Journal of Immunological Methods, 16 (1977) 351--359
351
© Elsevier/North-Holland Biomedical Press
MECHANIZATION OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR LARGE SCALE SCREENING OF SERA
E.J. RUITENBERG, J.A. van AMSTEL, B.J.M. BROSI and P.A. STEERENBERG National Institute of Public Health, P.O. Box 1, Bilthoven, The Netherlands
(Received 24 January 1977, accepted 12 February 1977 ) An on-line routing system (including dispensers, washing device, spectrophotometer, carts, elevators and identification device) for macro-ELISA is described. The system enables processing of 4,000 sera daily by 2 persons. Test results are presented on a data sheet as a combination of indentification number and extinction value.
INTRODUCTION T h e e n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) has been successfully a d a p t e d to screening o f p o r c i n e sera for the d e t e c t i o n o f a n t i b o d i e s to Trichinella spiralis ( R u i t e n b e r g et al., 1974). The assay can be p e r f o r m e d in polys t y r e n e t u b e s as antigen-carriers ( m a c r o - E L I S A ) or in p o l y s t y r e n e microplates c o n t a i n i n g 96 wells ( m i c r o - E L I S A ) ( R u i t e n b e r g et al., 1 9 7 5 ) . One o f t h e advantages o f E L I S A is its p o t e n t i a l for large scale screening, since m e c h a n i z a t i o n o f practically all steps o f t h e assay is feasible. Details on the assay itself and the a c c u r a c y o f the e q u i p m e n t have been published previously ( R u i t e n b e r g et al., 1976). In the p r e s e n t r e p o r t we describe the d e v e l o p m e n t o f an on-line m a c r o - E L I S A system for screening o f 4 , 0 0 0 sera daily. MATERIALS AND METHODS ELISA
In o r d e r to explain the on-line s y s t e m a brief description o f the e n z y m e i m m u n o a s s a y d e v e l o p e d f o r d e t e c t i o n o f antibodies t o T. spiralis in p o r c i n e sera is given. T h e p r o c e d u r e is described in detail elsewhere ( R u i t e n b e r g et al., 1976). Disposable p o l y s t y r e n e t u b e s (50 b y 11 m m , L K B - p r o d u k t e r AB, S t o c k h o l m , S w e d e n ) were c o a t e d with antigen (a c r u d e saline e x t r a c t o f T. spiralis muscle larvae) in the f o l l o w i n g w a y : one ml o f t h e antigen s o l u t i o n in s o d i u m c a r b o n a t e b u f f e r (0.1 M; p H 9.6) with NaN3 (0.02%) was added to each tube. T h e t u b e s were i n c u b a t e d at 37°C for 30 min and d i r e c t l y used f o r the assay w i t h o u t f u r t h e r storage. In a 1-min r u n t u b e s were flushed with
352 120 ml t a p w a t e r c o n t a i n i n g 0.05% T w e e n 20. Sera w e r e e x a m i n e d at a 1 : 10 dilution. F o r this p u r p o s e a n t i g e n - c o a t e d t u b e s a f t e r having b e e n w a s h e d were first filled w i t h 0.9 ml o f the d i l u e n t fluid ( p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 0.5% b o v i n e s e r u m a l b u m i n (BSA) a n d 0 . 0 5 % T w e e n 20) a n d n e x t , with 0.1 ml s e r u m . Prior to i n c u b a t i o n t u b e s w e r e s h a k e n f o r 15 sec. S e r u m i n c u b a t i o n was p e r f o r m e d ( w i t h o u t f u r t h e r r o t a t i o n ) at 3 7 ° C f o r 30 min. A f t e r washing ( 1 2 0 ml p e r t u b e ) 1 ml o f the c o n j u g a t e - - h o r s e radish p e r o x i d a s e c o n j u g a t e d to r a b b i t anti p o r c i n e IgG (H + L) - - o p t i m a l l y d i l u t e d in a 4% BSA s o l u t i o n c o n t a i n i n g 0.05% T w e e n 20 was a d d e d . A f t e r shaking for 15 sec t u b e s w e r e i n c u b a t e d ( w i t h o u t f u r t h e r r o t a t i o n ) at 3 7 ° C for 30 min. Excess c o n j u g a t e was r e m o v e d b y washing ( 1 2 0 ml per t u b e ) . Finally, the a m o u n t o f e n z y m e b o u n d to t h e t u b e s was d e t e r m i n e d b y a d d i n g 1 ml o f the specific s u b s t r a t e (5 a m i n o salicylic acid (5AS) and H202). I m m e d i a t e l y a f t e r i n c u b a t i o n at r o o m t e m p e r a t u r e f o r 1 h t h e t u b e s w e r e s h a k e n for 15 sec and t h e o p t i c a l d e n s i t y (O.D.) o f t h e r e a c t i o n p r o d u c t was m e a s u r e d in a spect r o p h o t o m e t e r at 4 4 9 n m . C o n t r a r y to t h e initially d e s c r i b e d p r o c e d u r e , the r e a c t i o n was n o t stopped with N a O H . T h e r e a s o n f o r this was t h e high speed w i t h w h i c h t h e readings c o u l d be p e r f o r m e d . T h e assays c o u l d be s y n c h r o n i z e d in such a w a y t h a t n o v a r i a t i o n s in O.D. in the individual t u b e s d u e to variable s u b s t r a t e incubation times occurred.
Available equipment S y s t e m Olli 3 0 0 0 (Olli Medical E l e c t r o n i c s , K i v e n l a h t i , F i n l a n d ) consisting of: a) a d i s p e n s e r with 24 d i s p o s a b l e plastic p i p e t t e s each linked to indi-
Fig. 1. Left: block with 3 × 8 polystyrene tubes (working block). Right: block with 3 x 8 polyethylene tubes for transport of samples (sample block) with strips and identification tags.
353 vidual syringes (range o f v o l u m e 2 5 - - 2 0 0 0 pl). b) Working blocks with a c a p a c i t y o f 24 p o l y s t y r e n e t u b e s ( L K B p r o d u k t e r , S t o c k h o l m , Sweden). T u b e s are used for the assay itself and as cuvettes in the s p e c t r o p h o t o m e t e r (Fig. 1). c) A p h o t o m e t e r (Olli 234), which measures 24 separate samples at a speed o f 24 samples/25 sec. d) Processor ( m i n i - c o m p u t e r ) f o r c o n t r o l o f p h o t o m e t e r and d a t a handling.
Alteration of existing equipment and developments of additional equipment In fig. 2 t h e o n 4 i n e m a c r o - E L I S A system is s h o w n . T h e system consists o f
a) Sample blocks Sample blocks with a c a p a c i t y o f 3 × 8 p o l y e t h y l e n e t u b e s (covered with strip per r o w o f 8 tubes) for t r a n s p o r t o f samples b o t h outside and within t h e s y s t e m (fig. 1).
b) Identification device T h e individual sample t u b e s possess an i d e n t i f i c a t i o n tag, the n u m b e r o f w h i c h - - t o g e t h e r with the sample b l o c k n u m b e r -- is stored in the m e m o r y of the c o m p u t e r . T h e n u m b e r o f t h e sample b l o c k is used t o i d e n t i f y t h e c o r r e s p o n d i n g w o r k i n g block. (fig. 2 hr. 4).
c) Dispenser In t h e on-line system the position o f the liquid c o n t a i n e r was changed to
Fig. 2. On-line system for macro-ELISA. 1 = elevator with cart; 2 = washing device; 3 = dispenser; 4 = identification device; 5 = spectrophotometer with processor.
354 p e r m i t h o r i z o n t a l m o v e m e n t o f w o r k i n g b l o c k in the dispenser (fig. 2 nr. 3; fig. 3)
d) Washing device T o facilitate washing p r o c e d u r e s a washing device was d e v e l o p e d enabling individual flushing o f t h e w o r k i n g t u b e s (fig. 2 hr. 2; fig. 4). T h e principle is a c o n t i n u o u s a d m i n i s t r a t i o n a n d r e m o v a l o f flushing liquid t h r o u g h a d o u b l e - w a l l e d t u b e , consisting of an inlet and an o u t l e t . Flushing w i t h a t o t a l v o l u m e o f 120 ml per t u b e in a 1-min r u n is achieved b y 4 sequential u p - and d o w n w a r d m o v e m e n t s . At t h e e n d o f the p r o c e d u r e t h e t u b e s are t o t a l l y d r y . This principle was used to d e v e l o p a washing device enabling flushing o f 4 X 24 t u b e s at t h e s a m e t i m e . T w e e n 20 (0.05%) is a d d e d to t h e flushing fluid b y a p u m p .
Fig. 3. Two dispensers. The right one shows the pipette holder for 24 pipettes and a container for liquids. In the left dispenser the liquid container is raised for filling of pipettes.
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Fig. 4, Washing device with 96 double walled tubes for flushing of 4 X 24 working tubes.
e) Carts and elevators In o r d e r to process a large n u m b e r o f samples in the on-line s y s t e m carts were d e v e l o p e d with a c a p a c i t y o f 10 X 4 sample or w o r k i n g blocks. Carts can be r e m o v e d f r o m the on-line s y s t e m and p u s h e d (on 4 wheels) t o the incubator. F u r t h e r m o r e , carts can be raised in the elevator s y s t e m (fig. 2 nr. 1; fig. 5).
f) Shakers A f t e r adding the d i l u e n t fluid and the serum sample t o the tubes, blocks with t u b e s are shaken o n a specially designed shaker (time-interval is variable) (fig. 6). T h e same shaking device is used for r o t a t i n g the t u b e s a f t e r the substrate i n c u b a t i o n period just p r i o r to m e a s u r e m e n t on the s p e c t r o p h o t o meter.
g) S p e c t r o p h o t o m e t e r and processor T h e w o r k i n g b l o c k with 24 p o l y s t y r e n e t u b e s c o n t a i n i n g the r e a c t i o n p r o d u c t are placed in the c u v e t t e b l o c k holder. Due t o the high optical quality o f the t u b e s the optical d e n s i t y is d i r e c t l y measured. Prior to measuring the b l o c k i d e n t i f i c a t i o n n u m b e r is i n t r o d u c e d via a t h u m b - w h e e l switch to the c o m p u t e r . Results are p r e s e n t e d o n a d a t a sheet as a c o m b i n a t i o n o f t w o figures: t h e sample n u m b e r (i.e. t h e n u m b e r o f the i d e n t i f i c a t i o n tag f r o m t h e sample t u b e ) and the e x t i n c t i o n value (the optical d e n s i t y ) o f t h e r e a c t i o n p r o d u c t
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Fig. 5. Cart and elevator. A four-wheeled cart with a capacity of 10 x 4 blocks in elevator.
in t h e w o r k i n g t u b e . T h e s p e c t r o p h o t o m e t e r was a d a p t e d to t h e on-line sys. t e m (fig. 2 nr. 5; fig. 7). T h e t o t a l e q u i p m e n t o c c u p i e s 5.16 X 0.75 m 2 f l o o r space. RESULTS
Routing of macro-ELISA In fig. 8 a b l o c k d i a g r a m o f t h e r o u t i n g o f t h e m a c r o - E L I S A s y s t e m is presented. T h e assay involves t h e f o l l o w i n g steps 1) C o a t i n g o f t h e / J o l y s t y r e n e t u b e s w i t h antigen
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Fig. 6. T w o shakers are s h o w n n e x t to t h e dispensers.
Fig. 7. S p e c t r o p h o t o m e t e r w i t h s h a k e r a n d p r i n t e r .
358 BLOCKDIAGRAM OF ROUTING FOR ELISA 1
2
3
4
8
5
9
6
10
11
12
Fig. 8. Blockdiagram of routing for macro-ELISA (see text). Cart with working blocks and tubes (cart A) from 12 (stc~age) -~ 3 (elevator) -~ 4 (dispenser, antigen in liquid container) -~ 5 (elevator) ~ 11 (inc u b a t o r at 37 ° C). 2) Addition o f diluent to working tubes after washing Cart A with working blocks and tubes from 11 (incubator)-~ 1 (elevator) -~ 2 (washing device) -* 3 (elevator)-~ 4 (dispenser, diluent in liquid container) -~ 5 (elevator) -~ 10 (parking at r o o m temperature). 3) Addition o f serum sample to working tubes containing diluent followed by serum incubation a) Cart with sample blocks and tubes (cart B) from 8 (storage at 4°C)-~ 9 (identification device)-* 3 (elevator)-~ 4 (dispenser, serum sample taken from sample tube remains in pipette until step 3 b) -~ 3 (elevator)-+ 8 (storage at 4 ° C). b) Cart A with working blocks and tubes containing diluent from 10 (parking) -~ 5 (elevator) -~ 4 (dispenser, serum sample from pipette added to working tube) -+ 5 (elevator) -~ 11 (incubator at 37°C). N.B. Since two dispensers are used, serum samples are transferred from 2 sample blocks to the corresponding 2 working blocks at the same time. Pipettes are changed after transfer of serum samples to working tubes in order to prevent contamination. 4) Addition of conjugate to working tubes after washing followed by conjugate incubation Cart A with working blocks and tubes containing the serum dilution from 11 (incubator at 37°C) ~ 1 (elevator) ~ 2 (washing device) ~ 3 (elevator) -~ 4 (dispenser, conjugate in liquid container) -~ 5 (elevator) -~ 11 (incubator at 37°C). 5) Addition o f substrate to working tubes after washing followed by substrate incubation Cart A with working blocks and tubes containing conjugate from 11 (incubator) -~ 1 (elevator) -~ 2 (washing device) -~ 3 (elevator) -~ 4 (dispenser, substrate in liquid container) -~ 5 (elevator) -~ 10 (parking at r o o m temperature). 6) Measurement of optical density of reaction p r o d u c t in working tubes
359
Cart A with w o r k i n g blocks and t u b e s c o n t a i n i n g substrate r e a c t i o n p r o d u c t f r o m 10 (parking) -~ 5 (elevator) -~ 6 ( p h o t o m e t e r ) -~ 7 (elevator). DISCUSSION AND CONCLUSIONS
With the d e s c r i b e d on-line s y s t e m f o r m a c r o - E L I S A a n u m b e r o f 4 , 0 0 0 sera can be processed daily b y 2 persons. Due to the a c c u r a c y o f b o t h the dispensers and the s p e c t r o p h o t o m e t e r highly r e p r o d u c i b l e results were o b t a i n e d as was described earlier ( R u i t e n b e r g et al., 1976). An i m p o r t a n t advantage is t h e p r e s e n t a t i o n o f the test results on a data-sheet as a c o m b i n a t i o n o f i d e n t i f i c a t i o n n u m b e r and e x t i n c t i o n value. E x t i n c t i o n values can be p r e s e n t e d as m e a s u r e d against blank values (i.e. substrate c o n t r o l ) or a f t e r s u b t r a c t i o n o f a previously d e t e r m i n e d c o n f i d e n c e limit. REFERENCES Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J. Buys, 1974, Bull. Wld. Hlth. Org. 51, 108. Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J. Buys, 1976, J. Immunol. Methods 10, 67. Ruitenberg, E.J., P.A. Steerenberg and B.J.M. Brosi, 1975, Medikon Nederland 4, 30.
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© Elsevier/North-Holland Biomedical Press
MECHANIZATION OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR LARGE SCALE SCREENING OF SERA
E.J. RUITENBERG, J.A. van AMSTEL, B.J.M. BROSI and P.A. STEERENBERG National Institute of Public Health, P.O. Box 1, Bilthoven, The Netherlands
(Received 24 January 1977, accepted 12 February 1977 ) An on-line routing system (including dispensers, washing device, spectrophotometer, carts, elevators and identification device) for macro-ELISA is described. The system enables processing of 4,000 sera daily by 2 persons. Test results are presented on a data sheet as a combination of indentification number and extinction value.
INTRODUCTION T h e e n z y m e - l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) has been successfully a d a p t e d to screening o f p o r c i n e sera for the d e t e c t i o n o f a n t i b o d i e s to Trichinella spiralis ( R u i t e n b e r g et al., 1974). The assay can be p e r f o r m e d in polys t y r e n e t u b e s as antigen-carriers ( m a c r o - E L I S A ) or in p o l y s t y r e n e microplates c o n t a i n i n g 96 wells ( m i c r o - E L I S A ) ( R u i t e n b e r g et al., 1 9 7 5 ) . One o f t h e advantages o f E L I S A is its p o t e n t i a l for large scale screening, since m e c h a n i z a t i o n o f practically all steps o f t h e assay is feasible. Details on the assay itself and the a c c u r a c y o f the e q u i p m e n t have been published previously ( R u i t e n b e r g et al., 1976). In the p r e s e n t r e p o r t we describe the d e v e l o p m e n t o f an on-line m a c r o - E L I S A system for screening o f 4 , 0 0 0 sera daily. MATERIALS AND METHODS ELISA
In o r d e r to explain the on-line s y s t e m a brief description o f the e n z y m e i m m u n o a s s a y d e v e l o p e d f o r d e t e c t i o n o f antibodies t o T. spiralis in p o r c i n e sera is given. T h e p r o c e d u r e is described in detail elsewhere ( R u i t e n b e r g et al., 1976). Disposable p o l y s t y r e n e t u b e s (50 b y 11 m m , L K B - p r o d u k t e r AB, S t o c k h o l m , S w e d e n ) were c o a t e d with antigen (a c r u d e saline e x t r a c t o f T. spiralis muscle larvae) in the f o l l o w i n g w a y : one ml o f t h e antigen s o l u t i o n in s o d i u m c a r b o n a t e b u f f e r (0.1 M; p H 9.6) with NaN3 (0.02%) was added to each tube. T h e t u b e s were i n c u b a t e d at 37°C for 30 min and d i r e c t l y used f o r the assay w i t h o u t f u r t h e r storage. In a 1-min r u n t u b e s were flushed with
352 120 ml t a p w a t e r c o n t a i n i n g 0.05% T w e e n 20. Sera w e r e e x a m i n e d at a 1 : 10 dilution. F o r this p u r p o s e a n t i g e n - c o a t e d t u b e s a f t e r having b e e n w a s h e d were first filled w i t h 0.9 ml o f the d i l u e n t fluid ( p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 0.5% b o v i n e s e r u m a l b u m i n (BSA) a n d 0 . 0 5 % T w e e n 20) a n d n e x t , with 0.1 ml s e r u m . Prior to i n c u b a t i o n t u b e s w e r e s h a k e n f o r 15 sec. S e r u m i n c u b a t i o n was p e r f o r m e d ( w i t h o u t f u r t h e r r o t a t i o n ) at 3 7 ° C f o r 30 min. A f t e r washing ( 1 2 0 ml p e r t u b e ) 1 ml o f the c o n j u g a t e - - h o r s e radish p e r o x i d a s e c o n j u g a t e d to r a b b i t anti p o r c i n e IgG (H + L) - - o p t i m a l l y d i l u t e d in a 4% BSA s o l u t i o n c o n t a i n i n g 0.05% T w e e n 20 was a d d e d . A f t e r shaking for 15 sec t u b e s w e r e i n c u b a t e d ( w i t h o u t f u r t h e r r o t a t i o n ) at 3 7 ° C for 30 min. Excess c o n j u g a t e was r e m o v e d b y washing ( 1 2 0 ml per t u b e ) . Finally, the a m o u n t o f e n z y m e b o u n d to t h e t u b e s was d e t e r m i n e d b y a d d i n g 1 ml o f the specific s u b s t r a t e (5 a m i n o salicylic acid (5AS) and H202). I m m e d i a t e l y a f t e r i n c u b a t i o n at r o o m t e m p e r a t u r e f o r 1 h t h e t u b e s w e r e s h a k e n for 15 sec and t h e o p t i c a l d e n s i t y (O.D.) o f t h e r e a c t i o n p r o d u c t was m e a s u r e d in a spect r o p h o t o m e t e r at 4 4 9 n m . C o n t r a r y to t h e initially d e s c r i b e d p r o c e d u r e , the r e a c t i o n was n o t stopped with N a O H . T h e r e a s o n f o r this was t h e high speed w i t h w h i c h t h e readings c o u l d be p e r f o r m e d . T h e assays c o u l d be s y n c h r o n i z e d in such a w a y t h a t n o v a r i a t i o n s in O.D. in the individual t u b e s d u e to variable s u b s t r a t e incubation times occurred.
Available equipment S y s t e m Olli 3 0 0 0 (Olli Medical E l e c t r o n i c s , K i v e n l a h t i , F i n l a n d ) consisting of: a) a d i s p e n s e r with 24 d i s p o s a b l e plastic p i p e t t e s each linked to indi-
Fig. 1. Left: block with 3 × 8 polystyrene tubes (working block). Right: block with 3 x 8 polyethylene tubes for transport of samples (sample block) with strips and identification tags.
353 vidual syringes (range o f v o l u m e 2 5 - - 2 0 0 0 pl). b) Working blocks with a c a p a c i t y o f 24 p o l y s t y r e n e t u b e s ( L K B p r o d u k t e r , S t o c k h o l m , Sweden). T u b e s are used for the assay itself and as cuvettes in the s p e c t r o p h o t o m e t e r (Fig. 1). c) A p h o t o m e t e r (Olli 234), which measures 24 separate samples at a speed o f 24 samples/25 sec. d) Processor ( m i n i - c o m p u t e r ) f o r c o n t r o l o f p h o t o m e t e r and d a t a handling.
Alteration of existing equipment and developments of additional equipment In fig. 2 t h e o n 4 i n e m a c r o - E L I S A system is s h o w n . T h e system consists o f
a) Sample blocks Sample blocks with a c a p a c i t y o f 3 × 8 p o l y e t h y l e n e t u b e s (covered with strip per r o w o f 8 tubes) for t r a n s p o r t o f samples b o t h outside and within t h e s y s t e m (fig. 1).
b) Identification device T h e individual sample t u b e s possess an i d e n t i f i c a t i o n tag, the n u m b e r o f w h i c h - - t o g e t h e r with the sample b l o c k n u m b e r -- is stored in the m e m o r y of the c o m p u t e r . T h e n u m b e r o f t h e sample b l o c k is used t o i d e n t i f y t h e c o r r e s p o n d i n g w o r k i n g block. (fig. 2 hr. 4).
c) Dispenser In t h e on-line system the position o f the liquid c o n t a i n e r was changed to
Fig. 2. On-line system for macro-ELISA. 1 = elevator with cart; 2 = washing device; 3 = dispenser; 4 = identification device; 5 = spectrophotometer with processor.
354 p e r m i t h o r i z o n t a l m o v e m e n t o f w o r k i n g b l o c k in the dispenser (fig. 2 nr. 3; fig. 3)
d) Washing device T o facilitate washing p r o c e d u r e s a washing device was d e v e l o p e d enabling individual flushing o f t h e w o r k i n g t u b e s (fig. 2 hr. 2; fig. 4). T h e principle is a c o n t i n u o u s a d m i n i s t r a t i o n a n d r e m o v a l o f flushing liquid t h r o u g h a d o u b l e - w a l l e d t u b e , consisting of an inlet and an o u t l e t . Flushing w i t h a t o t a l v o l u m e o f 120 ml per t u b e in a 1-min r u n is achieved b y 4 sequential u p - and d o w n w a r d m o v e m e n t s . At t h e e n d o f the p r o c e d u r e t h e t u b e s are t o t a l l y d r y . This principle was used to d e v e l o p a washing device enabling flushing o f 4 X 24 t u b e s at t h e s a m e t i m e . T w e e n 20 (0.05%) is a d d e d to t h e flushing fluid b y a p u m p .
Fig. 3. Two dispensers. The right one shows the pipette holder for 24 pipettes and a container for liquids. In the left dispenser the liquid container is raised for filling of pipettes.
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Fig. 4, Washing device with 96 double walled tubes for flushing of 4 X 24 working tubes.
e) Carts and elevators In o r d e r to process a large n u m b e r o f samples in the on-line s y s t e m carts were d e v e l o p e d with a c a p a c i t y o f 10 X 4 sample or w o r k i n g blocks. Carts can be r e m o v e d f r o m the on-line s y s t e m and p u s h e d (on 4 wheels) t o the incubator. F u r t h e r m o r e , carts can be raised in the elevator s y s t e m (fig. 2 nr. 1; fig. 5).
f) Shakers A f t e r adding the d i l u e n t fluid and the serum sample t o the tubes, blocks with t u b e s are shaken o n a specially designed shaker (time-interval is variable) (fig. 6). T h e same shaking device is used for r o t a t i n g the t u b e s a f t e r the substrate i n c u b a t i o n period just p r i o r to m e a s u r e m e n t on the s p e c t r o p h o t o meter.
g) S p e c t r o p h o t o m e t e r and processor T h e w o r k i n g b l o c k with 24 p o l y s t y r e n e t u b e s c o n t a i n i n g the r e a c t i o n p r o d u c t are placed in the c u v e t t e b l o c k holder. Due t o the high optical quality o f the t u b e s the optical d e n s i t y is d i r e c t l y measured. Prior to measuring the b l o c k i d e n t i f i c a t i o n n u m b e r is i n t r o d u c e d via a t h u m b - w h e e l switch to the c o m p u t e r . Results are p r e s e n t e d o n a d a t a sheet as a c o m b i n a t i o n o f t w o figures: t h e sample n u m b e r (i.e. t h e n u m b e r o f the i d e n t i f i c a t i o n tag f r o m t h e sample t u b e ) and the e x t i n c t i o n value (the optical d e n s i t y ) o f t h e r e a c t i o n p r o d u c t
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Fig. 5. Cart and elevator. A four-wheeled cart with a capacity of 10 x 4 blocks in elevator.
in t h e w o r k i n g t u b e . T h e s p e c t r o p h o t o m e t e r was a d a p t e d to t h e on-line sys. t e m (fig. 2 nr. 5; fig. 7). T h e t o t a l e q u i p m e n t o c c u p i e s 5.16 X 0.75 m 2 f l o o r space. RESULTS
Routing of macro-ELISA In fig. 8 a b l o c k d i a g r a m o f t h e r o u t i n g o f t h e m a c r o - E L I S A s y s t e m is presented. T h e assay involves t h e f o l l o w i n g steps 1) C o a t i n g o f t h e / J o l y s t y r e n e t u b e s w i t h antigen
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Fig. 6. T w o shakers are s h o w n n e x t to t h e dispensers.
Fig. 7. S p e c t r o p h o t o m e t e r w i t h s h a k e r a n d p r i n t e r .
358 BLOCKDIAGRAM OF ROUTING FOR ELISA 1
2
3
4
8
5
9
6
10
11
12
Fig. 8. Blockdiagram of routing for macro-ELISA (see text). Cart with working blocks and tubes (cart A) from 12 (stc~age) -~ 3 (elevator) -~ 4 (dispenser, antigen in liquid container) -~ 5 (elevator) ~ 11 (inc u b a t o r at 37 ° C). 2) Addition o f diluent to working tubes after washing Cart A with working blocks and tubes from 11 (incubator)-~ 1 (elevator) -~ 2 (washing device) -* 3 (elevator)-~ 4 (dispenser, diluent in liquid container) -~ 5 (elevator) -~ 10 (parking at r o o m temperature). 3) Addition o f serum sample to working tubes containing diluent followed by serum incubation a) Cart with sample blocks and tubes (cart B) from 8 (storage at 4°C)-~ 9 (identification device)-* 3 (elevator)-~ 4 (dispenser, serum sample taken from sample tube remains in pipette until step 3 b) -~ 3 (elevator)-+ 8 (storage at 4 ° C). b) Cart A with working blocks and tubes containing diluent from 10 (parking) -~ 5 (elevator) -~ 4 (dispenser, serum sample from pipette added to working tube) -+ 5 (elevator) -~ 11 (incubator at 37°C). N.B. Since two dispensers are used, serum samples are transferred from 2 sample blocks to the corresponding 2 working blocks at the same time. Pipettes are changed after transfer of serum samples to working tubes in order to prevent contamination. 4) Addition of conjugate to working tubes after washing followed by conjugate incubation Cart A with working blocks and tubes containing the serum dilution from 11 (incubator at 37°C) ~ 1 (elevator) ~ 2 (washing device) ~ 3 (elevator) -~ 4 (dispenser, conjugate in liquid container) -~ 5 (elevator) -~ 11 (incubator at 37°C). 5) Addition o f substrate to working tubes after washing followed by substrate incubation Cart A with working blocks and tubes containing conjugate from 11 (incubator) -~ 1 (elevator) -~ 2 (washing device) -~ 3 (elevator) -~ 4 (dispenser, substrate in liquid container) -~ 5 (elevator) -~ 10 (parking at r o o m temperature). 6) Measurement of optical density of reaction p r o d u c t in working tubes
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Cart A with w o r k i n g blocks and t u b e s c o n t a i n i n g substrate r e a c t i o n p r o d u c t f r o m 10 (parking) -~ 5 (elevator) -~ 6 ( p h o t o m e t e r ) -~ 7 (elevator). DISCUSSION AND CONCLUSIONS
With the d e s c r i b e d on-line s y s t e m f o r m a c r o - E L I S A a n u m b e r o f 4 , 0 0 0 sera can be processed daily b y 2 persons. Due to the a c c u r a c y o f b o t h the dispensers and the s p e c t r o p h o t o m e t e r highly r e p r o d u c i b l e results were o b t a i n e d as was described earlier ( R u i t e n b e r g et al., 1976). An i m p o r t a n t advantage is t h e p r e s e n t a t i o n o f the test results on a data-sheet as a c o m b i n a t i o n o f i d e n t i f i c a t i o n n u m b e r and e x t i n c t i o n value. E x t i n c t i o n values can be p r e s e n t e d as m e a s u r e d against blank values (i.e. substrate c o n t r o l ) or a f t e r s u b t r a c t i o n o f a previously d e t e r m i n e d c o n f i d e n c e limit. REFERENCES Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J. Buys, 1974, Bull. Wld. Hlth. Org. 51, 108. Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J. Buys, 1976, J. Immunol. Methods 10, 67. Ruitenberg, E.J., P.A. Steerenberg and B.J.M. Brosi, 1975, Medikon Nederland 4, 30.
