m I U / t u b e in the present method as compared with 0.12 m I U / t u b e in the standard method. The precision of replicated plasma samples covering a range of concentrations and assayed in consecutive batches is shown in Table 2. Except at the lowest concentration which is near the limit of detection, the precision (1 C.V.) beteen batches was approximately 13%. Specificity was measured against the standard method. 20 plasma samples were assayed by both methods (Fig. 5). The regression line is given by: rapid method - 0.94 (standard method) - 2 . 0 ; r = 0.93.
4. Stability of anti-hLH precipitate. When preincubated first and second antibody was lyophilized in small aliquots and stored at 5 ° for 7 weeks, the assay results obtained with this preparation were no different from those obtained with fresh precipitate (Table 3).
5. Binding constants. Scatchard plots ~1 prepared from standard curves for the standard and the short method are shown in Fig. 6. The concentration of binding sites were 2.4 n g / m l and 9.6 n g / m l respectively in keeping with a four fold increase in antibody concentration in the short method. The apparent equilibrium constant, which is nut dilution dependent, changed from 5 X 109 1/M in the standard method to 0.8 X 109 1/M in the short method, in consequence of the failure to achieve equilibrium in the short method 12.
RIA TABLE Plasma Sample
mean h L H mIU/ml
MRTHOD
FOR
2
hLH
139 TABLE 3
hLH mIU/ml hLH mIU/ml ( ± 1SD) Plasma using lyophilized using fresh Sample precipitate precipitate"
Standard No. of deviation batches
A B C
2.8 8.7 12.1
0.57 1.04 1.17
10 9 11
D E
24.2 39.0
3.34 4.63
6 6
A
2.9 2.8 ~- 0.6 9.8 8.7 ~ 1.8 C 11.0 12.0 ~ 1.2 D 35.8 38.9 ~ 4.6 Assay results using lyophilized anti-hLH precipitate after 7 weeks compared with results using freshly prepared precipitate. B
Between assay precision of plasma samples using rapid method.
110 LH 90 mIU/ml 70
o
~
50 STANDARD METHOD3010
0 10 20 3o ' 40 50 60 "70 8o o 9' 100 110 ' ' ' ' ' ' RAPIDMETHOD LHmIU/ml y ----0.94x -- 2. r = 0.93 Fig. 5 - - Correlation between values obtained on 20 plasma samples by rapid hLH method and the standard met.hod.
DISCUSSION The present method uses an incubation period that limits the hormonea n t i b o d y i n t e r a c t i o n to 8 0 % o f t h e b i n d i n g t h a t w o u l d b e a c h i e v e d a t e q u i l i b r i u m . T h e m a j o r e f f e c t o f t h i s is to r e d u c e s e n s i t i v i t y a n d t h r e e a s s a y m o d f i c a t i o n s a r e e m p l o y e d to r e t r i e v e m u c h o f t h e l o s t s e n s i t i v i t y without extending the incubation time. 1. S i n c e t h e a n t i b o d y h a s a b i n d i n g t e m p e r a t u r e o v e r t h e r a n g e 5 ° to a d v a n t a g e in i n c u b a t i n g a t a l o w is h i g h e r a t e l e v a t e d t e m p e r a t u r e
e n e r g y w h i c h is u n a f f e c t e d b y 37 ° , t h e r e is no t h e r m o d y n a m i c temperature. The reaction rate a n d m o r e r a p i d e q u i l i b r i u m is
Addendum: We have since tried the identical procedure for human follide stimulating hormones and the method works just as well.
140
G U P T A et. al
o,[ 0.4~ 0.3
A
0"
.
0.1 |
! Ab I
I
I
I
I
B conc° ng/ml
I
!
! lO Ab 2
Fig. 6 - - Scatchard plots of standard curves using the rapid method (o) and the standard method ( A ) . The intercepts on the abscissa represent the binding capacities AI~ and Ab2 f o r standard and rapid methods. Abl ---- 9.6 n g / m l ; Ab= -- 2.4 ng/ml. Equilibrium constants are K1 ~ 5 X 10 9 1/M and K= = 0.8 X 109 1/M f o r standard and rapid methods respectively.
thereby achieved with no loss of sensitivity. 25 ° was chosen in preference to 37 ° because the slight deterioration in binding with prolonged incubation at 25 ° is more marked at 37 ° . This may be due to denaturation of the antibody 1~. 2. Delayed addition of the labelled hormone until 5 hours after the unlabelled hormone addition improves sensitivity and precision especially in the lower assay range 12. 3. The volume of the incubation mixture was reduced from 800 pl in the standard method to 500 pl without reduction of individual pipetring volumes to less than 100 pl. At this level, pipetting precision is about 1% (1 C.V.). Sensitivity improves in direct proportion to the volume reduction. The incubation time of the standard method was further shortened by preincubation of first and second antibody. An optimum concentration of second antibody was found, slightly different for the 2 first antibody concentrations tested. Deterioration of hLH binding when the second antibody was increased above this optimum may be due to masking of hLH binding sites. The stability of the preincubated mixture, lyophilized in small aliquots allowed batches to be prepared at monthly intervals. Although plasma matrix effects interfering with the specificity of assays has been described with some preincubation methods z3, such an effect was not apparent in the present work. Indeed, non-specific effects on
RIA MEPrHOD FOR hLH
141
the precipitation stage due to the presence of plasma samples may be removed by completing this reaction under separate uniform conditions. The short method has sensitivity, precision and accuracy as measured against the standard method t h a t render it acceptable for clinical use with a turn round time of less t h a n 2 days. ACKNOWLEGMENT
The assistance of Mrs. L. Miller at different stages of this work is acknowledged. Thanks are due to NIAMDD, Bethesda, U.S.A., for the reagents used in this study.
REFERENCES 1. Faiman, C. and Ryan, R. J., (1967). J. Clin. Endocrinol. Metab. 27, 444-447. 2. Midgely, A. R., (1967). J. Clin. Endocrinol. Metab. 27, 295-299. 3. ~VIidgely, A. R., Jr., Rebar, R. W. and Niswender, G. D., (1969). Ac~a Endocrinol. Suppl. 142, 247-256. 4. Odell, W. D., Rayford, P. L., and Ress, G. T., (1967). J. Lab. Clin. Med. 79, 973-980. 5. Odell, W. D., Ross, G. T., and Rayford, P. L., (1966). Met~b. Clin. Exp. 15, 287289. 6. Levene, R. A., Donabedion, R. K., and Sobrinko, L. G., (1971). C'lin. Chem. 17, 931-935. 7. Sadayuki, K., Sansone, A. and Hreschyshyn, M. M., (1971). Am~r. $. Obstst. Crynecol. 109, 352-957. 8. Wide, L., (1969). Aet~ Hndo~r. Suppl. 142, 207-221. 9. Hales, C. N., and Randle, P. J., (1963). BiGh~a J. 88, 137-146. 10. Greenwood, F. C., Hunter. W. M., and Glover, J. S., (1963). Bioehem. J. 89, 114-10~. 11. Scatchard, G., (1949). Ann. N Y Ae~d. Sd. ~1, 660-672. 12. Rodbard, D., Ruder, H. J., Vaitukaitis, J., and Jaeobs, H. S., (1971). J. Cl/n. Hndo~.-r. 33, 343-355. 18. Ratcliffe, J. G., (1974). Br. M~t. Bull., 30, 32~7.
A RAPID RADIOIMMUNOASSAY METHOD FOR PLASMA LUTEINIZING HORMONE K. GUPTA, H. BUTTERFIELD, W. H. C. WALKER and E. V. YOUNGLAI Depart~nen~s of Nuclear Medicine, Pathology and Obstet~'ics and Gynecology, MeMaster University Medical Centre, Hamilton, Ontario, LSS 1~J9 (Accepted October ~0, 1975)
CDBIA, 8 (2) 133-141 (1975 Clin. Bioche~. Gupta, K., Butterfield, H., Walker, W. H. C. and YoungLai, E. V. Departmvnts of Nuclear Medicine, Pathology and Obstetrics and Gynecology, McMaster University Medical Centre, Hamilton, Ontario LSS $J9
A RAPID RADIOIMMUN.OASSAY METHOD FOR PLASMA LUTEINIZING HORMONE 1. A rapid double antibody radioimmunoassay method for the determination of plasma luteinizing hormone (hLH) is described. 2. The method involves preincubation of the anti-LH serum with the precipitating second antibody and incubation of this mixture with unknowns and standards at room temperature overnight. The incubation tubes are then centrifuged, supernatants decanted and the precipitates counted. 3. A total of two working days is required to complete the assay and report the results. 4. This method is more rapid than the conventional ones currently used for the estimation of LH in blood.
SEVERAL RADIOIMMUNOASSAY METHODS for measuring gonadotrophins h a v e b e e n d e s c r i b e d 1"s. A l t h o u g h t h e p r i n c i p l e o f t h e m e t h o d s is t h e same, some laboratories use precipitating second antibody to separate free h o r m o n e f r o m b o u n d , w h i l e o t h e r s h a v e u s e d talc6 a n d alcoholT. Rad~oi m m u n o a s s a y s e m p l o y i n g i m m u n o s o r b e n t s s h a v e also f o u n d s o m e a p p l i c a tion. All t h e s e m e t h o d s h a v e one o r m o r e d i s a d v a n t a g e s f o r clinical p u r poses, t h e m a j o r o n e b e i n g t h e l e n g t h y t u r n r o u n d t i m e . P r e s e n t a s s a y s r e q u i r e f i v e to seven d a y s of i n c u b a t i o n . H a l e s a n d R a n d l e 9 f o u n d t h a t p r e - p r e c i p i t a t e d i n s u l i n a n t i s e r u m w a s still c a p a b l e of r e a c t i n g w i t h a n t i g e n . I n t h i s c o m m u n i c a t i o n , t h e s a m e a p p r o a c h h a s b e e n a p p l i e d to t h e a s s a y o f h u m a n l u t e i n i n z i n g h o r m o n e ( h L H ) . C o n d i t i o n s h a v e been selected to r e d u c e t h e i n c u b a t i o n t i m e of t h e a s s a y to a m i n i m u m . Satisf a c t o r y p e r f o r m a n c e is o b t a i n e d w i t h a t u r n r o u n d t i m e of less t h a n 2 days. Correspondence: Dr. E. V. YoungLai, Department of Obstetrics and Gynecology, McMaster University Medical Centre, Hamilton, Ontario LSS 4J9.
134
G U P T A et al
12000
a Z
8000
0 en IZ 0 0
4000
0 ....
I 0.5
I 1
I 5
I 10
I 100
LH n g / t u b e
Fig. 1 - - Mean ± 1 S.D. values for 6 d i f f e r e n t standard curves using the rapid h L H procedure. MATERIALS AND METHODS
L E R 907, h u m a n p i t u i t a r y gonadotrophin s t a n d a r d and a n t i - h L H were supplied by the Hormone Distribution P r o g r a m of the National Institutes of Arthritis and M e t a b o l i c Disease ( N I A M D ) , Bethesda, Maryland, U.S.A. (1 ng L E R 907 -- 0.23 MIU hLH). Goat Anti Rabbit G a m m a Globulin was obtained f r o m Antibodies Incorporated, Davis, California 9'5616. N o r m a l rabbit serum was bought f r o m Grand Island Biological Company. 11~ H i g h Specific Activity (NEZ-0334) was obtained f r o m New England Nuclear, Massachusetts 01862. Bovine Serum Albumin Fraction V was purchased f r o m Sigma Chemical Company. Quality Controls were p r e p a r e d in the l a b o r a t o r y by pooling h u m a n plasma.
Reagents PBS
--
0.15 M.NaCI in 0.01 M. phosphate buffer, p H 7.8 containing 0.1% sodium
azide -diluted with 2% normal rabbit serum in PBS containing 0.5 M. E D T A to a working dilution of 1:12,000 and final dilution in the a s s a y tubes of 1:30,000.
Anti-hLH
hLH-I
hLH
1~ ~ h L H ( L E R 960) was iodinated with 11'~ according to a modification of Greenwood et al (10) and diluted with 2% bovine serum albumin in PBS. standards
~
p r e p a r e d in 1% bovine serum albumin in PBS.
RIA
METHOD
FOR
hLH
135
TABLE 1 PROTOCOL FOR LUTEINIZING HORMONE ASSAY
Tube No.
Specification
1,2,3
Background
4,56
Zero tube
, ....
Volume
LH Antbody Precipitate
NRS - + EDTA
(200~1)
Total Volume
200~1
400 BI
200~1
200~1
200~i
100~1
Standards in duplicate (0.5, 1.0, 2.0, 5.0 10.0, 20.0 50.0, & 100 nanograms)
& 200~1
Quality Controls
200~1
200~1
Unknowns
lOOv.l*
200~1
IOOBI
Buffer PBS
hLH-II~5 (lO,O00cpm) ~"
100~1 100~1
"
.~
100~1
L~
200~I
~
i00~i
"
•..22 f.,J
23-29 29, 30
•Q
I00~.1
~4
IOOBI
"
or
to end
200~.1
200~1
"
~.,
lOOp.1 lOOp.1
Incubated for 20 to 24 hours Added 500 ~1 of PBS *The volume may be varied as long as the total volume of unknown or standard + PBS is 200 ~1. + NRS-EDTA: 0.5M EDTA, 2% normal rabbit serum in PBS.
Method An optimum ratio of working dilution anti-hLH to goat anti rabbit gamma globulin (second antibody) was preineubated for 24 hours at 4 °. With the reagents employed this corresponded to 39 ml anti-hLH (1:12,000) and 1 ml second antibody. The assay protocol is shown in Table 1. After incubation at room temperature for 20 hours and addition of 0.5 ml PBS the tubes were centrifuged for 25 rain at 2500 rpm at 4 °. The supernatants were decanted and the deposits counted in a well type Nuclear Chicago gamma counter. Standard curves were plotted as counts bound versus concentration of LH (Fig. 1). The method was compared with the 5 day method of Faiman and Ryan ~ (Standard method) which has been in regular use in our laboratory for the past 3 years. RESULTS
1. Second antibody. T h e e f f e c t s of v a r y i n g a m o u n t s of second a n t i b o d y in t h e p r e i n c u b a tion mixture were studied, using anti hLH at an initial dilution of 1 i n 12,000 a n d 1 i n 24,000. A t b o t h a n t i b o d y d i l u t i o n s , a n i n c r e a s e
G U P T A et al
136 2260~)-
A2 °'-..... ° 2000
A1o ~
a Z
A3 •
1500 °
o
o
O ea m IZ O
o
1000
.:j
5~
0
.... 0
I 0.8
! 1
l 5
, ! 10
I 50
I 100
LH ng/tube
Fig. 2 - - S t a n d a r d curves obtained with varying anti-hLH and varying second antibody concentrations.
anti hLH dilution anti hLH/tube (~l)_
A1
A2
A3
A4
B1
B2
B3
12000 190
12000 195
12000 196
12000 198
24000 192
24000 198
10
5
4
2
8
24000 196 ~ 4
second antibody/tube (~l)
2
All other assay conditions constant as in the protocol.
in counts bound occurred as second antibody was increased up to an o p t i m u m beyond which further increase of second antibody reduced binding. The results over the whole range of the standard curve are shown in Fig. 2 and the effect on the binding in the zero standard tube is seen in more detail in Fig. 3.
RIA
MF_~HOD
FOR
137
hLH
30
25 D Z 0 ¢B I.zw 0 e-
20
15
10
5
! 4
I 5
I 6
! 7
SECOND ANTIBODY
I 8
I 9
~l/tube
F i g . 3 - - Percentage bound in zero standard tube using a n t i - h L H 1:30,000 with v a r y i n g amounts of second antibody.
4
A ~ x
•
11 0
I 50
I 100
I 150
I 200
T I M E IN H O U R S
Fig. 4 m Rate of progression of binding in r a p i d a s s a y using procedure of protocol a t 25 ° ( . ) and a t 5 ° ( X ) .
138
GUPTA
el, aZ
2. Temperature and time. The rate of progression of binding was measured using a series of zero-standard tubes in which the reaction was terminated after increasing time intervals. The increase in percent counts bound was followed at 5 °, 25 °, and 37 °, until equilibrium was reached. The equilibrium position was unaffected by temperature from 5 ° to 25 ° (Fig. 4). Results at 37 ° were unsatisfactory, with a marked and variable decrease in binding on prolonged incubation which may be due to instability of the antibody at elevated temperature. The forward reaction rate was temperature dependent, being almost doubled by increasing the temperature from 5 ° to 25 ° . Under the conditions selected for the assay, the reaction reaches 85 % completion in the zero-standard tube.
3. Sensitivity, precision and specificity. Sensitivity, defined as the least amount distinguishable from zero was 0.25 m I U / t u b e in the present method as compared with 0.12 m I U / t u b e in the standard method. The precision of replicated plasma samples covering a range of concentrations and assayed in consecutive batches is shown in Table 2. Except at the lowest concentration which is near the limit of detection, the precision (1 C.V.) beteen batches was approximately 13%. Specificity was measured against the standard method. 20 plasma samples were assayed by both methods (Fig. 5). The regression line is given by: rapid method - 0.94 (standard method) - 2 . 0 ; r = 0.93.
4. Stability of anti-hLH precipitate. When preincubated first and second antibody was lyophilized in small aliquots and stored at 5 ° for 7 weeks, the assay results obtained with this preparation were no different from those obtained with fresh precipitate (Table 3).
5. Binding constants. Scatchard plots ~1 prepared from standard curves for the standard and the short method are shown in Fig. 6. The concentration of binding sites were 2.4 n g / m l and 9.6 n g / m l respectively in keeping with a four fold increase in antibody concentration in the short method. The apparent equilibrium constant, which is nut dilution dependent, changed from 5 X 109 1/M in the standard method to 0.8 X 109 1/M in the short method, in consequence of the failure to achieve equilibrium in the short method 12.
RIA TABLE Plasma Sample
mean h L H mIU/ml
MRTHOD
FOR
2
hLH
139 TABLE 3
hLH mIU/ml hLH mIU/ml ( ± 1SD) Plasma using lyophilized using fresh Sample precipitate precipitate"
Standard No. of deviation batches
A B C
2.8 8.7 12.1
0.57 1.04 1.17
10 9 11
D E
24.2 39.0
3.34 4.63
6 6
A
2.9 2.8 ~- 0.6 9.8 8.7 ~ 1.8 C 11.0 12.0 ~ 1.2 D 35.8 38.9 ~ 4.6 Assay results using lyophilized anti-hLH precipitate after 7 weeks compared with results using freshly prepared precipitate. B
Between assay precision of plasma samples using rapid method.
110 LH 90 mIU/ml 70
o
~
50 STANDARD METHOD3010
0 10 20 3o ' 40 50 60 "70 8o o 9' 100 110 ' ' ' ' ' ' RAPIDMETHOD LHmIU/ml y ----0.94x -- 2. r = 0.93 Fig. 5 - - Correlation between values obtained on 20 plasma samples by rapid hLH method and the standard met.hod.
DISCUSSION The present method uses an incubation period that limits the hormonea n t i b o d y i n t e r a c t i o n to 8 0 % o f t h e b i n d i n g t h a t w o u l d b e a c h i e v e d a t e q u i l i b r i u m . T h e m a j o r e f f e c t o f t h i s is to r e d u c e s e n s i t i v i t y a n d t h r e e a s s a y m o d f i c a t i o n s a r e e m p l o y e d to r e t r i e v e m u c h o f t h e l o s t s e n s i t i v i t y without extending the incubation time. 1. S i n c e t h e a n t i b o d y h a s a b i n d i n g t e m p e r a t u r e o v e r t h e r a n g e 5 ° to a d v a n t a g e in i n c u b a t i n g a t a l o w is h i g h e r a t e l e v a t e d t e m p e r a t u r e
e n e r g y w h i c h is u n a f f e c t e d b y 37 ° , t h e r e is no t h e r m o d y n a m i c temperature. The reaction rate a n d m o r e r a p i d e q u i l i b r i u m is
Addendum: We have since tried the identical procedure for human follide stimulating hormones and the method works just as well.
140
G U P T A et. al
o,[ 0.4~ 0.3
A
0"
.
0.1 |
! Ab I
I
I
I
I
B conc° ng/ml
I
!
! lO Ab 2
Fig. 6 - - Scatchard plots of standard curves using the rapid method (o) and the standard method ( A ) . The intercepts on the abscissa represent the binding capacities AI~ and Ab2 f o r standard and rapid methods. Abl ---- 9.6 n g / m l ; Ab= -- 2.4 ng/ml. Equilibrium constants are K1 ~ 5 X 10 9 1/M and K= = 0.8 X 109 1/M f o r standard and rapid methods respectively.
thereby achieved with no loss of sensitivity. 25 ° was chosen in preference to 37 ° because the slight deterioration in binding with prolonged incubation at 25 ° is more marked at 37 ° . This may be due to denaturation of the antibody 1~. 2. Delayed addition of the labelled hormone until 5 hours after the unlabelled hormone addition improves sensitivity and precision especially in the lower assay range 12. 3. The volume of the incubation mixture was reduced from 800 pl in the standard method to 500 pl without reduction of individual pipetring volumes to less than 100 pl. At this level, pipetting precision is about 1% (1 C.V.). Sensitivity improves in direct proportion to the volume reduction. The incubation time of the standard method was further shortened by preincubation of first and second antibody. An optimum concentration of second antibody was found, slightly different for the 2 first antibody concentrations tested. Deterioration of hLH binding when the second antibody was increased above this optimum may be due to masking of hLH binding sites. The stability of the preincubated mixture, lyophilized in small aliquots allowed batches to be prepared at monthly intervals. Although plasma matrix effects interfering with the specificity of assays has been described with some preincubation methods z3, such an effect was not apparent in the present work. Indeed, non-specific effects on
RIA MEPrHOD FOR hLH
141
the precipitation stage due to the presence of plasma samples may be removed by completing this reaction under separate uniform conditions. The short method has sensitivity, precision and accuracy as measured against the standard method t h a t render it acceptable for clinical use with a turn round time of less t h a n 2 days. ACKNOWLEGMENT
The assistance of Mrs. L. Miller at different stages of this work is acknowledged. Thanks are due to NIAMDD, Bethesda, U.S.A., for the reagents used in this study.
REFERENCES 1. Faiman, C. and Ryan, R. J., (1967). J. Clin. Endocrinol. Metab. 27, 444-447. 2. Midgely, A. R., (1967). J. Clin. Endocrinol. Metab. 27, 295-299. 3. ~VIidgely, A. R., Jr., Rebar, R. W. and Niswender, G. D., (1969). Ac~a Endocrinol. Suppl. 142, 247-256. 4. Odell, W. D., Rayford, P. L., and Ress, G. T., (1967). J. Lab. Clin. Med. 79, 973-980. 5. Odell, W. D., Ross, G. T., and Rayford, P. L., (1966). Met~b. Clin. Exp. 15, 287289. 6. Levene, R. A., Donabedion, R. K., and Sobrinko, L. G., (1971). C'lin. Chem. 17, 931-935. 7. Sadayuki, K., Sansone, A. and Hreschyshyn, M. M., (1971). Am~r. $. Obstst. Crynecol. 109, 352-957. 8. Wide, L., (1969). Aet~ Hndo~r. Suppl. 142, 207-221. 9. Hales, C. N., and Randle, P. J., (1963). BiGh~a J. 88, 137-146. 10. Greenwood, F. C., Hunter. W. M., and Glover, J. S., (1963). Bioehem. J. 89, 114-10~. 11. Scatchard, G., (1949). Ann. N Y Ae~d. Sd. ~1, 660-672. 12. Rodbard, D., Ruder, H. J., Vaitukaitis, J., and Jaeobs, H. S., (1971). J. Cl/n. Hndo~.-r. 33, 343-355. 18. Ratcliffe, J. G., (1974). Br. M~t. Bull., 30, 32~7.
