Journal o f Immunological Methods, 31 (1979) 231--236 © Elsevier/North-Holland Biomedical Press
231
T H E R M A L C H A R A C T E R I S T I C S O F M I C R O T I T R E P L A T E S USED IN IMMUNOLOGICAL ASSAYS
SUSANNAH M. BURT a, TIMOTHY J.N. CARTER b,* and LARRY J. KRICKA a a Department of Clinical Chemistry, University of Birmingham, Birmingham B15 2TH, and b Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham B15 2TH, U.K.
(Received 9 July 1979, accepted 1 August 1979)
Thermal studies of various types of microtitre plate have shown that wells at the edges of a plate have different thermal characteristics from those in the centre. This may contribute to the 'edge effect' observed when such plates are used as solid supports for protein immobilisation in immunoassays.
INTRODUCTION Microtitre plates (MTPs) are widely used as solid supports for the immobilisation o f antigens and antibodies in b o t h qualitative and quantitative immunoassays (Voller et al., 1976; Saunders and Bartlett, 1977; MacDonald and Kelly, 1978). Several authors (Denmark and Chessum, 1978a, b; Hallid a y and Wisdom, 1978) have n o t e d variability in the apparent physical adsorption o f p r ot ei n o n t o the inside surface of MTP wells on di fferent parts o f a plate. This ef f ect has been r e p o r t e d to be m o s t p r o n o u n c e d at t he edges o f a plate -- the so~called 'edge effect'. One fact or which m ay c o n t r i b u t e to such effects is inter-well variation in t e m p e r a t u r e during incubation periods. This study describes investigations of t he thermal characteristics of microtitre plates. MATERIALS
Microtitre plates
Examples o f the following 96 well p o l y s t y r e n e plates were used: N u n c l o n (Nunc Co. Ltd., St af f or d ST16 2JU, U.K.), M129A ( D y n a t e c h Labs. Ltd., Billingshurst, Sussex, U.K.) and M24A (Sterilin Ltd., Teddington, Middlesex T W l l 8QT, U.K.).
* To w h o m correspondence should be addressed.
232
Temperature measuring devices (a) The temperature of liquid in MTP wells was measured using a thermistor probe {type 44018, Yellow Springs Inst. Co., Yellow Springs, OH 45387, U.S.A.) connected in a bridge circuit. The o u t p u t was monitored using a Phillips t y p e 8251 potentiometric recorder (Pye Unicam Ltd., York Street, Cambridge, U.K.) and calibrated using a t h e r m o m e t e r which had been accurately standardised by the National Physical Laboratory (Gallenkamp and Co. Ltd., Birmingham B1 3HT, U.K.). (b) In view of possible problems arising from the influence of the thermal capacity of a thermistor on the temperature of fluid in MTP wells during measurements, temperature was also measured indirectly by means of an AGA Thermovision 680 infra-red camera (AGA Aktievolag, Infra-red Instruments, Lidingo, Sweden). The o u t p u t , in the form of a grey scale, was recorded photographically. METHODS
Temperature studies The wells of a microtitre plate (Nunclon) were filled with distilled water (250 pl) using a Micromedic automatic dispenser. The plate was then covered with a plastic lid and incubated at 37°C for 3 h. After removal from the oven, temperature measurements were made during a 15 min cooling period in various inner and outer wells. In other experiments, wells of various types of MTP, equilibrated to ambient temperature for at least 2 h, were each filled with 250 pl of distilled
23.
o~
u~ oc
22.
o.. u.a
21 A
B
C
D ROW
F
E 1
G
I
H
A
B
C
D ROW
E
F
G
H
2
Fig. 1. Cooling studies on a microtitre plate. Temperature profile of t w o adjacent c o l u m n s of wells (A1--H1) and (A2--H2) on a microtitre plate incubated at 37°C and t h e n allowed to cool at ambient temperature (20°C) for 15 rain.
19.71 19.45 19.37 19.25 19.21 19.17 19.21 19.34
19.44 19.18 19.07 18.96 18.90 18.85 18.89 19.12
2
19.32 19.04 18.91 18.80 18.77 18.72 18.76 18.97
3 19.23 19.86 18.83 18.72 18.69 18.66 18.71 18.94
4 19.20 18.91 18.76 18.68 18.66 18.66 18.69 18.92
5 19.16 18.87 18.73 18.67 18.64 18.65 18.69 18.96
6 19.14 18.84 18.67 18.64 18.60 18.62 19.67 18.93
7 19.11 18.83 18.67 18.62 18.59 18.63 18.65 18.91
8 19.12 18.84 18.68 18.64 18.59 18.63 18.67 18.94
9 19.20 18.86 18.72 18.68 18.65 18.65 18.71 19.00
10 19.28 18.96 18.84 18.79 18.78 18.80 18.84 19.17
11
19.50 19.27 19.12 19.10 19.11 19.15 19.18 19.42
12
Fig. 2. T h e r m a l profile (°C) of a m i c r o t i t r e plate following i n c u b a t i o n at a m b i e n t t e m p e r a t u r e (21.6°C). Each well was filled w i t h 250 gl distilled water.
A B C D E F G H
1
b~ 50
234 w a t e r , also a t a m b i e n t t e m p e r a t u r e . T h e s e MTPs were t h e n left either c o v e r e d o r u n c o v e r e d at a m b i e n t t e m p e r a t u r e in a m a t t - b l a c k d r a u g h t - s h i e l d o n an o p e n l a b o r a t o r y b e n c h . T e m p e r a t u r e m e a s u r e m e n t s in t h e wells were t a k e n , b o t h d i r e c t l y and using an infra-red c a m e r a , o v e r a p e r i o d o f 2.5 h. F o r c o m p a r i s o n , e x a m p l e s o f t h e s a m e t y p e s o f MTP w e r e also left u n d e r t h e s a m e c o n d i t i o n s e i t h e r w i t h all wells e m p t y or w i t h o n l y selected wells filled. RESULTS A c o o l i n g s t u d y o n a p l a t e i n c u b a t e d at 3 7 ° C revealed a d i s t i n c t t h e r m a l p r o f i l e a l o n g a d j a c e n t c o l u m n s . Wells at t h e end o f a c o l u m n c o o l e d f a s t e r t h a n t h o s e in t h e c e n t r e . G e n e r a l l y t h e t e m p e r a t u r e o f wells in o u t e r r o w s was l o w e r t h a n t h e t e m p e r a t u r e o f i n n e r wells (Fig. 1). T h e t e m p e r a t u r e s o f fluids in wells in a d j a c e n t c o l u m n s o f a m i c r o t i t r e p l a t e i n c u b a t e d at a m b i e n t t e m p e r a t u r e w e r e highest at t h e edges, a n d t h e o u t e r c o l u m n was at a higher t e m p e r a t u r e t h a n a d j a c e n t i n t e r i o r c o l u m n s . T h e t e m p e r a t u r e p r o f i l e across a p l a t e , w h e n e a c h well was filled w i t h 2 5 0 pl o f distilled w a t e r , is s h o w n in Fig. 2.
Fig. 3. Infra-red photograph of a microtitre plate following incubation at ambient temperature (21.1°C) for 2 h. Each well was filled with 250 pl distilled water. Tempera. ture scale: transition from white (cold) to black (hot) represents 2 C. ,
.
O
235
Fig. 4. Infra-red photograph of a partially filled microtitre plate following incubation at ambient temperature (21.1°C) overnight. Wells in rows D, G and H and columns 1, 2 and 7 were filled with 250 pl distilled water. Temperature scale: transition from white (cold) to black (hot) represents 2°C.
G e n e r a l l y , t h e t e m p e r a t u r e o f fluid in t h e wells o f a p l a t e i n c u b a t e d at a m b i e n t t e m p e r a t u r e was several degrees b e l o w a m b i e n t . In an e m p t y or p a r t i a l l y filled p l a t e e m p t y wells w e r e at a m b i e n t t e m p e r a t u r e w h e r e a s filled wells w e r e several degrees b e l o w a m b i e n t . T h e results o f infra-red p h o t o g r a p h y o f a filled a n d p a r t i a l l y filled M T P are s h o w n in Figs. 3 a n d 4. A m a r k e d edge e f f e c t was n o t e d f o r all t y p e s o f p l a t e t e s t e d , t h e t e m p e r a t u r e at t h e edge, a n d in p a r t i c u l a r t h e c o m e r o f a plate, was v e r y m u c h higher t h a n t h e c e n t r e f o l l o w i n g i n c u b a t i o n at r o o m temperature for either 2 h or overnight. Covering of a plate eliminated the t e m p e r a t u r e v a r i a t i o n across t h e p l a t e b u t it was re-established if t h e p l a t e w a s l e f t u n c o v e r e d f o r m o r e t h a n 30 rain. H a n d l i n g o f an e q u i l i b r a t e d p l a t e c a u s e d a p r o n o u n c e d d i s t u r b a n c e in t h e t e m p e r a t u r e o f t h e p l a t e a n d t h e t h e r m a l r e c o r d o f a n y h a n d l i n g r e m a i n e d f o r u p t o 10 m i n . DISCUSSION This s t u d y has d e m o n s t r a t e d t h a t wells at t h e edges o f a m i c r o t i t r e p l a t e h a v e d i f f e r e n t t h e r m a l c h a r a c t e r i s t i c s f r o m o t h e r wells o n t h e plate. F o r
236 uncovered plates incubated under ambient conditions, the temperature of fluid in wells was generally lower than ambient. Moreover, fluid in wells at the edges of a plate was warmer than t h a t in wells in the interior of the plate. Other workers have noted this p h e n o m e n o n {Denmark and Chessum, 1978a), but the reason for the temperature gradient remains obscure. It is unlikely, for example, to be due to evaporational cooling since this would be expected to cool the outer wells more efficiently than the inner. It is our opinion that these temperature gradients are due to thermal exchange with the environment. We have, for example, shown that plates heated above ambient temperature tend to cool preferentially at the edges. The reverse effect might occur when plates are initially below ambient temperature. Why this effect is abolished by covering the plate, however, is difficult to reconcile with this hypothesis. Microtitre plates are c o m m o n l y employed for ELISA type assays and the inter-well variation in temperature demonstrated in this study may influence two stages of such assay. Firstly, the coating of the inside surface of wells with antigen or antibody and secondly, the colorimetric assay for bound enzyme-labelled antigen or antibody. An enhanced enzymatic rate of reaction may account in part for the so-called 'edge effect' observed in assays carried o u t on microtitre plates. ACKNOWLEDGEMENT We would like to acknowledge Dr. M. Marquis, Materials Science Department, University of Birmingham, for assistance with the thermal photography. REFERENCES
Denmark, J.R. and B.S. Chessum, 1978a, Med. Lab. Sci. 35,227. Denmark, J.R. and B.S. Chessum, 1978b, Lancet i, 161. Halliday, M.I. and G.B. Wisdom, 1978, FEBS Lett. 96,298. MacDonald, D.J. and A.M. Kelly, 1978, Clin. Chim. Acta 87,367. Saunders, G.C. and M.L. Bartlett, 1977, Appl. Environ. Microbiol. 34,518. Voller, A., D.E. Bidwell and A. Bartlett, 1976, Bull. W.H.O. 53, 55.
231
T H E R M A L C H A R A C T E R I S T I C S O F M I C R O T I T R E P L A T E S USED IN IMMUNOLOGICAL ASSAYS
SUSANNAH M. BURT a, TIMOTHY J.N. CARTER b,* and LARRY J. KRICKA a a Department of Clinical Chemistry, University of Birmingham, Birmingham B15 2TH, and b Department of Clinical Chemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Birmingham B15 2TH, U.K.
(Received 9 July 1979, accepted 1 August 1979)
Thermal studies of various types of microtitre plate have shown that wells at the edges of a plate have different thermal characteristics from those in the centre. This may contribute to the 'edge effect' observed when such plates are used as solid supports for protein immobilisation in immunoassays.
INTRODUCTION Microtitre plates (MTPs) are widely used as solid supports for the immobilisation o f antigens and antibodies in b o t h qualitative and quantitative immunoassays (Voller et al., 1976; Saunders and Bartlett, 1977; MacDonald and Kelly, 1978). Several authors (Denmark and Chessum, 1978a, b; Hallid a y and Wisdom, 1978) have n o t e d variability in the apparent physical adsorption o f p r ot ei n o n t o the inside surface of MTP wells on di fferent parts o f a plate. This ef f ect has been r e p o r t e d to be m o s t p r o n o u n c e d at t he edges o f a plate -- the so~called 'edge effect'. One fact or which m ay c o n t r i b u t e to such effects is inter-well variation in t e m p e r a t u r e during incubation periods. This study describes investigations of t he thermal characteristics of microtitre plates. MATERIALS
Microtitre plates
Examples o f the following 96 well p o l y s t y r e n e plates were used: N u n c l o n (Nunc Co. Ltd., St af f or d ST16 2JU, U.K.), M129A ( D y n a t e c h Labs. Ltd., Billingshurst, Sussex, U.K.) and M24A (Sterilin Ltd., Teddington, Middlesex T W l l 8QT, U.K.).
* To w h o m correspondence should be addressed.
232
Temperature measuring devices (a) The temperature of liquid in MTP wells was measured using a thermistor probe {type 44018, Yellow Springs Inst. Co., Yellow Springs, OH 45387, U.S.A.) connected in a bridge circuit. The o u t p u t was monitored using a Phillips t y p e 8251 potentiometric recorder (Pye Unicam Ltd., York Street, Cambridge, U.K.) and calibrated using a t h e r m o m e t e r which had been accurately standardised by the National Physical Laboratory (Gallenkamp and Co. Ltd., Birmingham B1 3HT, U.K.). (b) In view of possible problems arising from the influence of the thermal capacity of a thermistor on the temperature of fluid in MTP wells during measurements, temperature was also measured indirectly by means of an AGA Thermovision 680 infra-red camera (AGA Aktievolag, Infra-red Instruments, Lidingo, Sweden). The o u t p u t , in the form of a grey scale, was recorded photographically. METHODS
Temperature studies The wells of a microtitre plate (Nunclon) were filled with distilled water (250 pl) using a Micromedic automatic dispenser. The plate was then covered with a plastic lid and incubated at 37°C for 3 h. After removal from the oven, temperature measurements were made during a 15 min cooling period in various inner and outer wells. In other experiments, wells of various types of MTP, equilibrated to ambient temperature for at least 2 h, were each filled with 250 pl of distilled
23.
o~
u~ oc
22.
o.. u.a
21 A
B
C
D ROW
F
E 1
G
I
H
A
B
C
D ROW
E
F
G
H
2
Fig. 1. Cooling studies on a microtitre plate. Temperature profile of t w o adjacent c o l u m n s of wells (A1--H1) and (A2--H2) on a microtitre plate incubated at 37°C and t h e n allowed to cool at ambient temperature (20°C) for 15 rain.
19.71 19.45 19.37 19.25 19.21 19.17 19.21 19.34
19.44 19.18 19.07 18.96 18.90 18.85 18.89 19.12
2
19.32 19.04 18.91 18.80 18.77 18.72 18.76 18.97
3 19.23 19.86 18.83 18.72 18.69 18.66 18.71 18.94
4 19.20 18.91 18.76 18.68 18.66 18.66 18.69 18.92
5 19.16 18.87 18.73 18.67 18.64 18.65 18.69 18.96
6 19.14 18.84 18.67 18.64 18.60 18.62 19.67 18.93
7 19.11 18.83 18.67 18.62 18.59 18.63 18.65 18.91
8 19.12 18.84 18.68 18.64 18.59 18.63 18.67 18.94
9 19.20 18.86 18.72 18.68 18.65 18.65 18.71 19.00
10 19.28 18.96 18.84 18.79 18.78 18.80 18.84 19.17
11
19.50 19.27 19.12 19.10 19.11 19.15 19.18 19.42
12
Fig. 2. T h e r m a l profile (°C) of a m i c r o t i t r e plate following i n c u b a t i o n at a m b i e n t t e m p e r a t u r e (21.6°C). Each well was filled w i t h 250 gl distilled water.
A B C D E F G H
1
b~ 50
234 w a t e r , also a t a m b i e n t t e m p e r a t u r e . T h e s e MTPs were t h e n left either c o v e r e d o r u n c o v e r e d at a m b i e n t t e m p e r a t u r e in a m a t t - b l a c k d r a u g h t - s h i e l d o n an o p e n l a b o r a t o r y b e n c h . T e m p e r a t u r e m e a s u r e m e n t s in t h e wells were t a k e n , b o t h d i r e c t l y and using an infra-red c a m e r a , o v e r a p e r i o d o f 2.5 h. F o r c o m p a r i s o n , e x a m p l e s o f t h e s a m e t y p e s o f MTP w e r e also left u n d e r t h e s a m e c o n d i t i o n s e i t h e r w i t h all wells e m p t y or w i t h o n l y selected wells filled. RESULTS A c o o l i n g s t u d y o n a p l a t e i n c u b a t e d at 3 7 ° C revealed a d i s t i n c t t h e r m a l p r o f i l e a l o n g a d j a c e n t c o l u m n s . Wells at t h e end o f a c o l u m n c o o l e d f a s t e r t h a n t h o s e in t h e c e n t r e . G e n e r a l l y t h e t e m p e r a t u r e o f wells in o u t e r r o w s was l o w e r t h a n t h e t e m p e r a t u r e o f i n n e r wells (Fig. 1). T h e t e m p e r a t u r e s o f fluids in wells in a d j a c e n t c o l u m n s o f a m i c r o t i t r e p l a t e i n c u b a t e d at a m b i e n t t e m p e r a t u r e w e r e highest at t h e edges, a n d t h e o u t e r c o l u m n was at a higher t e m p e r a t u r e t h a n a d j a c e n t i n t e r i o r c o l u m n s . T h e t e m p e r a t u r e p r o f i l e across a p l a t e , w h e n e a c h well was filled w i t h 2 5 0 pl o f distilled w a t e r , is s h o w n in Fig. 2.
Fig. 3. Infra-red photograph of a microtitre plate following incubation at ambient temperature (21.1°C) for 2 h. Each well was filled with 250 pl distilled water. Tempera. ture scale: transition from white (cold) to black (hot) represents 2 C. ,
.
O
235
Fig. 4. Infra-red photograph of a partially filled microtitre plate following incubation at ambient temperature (21.1°C) overnight. Wells in rows D, G and H and columns 1, 2 and 7 were filled with 250 pl distilled water. Temperature scale: transition from white (cold) to black (hot) represents 2°C.
G e n e r a l l y , t h e t e m p e r a t u r e o f fluid in t h e wells o f a p l a t e i n c u b a t e d at a m b i e n t t e m p e r a t u r e was several degrees b e l o w a m b i e n t . In an e m p t y or p a r t i a l l y filled p l a t e e m p t y wells w e r e at a m b i e n t t e m p e r a t u r e w h e r e a s filled wells w e r e several degrees b e l o w a m b i e n t . T h e results o f infra-red p h o t o g r a p h y o f a filled a n d p a r t i a l l y filled M T P are s h o w n in Figs. 3 a n d 4. A m a r k e d edge e f f e c t was n o t e d f o r all t y p e s o f p l a t e t e s t e d , t h e t e m p e r a t u r e at t h e edge, a n d in p a r t i c u l a r t h e c o m e r o f a plate, was v e r y m u c h higher t h a n t h e c e n t r e f o l l o w i n g i n c u b a t i o n at r o o m temperature for either 2 h or overnight. Covering of a plate eliminated the t e m p e r a t u r e v a r i a t i o n across t h e p l a t e b u t it was re-established if t h e p l a t e w a s l e f t u n c o v e r e d f o r m o r e t h a n 30 rain. H a n d l i n g o f an e q u i l i b r a t e d p l a t e c a u s e d a p r o n o u n c e d d i s t u r b a n c e in t h e t e m p e r a t u r e o f t h e p l a t e a n d t h e t h e r m a l r e c o r d o f a n y h a n d l i n g r e m a i n e d f o r u p t o 10 m i n . DISCUSSION This s t u d y has d e m o n s t r a t e d t h a t wells at t h e edges o f a m i c r o t i t r e p l a t e h a v e d i f f e r e n t t h e r m a l c h a r a c t e r i s t i c s f r o m o t h e r wells o n t h e plate. F o r
236 uncovered plates incubated under ambient conditions, the temperature of fluid in wells was generally lower than ambient. Moreover, fluid in wells at the edges of a plate was warmer than t h a t in wells in the interior of the plate. Other workers have noted this p h e n o m e n o n {Denmark and Chessum, 1978a), but the reason for the temperature gradient remains obscure. It is unlikely, for example, to be due to evaporational cooling since this would be expected to cool the outer wells more efficiently than the inner. It is our opinion that these temperature gradients are due to thermal exchange with the environment. We have, for example, shown that plates heated above ambient temperature tend to cool preferentially at the edges. The reverse effect might occur when plates are initially below ambient temperature. Why this effect is abolished by covering the plate, however, is difficult to reconcile with this hypothesis. Microtitre plates are c o m m o n l y employed for ELISA type assays and the inter-well variation in temperature demonstrated in this study may influence two stages of such assay. Firstly, the coating of the inside surface of wells with antigen or antibody and secondly, the colorimetric assay for bound enzyme-labelled antigen or antibody. An enhanced enzymatic rate of reaction may account in part for the so-called 'edge effect' observed in assays carried o u t on microtitre plates. ACKNOWLEDGEMENT We would like to acknowledge Dr. M. Marquis, Materials Science Department, University of Birmingham, for assistance with the thermal photography. REFERENCES
Denmark, J.R. and B.S. Chessum, 1978a, Med. Lab. Sci. 35,227. Denmark, J.R. and B.S. Chessum, 1978b, Lancet i, 161. Halliday, M.I. and G.B. Wisdom, 1978, FEBS Lett. 96,298. MacDonald, D.J. and A.M. Kelly, 1978, Clin. Chim. Acta 87,367. Saunders, G.C. and M.L. Bartlett, 1977, Appl. Environ. Microbiol. 34,518. Voller, A., D.E. Bidwell and A. Bartlett, 1976, Bull. W.H.O. 53, 55.
