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Enzyme-immunoassay in the detection of hepatitis B surface antigen
Journal oflmmunological Methods, 15 (1977) 343--353 © Elsevier/North-Holland Biomedical Press
343
E N Z Y M E - I M M U N O A S S A Y IN T H E D E T E C T I O N O F H E P A T I T I S B SURFACE ANTIGEN
P. UKKONEN, V. KOISTINEN 1 and K. PENTTINEN
Laboratory of Viral Immunopathology, Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland (Received 25 November 1976, accepted 10 December 1976)
A new enzyme-immunoassay (EIA, Hepanostika Microelisa System) for the detection of hepatitis B surface antigen was evaluated against other methods, namely complement fixation, Hepanosticon, AusRIA II and Finnish Red Cross Radioimmunoassay (FRC-RIA). EIA detected the greatest number of positive samples in a serum panel consisting of 142 sera from clinical hepatitis patients. FRC-RIA was the most sensitive method for subtype ad, while EIA detected the ay specimen at the highest dilution. None of the test systems gave the 'optimal' result in the screening test, and it is proposed that a separate procedure for each antigen subtype should be carried out to detect the greatest number of positive samples.
INTRODUCTION Since the d i s c o v e r y o f hepatitis B surface antigen (HBsAg, Australia antigen), n u m e r o u s m e t h o d s have b e e n d e v e l o p e d for t h e d e m o n s t r a t i o n o f HB,Ag. I m m u n o d i f f u s i o n a n d c o u n t e r i m m u n o e l e c t r o p h o r e s i s have been f o u n d t o o insensitive for diagnostic purposes, especially in the case o f b l o o d d o n o r s . M e t h o d s based o n the principle o f h a e m a g g l u t i n a t i o n are simple, rapid and c h e a p and m o d e r a t e l y sensitive. R a d i o i m m u n o a s s a y s are t h e m o s t sensitive m e t h o d s available, b u t t h e y are expensive and have the disadvantage o f requiring radioactive material. In this s t u d y a n e w e n z y m e - i m m u n o a s s a y (Wolters et al., 1 9 7 6 b ) , is evaluated against various o t h e r m e t h o d s . MATERIAL AND METHODS
E n z y m e - i m m u n o a s s a y ( E I A , h e p a n o s t i k a microelisa s y s t e m ) This new test was supplied b y Dr. KaSaki f r o m the O r g a n o n Scientific D e v e l o p m e n t G r o u p (Wolters et al., 1 9 7 6 b ) , and was used as d i r e c t e d b y t h e m a n u f a c t u r e r ( O r g a n o n T e k n i k a , Holland). T h e assay is based o n t h e 'sand-
1 The Finnish Red Cross Blood Transfusion Service, Helsinki, Finland.
344 wich' principle. The b o t t o m s of the wells in a microtitre plate (Cooke) are pre-coated with sheep anti-HBs. Any HBsAg present in the serum or plasma under investigation is bound to the solid-phase ant i body, making an antigena n t i b o d y complex. Upon the addition of a second a n t i b o d y (sheep antiHB~*, conjugate), which is labelled with the e n z y m e horse radish peroxidase (HRP), a new solid-phase complex is formed, consisting of Ab-Ag-Ab*. The substrate o-phenyl-diamine (OPD • 2 HC1) is then added. In the case of a positive sample, the enzymatic reaction results in a yellow-orange coloration. The result can be read with the naked eye or in a p h o t o m e t e r . When read with the naked eye, a positive result is indicated by a more intense yelloworange colour than that of the negative control. Positivity in the preliminary test is then confirmed by a nedtralization procedure with anti-HB~ (Hepanostika C o n f i r m a t o r y test). In this study the test pr oc e dur e was as follows: 1) 0.1 ml of test sample was added to each well. One negative (--) and two positive controls (+ and ++) supplied by the m a n u f a c t u r e r were included in each plate. 2) The plate was incubated for a b o u t 16 h at r o o m t e m p e r a t u r e (an alternative incubation is 2 h at 37°C). 3) The wells were aspirated. 4) The wells were washed 3 times with wash fluid supplied by the manufacturer. 5) 0.1 ml of conjugate (anti-HBs*) was added to each well. 6) The plate was incubated for 2 h at 37°C. 7) The wells were emptied by aspiration. 8) The wells were washed 4 times with wash fluid. 9) 0.1 ml of freshly prepared substrate solution (OPD • 2 HC1 and peroxide dissolved in distilled water) was added to each well. 10) The plate was incubated for 50 min at room t e m p e r a t u r e in the dark. 11) The enzymatic reaction was stopped by adding 0.05 ml of 4 N H2SO4. 12) The plate was placed on a mirror device used for the reading of mierotitre plates. A white sheet was placed on the top of the plate, and the plate was lighted from above and read by naked eye by three observers. The colour of each well was compared with that of the controls in the same plate. The result was negative (--) if the colour was equal to or less intense than th at of the negative control, and weakly positive (+) if the colour was more intense than that of the negative control but less intense than that of the ++ control. A strongly positive result (++) was scored if the eolour was equal to or more intense than that of the ++ control. If there was discrepancy between the readings, the result obtained by the majority (2/3) of the readers was accepted. The samples giving discrepant results in the screening test were also neutralized or tested repeatedly. The positivity in screening was confirmed by neutralization with anti-HBs (Hepanostika C o n f i r m a t o r y Test). In this study the procedure included the following steps: 1) 0.1 ml of a sample positive in screening was added to three adjoining wells (a, b and e) in a mierotitre plate pre-coated with antiHB~. Positive and negative controls were included as in the screening test. 2) The plate was incubated for 16 h at r o o m temperature. 3) The contents of wells a and b were aspirated. 4) The wells a and b were washed 3 times with wash fluid. 5) 0.15 ml of human anti-HBs was added to well a. 6) 0.15 ml of
345 c o n tr o l fluid was added to well b. 7) The plate was incubated for 2 h at 37 ° C. 8) The contents of wells a, b and c were aspirated. 9) All the wells a, b and c were washed 3 times with wash fluid. 10) 0.1 ml of conjugate (anti-HB~*) was added to all the wells, and the pr oc e dure was cont i nued as in the startdard screening test. The result is a specific positive if the colour of well a is less intense than that of b and c, non-specific if the colour of well a is weaker than that of the ++ control and equal to or m ore intense than that of b, irrespective of the colour of well c, and inconclusive (?) if the colour of well a is weaker than th at of b while c is negative, or bot h a and b are negative while c is positive. The result is negative if all the wells a, b and c are negative. New 8-channelled micropipettes with disposable p o l y p r o p y l e n e tips (Labpipette, Labsystems Oy division of Finnpipette Ky, Finland) were used for delivering the reagents (volumes 50 and 100 pl) and for washing/aspirating (200 pl). The two-fold dilutions for the titrations were prepared in undiluted normal h u man serum free f r om HB~Ag and anti-HBs.
Finnish R e d Cross radioimmunoassay (FRC-RIA ) This m e t h o d will be described in m or e detail in a separate paper. Anti-HBs was p r o d u c e d in a sheep by immunizing with purified HBsAg of subtype ad. Gamma globulin was isolated f r om the sheep serum by caprylic acid precipitation (Steinbuch and Audran, 1969) and adsorbed non-covalently on the walls of p o l y s t y r e n e test tubes (12 X 55 m m) (Catt and Tregear, 1967). Antibodies to HBsAg were isolated by passing the sheep IgG through a col um n of HBsAg coupled to Sepharose 4B by the CNBr m e t h o d (Cuatrecasas, 1970) and eluting with 5 M NaI. The antibodies were labelled with l~sI using the Chloramin T m e t h o d (Hunter and G r eenw o od, 1962). The RIA test was perf o r m e d as follows: 0.2 ml o f the sample (serum or plasma) was pi pet t ed into the a n t i b o d y - c o a t e d tube, the tube was incubated overnight with stirring at r o o m t e m p e r a t u r e and washed with saline. 0.2 ml of labelled a n t i b o d y solution was added, the tube was stirred for a further 4 h at r o o m temperature, washed, and the radioactivity c o u n t e d in a gamma counter. A specimen was considered positive, as in AusRIA, if it gave a c o u n t rate m ore than 2.1 times the mean of the negative controls (10 negative and 6 positive controls were included in each series). If the results of FRC-RIA and the ot her m et hods were inconsistent, a neutralization e x p e r i m e n t was done: FRC-RIA was perfo rmed as usual, except t hat 0.02 ml of sheep antiserum to HB~Ag was added together with the labelled antibody. The same a m o u n t of normal sheep serum was added to the control tube. If the antiserum rendered the result negative, the specimen was considered to contain HBsAg. The two-fold dilutions for the titrations were made using undiluted normal human serum free f r om HB~Ag and anti-HBs.
346
AusRIA H The test was p e r f o r m e d according to the manufacturer's instructions ( A b b o t t Laboratories, U.S.A.) The two-fold dilutions for the titrations were prepared using undiluted normal hum a n serum free from HBsAg and anti-
HB~. Hepanosticon The test was p e r f o r m e d in accordance with the instructions of the manufacturer (Organon Teknika, Holland). The samples were heated at 56°C for 30 min, because this has been f ound to reduce the n u m b e r of false positives in the screening test (Reesink et al., 1973). All the preliminarily positive samples were retested with the Hepanosticon Absorbent. The two-fold dilutions for the titrations were prepared using undiluted normal human serum free f r o m HB~Ag and anti-HBs.
Complement fixation (CF) A m i c r o t e c h n i q u e with disposable U-microplates (Meda, Daniel & Co, Finland) was used. All the reagents and samples were diluted in veronal b u ff er diluent, the first dilution of the sample being 1 : 8. The haemolytic system consisted of 1% sheep e r y t h r o c y t e s and two full units of haemolysin. 1.5 to 1.7 units of c o m p l e m e n t was used. Anti-HB~ was serum from a haemophiliac. This antiserum was shown by i mm unodi ffusi on to contain antibodies only against the c o m m o n d e t e r m i n a n t a. The positive control was serum from a chronic carrier of HB~Ag, the titre of which varied from 128 to 512 in the CF test (the titre is expressed as the reciprocal of the highest dilution giving a positive result). The reagents were pipetted at r o o m temperature. 0.025 ml of diluted sample and anti-HBs were incubated for 45 rain at 4°C and c o m p l e m e n t was then added. The haemolytic system was added after incubation for 16 h at 4°C. The plates were further incubated for 1 h at 37°C and read by visual estimation of the degree of haemolysis.
hnmunodiffusion (ID) A micr o mo d if i cat i on of the O u c h t e r l o n y m e t h o d developed by Wadsw o rth (1957), Krause and Raunio (1967) and Salmi (1969) was used for the subtyping of the HBsAg positive sera. The reference antigens were serum f r o m a chronic a s y m p t o m a t i c carrier of HBsAg (subtype ad), and serum from a narcotics addict who had had hepatitis twice (subtype ay). Two sheep anti-HB~ antisera were obtained by immunizing with purified HB~Ag from a s y m p t o m a t i c blood donors (subtype ad) and from the narcotics addict mentioned above (subtype ay). The identity of these reagents was confirmed with the reagents supplied by National Institute of Allergy and Infectious
347 Diseases, National Institutes of Health, U.S.A. (catalog numbers V 801-002027, V 802-001-027, V 802-002-027, V 803-001-027, V 801-502-058, V 802-501-558 and V 803-501-565). The reference antigen of subtype ad (diluted 1 : 2 in phosphate buffered saline) was placed on the top of the sevenwell pattern, and of subtype ay (1 : 4) on the b o t t o m . Antiserum, anti-ad (1 : 20) or antimy (1 : 80), was placed in the center well. One sample was then added to the two wells on the right and another sample on the left. The subtype (ad or ay) was determined by the precipitate showing identity with one reference antigen and partial identity (spur formation) with the other reference antigen. Material The serum panel consisted of 142 sera from 136 patients with suspected hepatitis B infection or recovering from hepatitis B. The sera were sent by various hospitals to our routine diagnostic laboratory between 1974 and 1976, and CF and AusRIA II were performed on arrival. Some of the oldest samples (strongly positive) were tested with AusRIA I, since AusRIA II was not then available. The samples were then stored at --20°C until Spring 1976 when FRC-RIA, Hepanosticon, EIA and ID tests were carried out. Two plasmas were titrated to estimate the sensitivities of the different methods. One was taken from an asymptomatic blood donor (subtype ad), and the other from a narcotics addict who had had hepatitis twice (subtype
ay). RESULTS The subtypes of the serum panel Thirty-seven of the samples could be subtyped by ID. 11 were of subtype ad, and 26 of subtype ay. The average age of the patients with subtype ad was 42 years, and of those with subtype ay 23 years. Titration o f the ad and ay specimen (table 1) A distinct pattern of subtype sensitivity was found. Both radioimmunoassays detected the ad specimen at higher dilution than the ay, while with the other three techniques the reverse was true. FRC-RIA was the most sensitive m e t h o d for subtype ad, and EIA for subtype ay. For both subtypes AusRIA II was less sensitive than FRC-RIA. Hepanosticon and CF were approximately equal in sensitivity, both detecting the ay specimen at higher dilution. Detection o f HB~Ag in the serum panel (tables 2 and 3) As shown in table 2, EIA detected most positive samples, 50 out of 142. FRC-RIA gave one more positive (49) than AusRIA II {48}. By using Hepa-
348 TABLE 1 The relative sensitivities of different methods in the detection of HBsAg subtypes. Specimen (plasma) Subtype ad Subtype ay
Titres of specimen * Hepanosticon
CF
AusRIA II
FRC-RIA
EIA
64 256
64 512
6400 3200
12 800 6400
3200 12,800
* The specimen were diluted in veronal buffered saline in CF, while for the other methods the diluent was undiluted normal human serum free from HBsAg and anti-HB s.
nosticon 46 positive samples were detected compared with 44 by CF. T h e d i s c r e p a n c i e s o b s e r v e d b e t w e e n t e s t s a r e d e t a i l e d in t a b l e 3.
Neutralization with anti-HBs in EIA (Hepanostika Confirmatory test)(table 4) Thirty different sera were tested by the neutralization procedure. All f o u n d t o b e s t r o n g l y p o s i t i v e ( + + ) in t h e p r e l i m i n a r y t e s t in E I A w e r e c l e a r l y n e u t r a l i z e d , i.e. w e r e s p e c i f i c a l l y p o s i t i v e . O u t o f five s a m p l e s w e a k l y p o s i t i v e (+) i n s c r e e n i n g , o n e ( n o . 2) w a s c l e a r l y n e u t r a l i z e d , o n e ( 1 3 4 ) c o u l d not be resolved (both samples were from patients with previously proven hepatitis B infection), and three were entirely negative. Out of 10 sera showi n g a d i s c r e p a n c y b e t w e e n t h e r e a d e r s (_+) a n d u s u a l l y a l s o n e g a t i v i t y w i t h t h e o t h e r m e t h o d s , o n e ( 1 4 5 ) w a s n e u t r a l i z e d , o n e ( 1 4 1 ) ~vas i n c o n c l u s i v e , and eight were negative.
Reproducibility of the screening results in EIA (table 5) T h i r t y - t w o s e r a g i v i n g d i f f e r e n t r e s u l t s in t h e s c r e e n i n g t e s t o f E I A w e r e r e t e s t e d . A l l o f t h e 15 s a m p l e s s t r o n g l y p o s i t i v e in t h e f i r s t t e s t w e r e a l s o
TABLE 2 Detection of HBsAg among 142 serum samples by different methods. Result
CF
Hepanosticon
AusRIA II
FRC-RIA
EIA
Specific positive Negative ? Anticomplentary
44 94
46 96
48 94
49 91 2*
50 90 2**
4
* Sera nos. 75 and 63. See remarks in table 3. ** Sera nos. 134 and 141. See remarks in table 3.
349 TABLE 3 D e t e c t i o n of HBsAg. Detailed presentation of sera showing discrepancy b e t w e e n different methods. Ref. no. of serum
CF
Hepanosticon
AusRIA II
FRCRIA
EIA
78 133 139 3
-~64 * ~16 * --
+ + ---
+ + + +
+ + + +
+ + + +
143
--
--
--
+
--
2
.
.
.
.
+
145
.
.
.
.
+
134
.
.
.
.
?
141
w
--
75
9
63
44 76
~16 * ~16 *
m
?
Remarks
A n t i c o m p l e m e n t a r y serum A n t i c o m p l e m e n t a r y serum The previous sample (taken 10 days earlier) was also positive in CF F R C - R I A : repeatedly positive, could be neutralized The samples taken 2 m o n t h s earlier were positive in ID, CF, and A u s R I A II. The subtype was a y E I A : weakly positive in screening, could be neutralized EIA: weakly positive in screening, could n o t be clearly neutralized. A u s R I A II was positive but CF negative in a sample taken 16 days earlier E I A : weakly positive in screening, could not be neutralized F R C - R I A : weakly positive in screening, could not be neutralized F R C - R I A : weakly positive in screening, not available for neutralization A n t i c o m p l e m e n t a r y serum A n t i c o m p l e m e n t a r y serum
* The figure indicates the reciprocal of the first dilution giving a negative result when titrated in the absence of anti-HB s. strongly positive in the repeat test. Out of 6 samples weakly positive in the f i r s t t e s t , t w o w e r e r e g a r d e d as n e g a t i v e in t h e s e c o n d t e s t b y all t h e t h r e e readers, two showed discrepancy between the readers, and two were repeatedly positive (nos. 2 and 134, both from patients with previously proven hepatitis B infection). Discrepant reading results were obtained repeatedly with 10 samples, while one sample read discrepantly in the first test was e n t i r e l y n e g a t i v e in t h e s e c o n d t e s t . A l l t h e s a m p l e s g i v i n g d i s c r e p a n t r e a d i n g s in e i t h e r o f t h e t e s t s w e r e n e g a t i v e b y t h e o t h e r m e t h o d s i n c l u d i n g t h e Hepanostika Confirmatory test provided that the interpretation of two r e a d e r s w a s a c c e p t e d as t h e f i r s t r e s u l t .
35O TABLE 4 E n z y m e - i m m u n o a s s a y in t h e d e t e c t i o n of HBsAg. Results b y t h e H e p a n o s t i k a C o n f i r m a t o r y test. R e s u l t in t h e s c r e e n i n g test
R e s u l t in t h e C o n f i r m a t o r y test ++
++ + ±
15 1a
Total
16
a Serum b Serum c Serum d Serum
no. no. no. no.
+
Total
9
3 8
15 5 10
1]
30
]b
2. See r e m a r k s in 134. See r e m a r k s 145. See r e m a r k s 141. See r e m a r k s
1 c
1 d
1
2
table 3. in table 3. in table 3. in t a b l e 3.
Absorption with the Hepanosticon Absorbent (table 6) One clearly false positive result (no. 34) was eliminated with the Absorbent. All of those samples positive in the preliminary Hepanosticon test and also with other methods were positive after the absorption procedure, including sample number 138, which was repeatedly only weakly positive in the screening test, but strongly positive after absorption. Two samples weakly positive in screening (75 and 54) turned out to be negative after absorption; they were also negative according to the other methods. TABLE 5 E n z y m e - i m m u n o a s s a y in t h e d e t e c t i o n of HBsAg. R e p r o d u c i b i l i t y of the results w h e n t h e s c r e e n i n g test was r e p e a t e d . T h e reading was p e r f o r m e d w i t h t h e n a k e d eye by t h r e e readers. R e s u l t in t h e first test *
R e s u l t in t h e s e c o n d t e s t * +-t- ÷ +
++
Total
+ -~- +
"~-
++ ++ ++ + + + -±
15 1 **
1 ***
2 10
2 1
15 6 11
Total
16
1
12
3
32
* ++ ++ ++ : R e g a r d e d as s t r o n g l y positive b y all t h e readers. + + + : R e g a r d e d as weakly positive b y all t h e readers. : R e g a r d e d as negative b y all t h e readers. -+ : D i s c r e p a n c y b e t w e e n t h e readers. ** S e r u m no. 2. See r e m a r k s in t a b l e 3. *** S e r u m no. 134. See r e m a r k s in table 3.
351 TABLE 6 H e p a n o s t i c o n in the d e t e c t i o n o f HBsAg. T h e results o f a b s o r p t i o n w i t h H e p a n o s t i c o n A b s o r b e n t o f 49 sera positive or w e a k l y positive in t h e p r e l i m i n a r y H e p a n o s t i c o n test, and comparison with other methods. N u m b e r of sera
43 1(133) * 1(78) 1(34) 1(138) 1(75) 1(54)
Hepanosticon
CF
Before absorption
After absorption
+ + +
+ + +
+
.
++ -+
+ -.
AusRIA II
+ <64 --
FRCRIA
+ + +
.
+ + +
.
+ --
.
+ + +
.
+ -.
EIA
.
.
+ ?
+ --
.
* R e f e r e n c e n u m b e r o f serum. See r e m a r k s in t a b l e 3.
Anticomplementary sera (table 7) Four sera were found to be anticomplementary in the CF test. Two samples giving the lowest anticomplementary t i t r e s ( < 1 6 ; i.e. t h e d i l u t i o n 1 : 1 6 is t h e f i r s t d i l u t i o n g i v i n g n e g a t i v e r e s u l t w h e n t i t r a t e d i n t h e a b s e n c e o f a n t i - H B s ) w e r e n e g a t i v e i n all o t h e r t e c h n i q u e s . Sample number 139 (titre <16) was positive in the three immunoassays, but negative in Hepanosticon. Sample number 133, which had the highest anticomplementary titre (<64), w a s p o s i t i v e b y all t h e o t h e r m e t h o d s .
TABLE7 DetectionofHBsAginfouranticomplementarysera. R e f e r e n c e no. of serum
CF *
Hepanosticon
AusRIA II .
FRCRIA
.
EIA
44
<16
.
76
<16
.
139
<16
--
+
+
+
133
<64
+
+
+
+
.
.
.
.
.
* T h e figure in t h e CF test i n d i c a t e s t h e first negative d i l u t i o n o f t h e s a m p l e w h e n tit r a t e d in t h e a b s e n c e o f anti-HB s.
352 DISCUSSION The new enzyme-immunoassay Hepanostika Microelisa System proves promising. Its sensitivity is of at least the same order as that of the radioimmunological methods. Handling of radioactive material is avoided and expensive equipment is not needed. According to the instructions, three readers were used in this study. The reading agreed by two of them was taken as the final result, and by this criterion most of the discrepant readings turned out to be negative. The confirmatory test is esssential for all samples showing positivity or discrepant results in the EIA screening test. Most of those samples weakly positive or reading discrepantly on screening were negative after confirmation. In order to avoid such false positives, the strength of the negative control should be adjusted upwards, but this may, of course, diminish the sensitivity of the test. Reading with the naked eye is laborious, and differences in subjective interpretation are c o m m o n with weakly positive samples. A potential improvement may be the special multichannelled photometer (Finnpipette® Analyzer System) used by Leinikki and P~issil~ (1976). This measures absorbance vertically through the bottoms of the cuvettes, and is equipped with a programmable desk calculator. None of the test systems studied gave ideal results. In the FRC-RIA, a sheep anti-HBs against subtype ad was used, and this m e t h o d was the most sensitive for detection of the ad specimen. EIA gave the highest titre for the a~ specimen; similar findings have been reported by Wolters et al. (1976a,b). Most of the serum samples in this study were of subtype ay, and this may explain why EIA gave more positives than the other methods. It is possible that an immunoassay using a mixture of subtype specific antibodies is less sensitive to a particular subtype than a m e t h o d having only the subtype specific antibody against the antigen being tested. The optimal result in screening would be obtained with a test system which uses separate procedures for each antigen subtype. Many hepatitis patients have anti-complementary sera, which is partly due to the high frequency of immune complexes among these patients (Lambert et al., 1976). These anti-complementary samples cannot be resolved with the CF test. The standardization of the reagents and the m a n y controls needed in this technique make it unsatisfactory as compared, for instance, with Hepanosticon. The latter is simple, has about the same degree of sensitivity as CF, and anticomplementarity does n o t interfere with the outcome. Acknowledgements This study was partly supported by grants from The Finnish Medical Society Duodecim and The Sigrid Juselius Foundation. Equipment for the Hepanostika Microelisa System and Hepanosticon was supplied by Organon Teknika.
353 L a b s y s t e m s O y division o f F i n n p i p e t t e K y p r o v i d e d us with the new multichannelled Labpipettes. T h e t e c h n i c a l assistance o f Miss Ulla Viitanen, Miss H e l e n a Kainulainen, Mrs. Anja KallionpS,~i, Miss Satu Kangas and Miss Kirsi-Marja Pylkk~inen is gratefully a c k n o w l e d g e d . REFERENCES Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194,495. Krause, U. and V. Raunio, 1967, Acta Pathol. Microbio|. Scand. 71,328. Lambert, P-H., E. Tribollet, A. Celada, K. Madalinski, P.C. Frei and P-A. Miescher, 1976, J. Clin. Invest. (submitted). Leinikki, P. and Suvi P~ssil~, 1976, J. Clin. Pathol., 29 (in press). Reesink, H.W., W.J. Duimel and H.G.J. Brummelhuis, 1973, Lancet 2, 1351. Salmi, A., 1969, Acta Pathol. Microbiol. Scand. 76, 271. Steinbuch, M. and R. Audran, 1969, Arch. Biochem. Biophys. 134,279. Wadsworth, C., 1957, Int. Arch. All. 10,350. Wolters, G., L. Kuijpers, J. Ka~aki and A. Schuurs, 1976a, J. Inf. Dis. (in press). Wolters, G., L. Kuijpers, J. KaSaki and A. Schuurs, 1976b, J. Clin. Patho|. 29,873.
343
E N Z Y M E - I M M U N O A S S A Y IN T H E D E T E C T I O N O F H E P A T I T I S B SURFACE ANTIGEN
P. UKKONEN, V. KOISTINEN 1 and K. PENTTINEN
Laboratory of Viral Immunopathology, Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland (Received 25 November 1976, accepted 10 December 1976)
A new enzyme-immunoassay (EIA, Hepanostika Microelisa System) for the detection of hepatitis B surface antigen was evaluated against other methods, namely complement fixation, Hepanosticon, AusRIA II and Finnish Red Cross Radioimmunoassay (FRC-RIA). EIA detected the greatest number of positive samples in a serum panel consisting of 142 sera from clinical hepatitis patients. FRC-RIA was the most sensitive method for subtype ad, while EIA detected the ay specimen at the highest dilution. None of the test systems gave the 'optimal' result in the screening test, and it is proposed that a separate procedure for each antigen subtype should be carried out to detect the greatest number of positive samples.
INTRODUCTION Since the d i s c o v e r y o f hepatitis B surface antigen (HBsAg, Australia antigen), n u m e r o u s m e t h o d s have b e e n d e v e l o p e d for t h e d e m o n s t r a t i o n o f HB,Ag. I m m u n o d i f f u s i o n a n d c o u n t e r i m m u n o e l e c t r o p h o r e s i s have been f o u n d t o o insensitive for diagnostic purposes, especially in the case o f b l o o d d o n o r s . M e t h o d s based o n the principle o f h a e m a g g l u t i n a t i o n are simple, rapid and c h e a p and m o d e r a t e l y sensitive. R a d i o i m m u n o a s s a y s are t h e m o s t sensitive m e t h o d s available, b u t t h e y are expensive and have the disadvantage o f requiring radioactive material. In this s t u d y a n e w e n z y m e - i m m u n o a s s a y (Wolters et al., 1 9 7 6 b ) , is evaluated against various o t h e r m e t h o d s . MATERIAL AND METHODS
E n z y m e - i m m u n o a s s a y ( E I A , h e p a n o s t i k a microelisa s y s t e m ) This new test was supplied b y Dr. KaSaki f r o m the O r g a n o n Scientific D e v e l o p m e n t G r o u p (Wolters et al., 1 9 7 6 b ) , and was used as d i r e c t e d b y t h e m a n u f a c t u r e r ( O r g a n o n T e k n i k a , Holland). T h e assay is based o n t h e 'sand-
1 The Finnish Red Cross Blood Transfusion Service, Helsinki, Finland.
344 wich' principle. The b o t t o m s of the wells in a microtitre plate (Cooke) are pre-coated with sheep anti-HBs. Any HBsAg present in the serum or plasma under investigation is bound to the solid-phase ant i body, making an antigena n t i b o d y complex. Upon the addition of a second a n t i b o d y (sheep antiHB~*, conjugate), which is labelled with the e n z y m e horse radish peroxidase (HRP), a new solid-phase complex is formed, consisting of Ab-Ag-Ab*. The substrate o-phenyl-diamine (OPD • 2 HC1) is then added. In the case of a positive sample, the enzymatic reaction results in a yellow-orange coloration. The result can be read with the naked eye or in a p h o t o m e t e r . When read with the naked eye, a positive result is indicated by a more intense yelloworange colour than that of the negative control. Positivity in the preliminary test is then confirmed by a nedtralization procedure with anti-HB~ (Hepanostika C o n f i r m a t o r y test). In this study the test pr oc e dur e was as follows: 1) 0.1 ml of test sample was added to each well. One negative (--) and two positive controls (+ and ++) supplied by the m a n u f a c t u r e r were included in each plate. 2) The plate was incubated for a b o u t 16 h at r o o m t e m p e r a t u r e (an alternative incubation is 2 h at 37°C). 3) The wells were aspirated. 4) The wells were washed 3 times with wash fluid supplied by the manufacturer. 5) 0.1 ml of conjugate (anti-HBs*) was added to each well. 6) The plate was incubated for 2 h at 37°C. 7) The wells were emptied by aspiration. 8) The wells were washed 4 times with wash fluid. 9) 0.1 ml of freshly prepared substrate solution (OPD • 2 HC1 and peroxide dissolved in distilled water) was added to each well. 10) The plate was incubated for 50 min at room t e m p e r a t u r e in the dark. 11) The enzymatic reaction was stopped by adding 0.05 ml of 4 N H2SO4. 12) The plate was placed on a mirror device used for the reading of mierotitre plates. A white sheet was placed on the top of the plate, and the plate was lighted from above and read by naked eye by three observers. The colour of each well was compared with that of the controls in the same plate. The result was negative (--) if the colour was equal to or less intense than th at of the negative control, and weakly positive (+) if the colour was more intense than that of the negative control but less intense than that of the ++ control. A strongly positive result (++) was scored if the eolour was equal to or more intense than that of the ++ control. If there was discrepancy between the readings, the result obtained by the majority (2/3) of the readers was accepted. The samples giving discrepant results in the screening test were also neutralized or tested repeatedly. The positivity in screening was confirmed by neutralization with anti-HBs (Hepanostika C o n f i r m a t o r y Test). In this study the procedure included the following steps: 1) 0.1 ml of a sample positive in screening was added to three adjoining wells (a, b and e) in a mierotitre plate pre-coated with antiHB~. Positive and negative controls were included as in the screening test. 2) The plate was incubated for 16 h at r o o m temperature. 3) The contents of wells a and b were aspirated. 4) The wells a and b were washed 3 times with wash fluid. 5) 0.15 ml of human anti-HBs was added to well a. 6) 0.15 ml of
345 c o n tr o l fluid was added to well b. 7) The plate was incubated for 2 h at 37 ° C. 8) The contents of wells a, b and c were aspirated. 9) All the wells a, b and c were washed 3 times with wash fluid. 10) 0.1 ml of conjugate (anti-HB~*) was added to all the wells, and the pr oc e dure was cont i nued as in the startdard screening test. The result is a specific positive if the colour of well a is less intense than that of b and c, non-specific if the colour of well a is weaker than that of the ++ control and equal to or m ore intense than that of b, irrespective of the colour of well c, and inconclusive (?) if the colour of well a is weaker than th at of b while c is negative, or bot h a and b are negative while c is positive. The result is negative if all the wells a, b and c are negative. New 8-channelled micropipettes with disposable p o l y p r o p y l e n e tips (Labpipette, Labsystems Oy division of Finnpipette Ky, Finland) were used for delivering the reagents (volumes 50 and 100 pl) and for washing/aspirating (200 pl). The two-fold dilutions for the titrations were prepared in undiluted normal h u man serum free f r om HB~Ag and anti-HBs.
Finnish R e d Cross radioimmunoassay (FRC-RIA ) This m e t h o d will be described in m or e detail in a separate paper. Anti-HBs was p r o d u c e d in a sheep by immunizing with purified HBsAg of subtype ad. Gamma globulin was isolated f r om the sheep serum by caprylic acid precipitation (Steinbuch and Audran, 1969) and adsorbed non-covalently on the walls of p o l y s t y r e n e test tubes (12 X 55 m m) (Catt and Tregear, 1967). Antibodies to HBsAg were isolated by passing the sheep IgG through a col um n of HBsAg coupled to Sepharose 4B by the CNBr m e t h o d (Cuatrecasas, 1970) and eluting with 5 M NaI. The antibodies were labelled with l~sI using the Chloramin T m e t h o d (Hunter and G r eenw o od, 1962). The RIA test was perf o r m e d as follows: 0.2 ml o f the sample (serum or plasma) was pi pet t ed into the a n t i b o d y - c o a t e d tube, the tube was incubated overnight with stirring at r o o m t e m p e r a t u r e and washed with saline. 0.2 ml of labelled a n t i b o d y solution was added, the tube was stirred for a further 4 h at r o o m temperature, washed, and the radioactivity c o u n t e d in a gamma counter. A specimen was considered positive, as in AusRIA, if it gave a c o u n t rate m ore than 2.1 times the mean of the negative controls (10 negative and 6 positive controls were included in each series). If the results of FRC-RIA and the ot her m et hods were inconsistent, a neutralization e x p e r i m e n t was done: FRC-RIA was perfo rmed as usual, except t hat 0.02 ml of sheep antiserum to HB~Ag was added together with the labelled antibody. The same a m o u n t of normal sheep serum was added to the control tube. If the antiserum rendered the result negative, the specimen was considered to contain HBsAg. The two-fold dilutions for the titrations were made using undiluted normal human serum free f r om HB~Ag and anti-HBs.
346
AusRIA H The test was p e r f o r m e d according to the manufacturer's instructions ( A b b o t t Laboratories, U.S.A.) The two-fold dilutions for the titrations were prepared using undiluted normal hum a n serum free from HBsAg and anti-
HB~. Hepanosticon The test was p e r f o r m e d in accordance with the instructions of the manufacturer (Organon Teknika, Holland). The samples were heated at 56°C for 30 min, because this has been f ound to reduce the n u m b e r of false positives in the screening test (Reesink et al., 1973). All the preliminarily positive samples were retested with the Hepanosticon Absorbent. The two-fold dilutions for the titrations were prepared using undiluted normal human serum free f r o m HB~Ag and anti-HBs.
Complement fixation (CF) A m i c r o t e c h n i q u e with disposable U-microplates (Meda, Daniel & Co, Finland) was used. All the reagents and samples were diluted in veronal b u ff er diluent, the first dilution of the sample being 1 : 8. The haemolytic system consisted of 1% sheep e r y t h r o c y t e s and two full units of haemolysin. 1.5 to 1.7 units of c o m p l e m e n t was used. Anti-HB~ was serum from a haemophiliac. This antiserum was shown by i mm unodi ffusi on to contain antibodies only against the c o m m o n d e t e r m i n a n t a. The positive control was serum from a chronic carrier of HB~Ag, the titre of which varied from 128 to 512 in the CF test (the titre is expressed as the reciprocal of the highest dilution giving a positive result). The reagents were pipetted at r o o m temperature. 0.025 ml of diluted sample and anti-HBs were incubated for 45 rain at 4°C and c o m p l e m e n t was then added. The haemolytic system was added after incubation for 16 h at 4°C. The plates were further incubated for 1 h at 37°C and read by visual estimation of the degree of haemolysis.
hnmunodiffusion (ID) A micr o mo d if i cat i on of the O u c h t e r l o n y m e t h o d developed by Wadsw o rth (1957), Krause and Raunio (1967) and Salmi (1969) was used for the subtyping of the HBsAg positive sera. The reference antigens were serum f r o m a chronic a s y m p t o m a t i c carrier of HBsAg (subtype ad), and serum from a narcotics addict who had had hepatitis twice (subtype ay). Two sheep anti-HB~ antisera were obtained by immunizing with purified HB~Ag from a s y m p t o m a t i c blood donors (subtype ad) and from the narcotics addict mentioned above (subtype ay). The identity of these reagents was confirmed with the reagents supplied by National Institute of Allergy and Infectious
347 Diseases, National Institutes of Health, U.S.A. (catalog numbers V 801-002027, V 802-001-027, V 802-002-027, V 803-001-027, V 801-502-058, V 802-501-558 and V 803-501-565). The reference antigen of subtype ad (diluted 1 : 2 in phosphate buffered saline) was placed on the top of the sevenwell pattern, and of subtype ay (1 : 4) on the b o t t o m . Antiserum, anti-ad (1 : 20) or antimy (1 : 80), was placed in the center well. One sample was then added to the two wells on the right and another sample on the left. The subtype (ad or ay) was determined by the precipitate showing identity with one reference antigen and partial identity (spur formation) with the other reference antigen. Material The serum panel consisted of 142 sera from 136 patients with suspected hepatitis B infection or recovering from hepatitis B. The sera were sent by various hospitals to our routine diagnostic laboratory between 1974 and 1976, and CF and AusRIA II were performed on arrival. Some of the oldest samples (strongly positive) were tested with AusRIA I, since AusRIA II was not then available. The samples were then stored at --20°C until Spring 1976 when FRC-RIA, Hepanosticon, EIA and ID tests were carried out. Two plasmas were titrated to estimate the sensitivities of the different methods. One was taken from an asymptomatic blood donor (subtype ad), and the other from a narcotics addict who had had hepatitis twice (subtype
ay). RESULTS The subtypes of the serum panel Thirty-seven of the samples could be subtyped by ID. 11 were of subtype ad, and 26 of subtype ay. The average age of the patients with subtype ad was 42 years, and of those with subtype ay 23 years. Titration o f the ad and ay specimen (table 1) A distinct pattern of subtype sensitivity was found. Both radioimmunoassays detected the ad specimen at higher dilution than the ay, while with the other three techniques the reverse was true. FRC-RIA was the most sensitive m e t h o d for subtype ad, and EIA for subtype ay. For both subtypes AusRIA II was less sensitive than FRC-RIA. Hepanosticon and CF were approximately equal in sensitivity, both detecting the ay specimen at higher dilution. Detection o f HB~Ag in the serum panel (tables 2 and 3) As shown in table 2, EIA detected most positive samples, 50 out of 142. FRC-RIA gave one more positive (49) than AusRIA II {48}. By using Hepa-
348 TABLE 1 The relative sensitivities of different methods in the detection of HBsAg subtypes. Specimen (plasma) Subtype ad Subtype ay
Titres of specimen * Hepanosticon
CF
AusRIA II
FRC-RIA
EIA
64 256
64 512
6400 3200
12 800 6400
3200 12,800
* The specimen were diluted in veronal buffered saline in CF, while for the other methods the diluent was undiluted normal human serum free from HBsAg and anti-HB s.
nosticon 46 positive samples were detected compared with 44 by CF. T h e d i s c r e p a n c i e s o b s e r v e d b e t w e e n t e s t s a r e d e t a i l e d in t a b l e 3.
Neutralization with anti-HBs in EIA (Hepanostika Confirmatory test)(table 4) Thirty different sera were tested by the neutralization procedure. All f o u n d t o b e s t r o n g l y p o s i t i v e ( + + ) in t h e p r e l i m i n a r y t e s t in E I A w e r e c l e a r l y n e u t r a l i z e d , i.e. w e r e s p e c i f i c a l l y p o s i t i v e . O u t o f five s a m p l e s w e a k l y p o s i t i v e (+) i n s c r e e n i n g , o n e ( n o . 2) w a s c l e a r l y n e u t r a l i z e d , o n e ( 1 3 4 ) c o u l d not be resolved (both samples were from patients with previously proven hepatitis B infection), and three were entirely negative. Out of 10 sera showi n g a d i s c r e p a n c y b e t w e e n t h e r e a d e r s (_+) a n d u s u a l l y a l s o n e g a t i v i t y w i t h t h e o t h e r m e t h o d s , o n e ( 1 4 5 ) w a s n e u t r a l i z e d , o n e ( 1 4 1 ) ~vas i n c o n c l u s i v e , and eight were negative.
Reproducibility of the screening results in EIA (table 5) T h i r t y - t w o s e r a g i v i n g d i f f e r e n t r e s u l t s in t h e s c r e e n i n g t e s t o f E I A w e r e r e t e s t e d . A l l o f t h e 15 s a m p l e s s t r o n g l y p o s i t i v e in t h e f i r s t t e s t w e r e a l s o
TABLE 2 Detection of HBsAg among 142 serum samples by different methods. Result
CF
Hepanosticon
AusRIA II
FRC-RIA
EIA
Specific positive Negative ? Anticomplentary
44 94
46 96
48 94
49 91 2*
50 90 2**
4
* Sera nos. 75 and 63. See remarks in table 3. ** Sera nos. 134 and 141. See remarks in table 3.
349 TABLE 3 D e t e c t i o n of HBsAg. Detailed presentation of sera showing discrepancy b e t w e e n different methods. Ref. no. of serum
CF
Hepanosticon
AusRIA II
FRCRIA
EIA
78 133 139 3
-~64 * ~16 * --
+ + ---
+ + + +
+ + + +
+ + + +
143
--
--
--
+
--
2
.
.
.
.
+
145
.
.
.
.
+
134
.
.
.
.
?
141
w
--
75
9
63
44 76
~16 * ~16 *
m
?
Remarks
A n t i c o m p l e m e n t a r y serum A n t i c o m p l e m e n t a r y serum The previous sample (taken 10 days earlier) was also positive in CF F R C - R I A : repeatedly positive, could be neutralized The samples taken 2 m o n t h s earlier were positive in ID, CF, and A u s R I A II. The subtype was a y E I A : weakly positive in screening, could be neutralized EIA: weakly positive in screening, could n o t be clearly neutralized. A u s R I A II was positive but CF negative in a sample taken 16 days earlier E I A : weakly positive in screening, could not be neutralized F R C - R I A : weakly positive in screening, could not be neutralized F R C - R I A : weakly positive in screening, not available for neutralization A n t i c o m p l e m e n t a r y serum A n t i c o m p l e m e n t a r y serum
* The figure indicates the reciprocal of the first dilution giving a negative result when titrated in the absence of anti-HB s. strongly positive in the repeat test. Out of 6 samples weakly positive in the f i r s t t e s t , t w o w e r e r e g a r d e d as n e g a t i v e in t h e s e c o n d t e s t b y all t h e t h r e e readers, two showed discrepancy between the readers, and two were repeatedly positive (nos. 2 and 134, both from patients with previously proven hepatitis B infection). Discrepant reading results were obtained repeatedly with 10 samples, while one sample read discrepantly in the first test was e n t i r e l y n e g a t i v e in t h e s e c o n d t e s t . A l l t h e s a m p l e s g i v i n g d i s c r e p a n t r e a d i n g s in e i t h e r o f t h e t e s t s w e r e n e g a t i v e b y t h e o t h e r m e t h o d s i n c l u d i n g t h e Hepanostika Confirmatory test provided that the interpretation of two r e a d e r s w a s a c c e p t e d as t h e f i r s t r e s u l t .
35O TABLE 4 E n z y m e - i m m u n o a s s a y in t h e d e t e c t i o n of HBsAg. Results b y t h e H e p a n o s t i k a C o n f i r m a t o r y test. R e s u l t in t h e s c r e e n i n g test
R e s u l t in t h e C o n f i r m a t o r y test ++
++ + ±
15 1a
Total
16
a Serum b Serum c Serum d Serum
no. no. no. no.
+
Total
9
3 8
15 5 10
1]
30
]b
2. See r e m a r k s in 134. See r e m a r k s 145. See r e m a r k s 141. See r e m a r k s
1 c
1 d
1
2
table 3. in table 3. in table 3. in t a b l e 3.
Absorption with the Hepanosticon Absorbent (table 6) One clearly false positive result (no. 34) was eliminated with the Absorbent. All of those samples positive in the preliminary Hepanosticon test and also with other methods were positive after the absorption procedure, including sample number 138, which was repeatedly only weakly positive in the screening test, but strongly positive after absorption. Two samples weakly positive in screening (75 and 54) turned out to be negative after absorption; they were also negative according to the other methods. TABLE 5 E n z y m e - i m m u n o a s s a y in t h e d e t e c t i o n of HBsAg. R e p r o d u c i b i l i t y of the results w h e n t h e s c r e e n i n g test was r e p e a t e d . T h e reading was p e r f o r m e d w i t h t h e n a k e d eye by t h r e e readers. R e s u l t in t h e first test *
R e s u l t in t h e s e c o n d t e s t * +-t- ÷ +
++
Total
+ -~- +
"~-
++ ++ ++ + + + -±
15 1 **
1 ***
2 10
2 1
15 6 11
Total
16
1
12
3
32
* ++ ++ ++ : R e g a r d e d as s t r o n g l y positive b y all t h e readers. + + + : R e g a r d e d as weakly positive b y all t h e readers. : R e g a r d e d as negative b y all t h e readers. -+ : D i s c r e p a n c y b e t w e e n t h e readers. ** S e r u m no. 2. See r e m a r k s in t a b l e 3. *** S e r u m no. 134. See r e m a r k s in table 3.
351 TABLE 6 H e p a n o s t i c o n in the d e t e c t i o n o f HBsAg. T h e results o f a b s o r p t i o n w i t h H e p a n o s t i c o n A b s o r b e n t o f 49 sera positive or w e a k l y positive in t h e p r e l i m i n a r y H e p a n o s t i c o n test, and comparison with other methods. N u m b e r of sera
43 1(133) * 1(78) 1(34) 1(138) 1(75) 1(54)
Hepanosticon
CF
Before absorption
After absorption
+ + +
+ + +
+
.
++ -+
+ -.
AusRIA II
+ <64 --
FRCRIA
+ + +
.
+ + +
.
+ --
.
+ + +
.
+ -.
EIA
.
.
+ ?
+ --
.
* R e f e r e n c e n u m b e r o f serum. See r e m a r k s in t a b l e 3.
Anticomplementary sera (table 7) Four sera were found to be anticomplementary in the CF test. Two samples giving the lowest anticomplementary t i t r e s ( < 1 6 ; i.e. t h e d i l u t i o n 1 : 1 6 is t h e f i r s t d i l u t i o n g i v i n g n e g a t i v e r e s u l t w h e n t i t r a t e d i n t h e a b s e n c e o f a n t i - H B s ) w e r e n e g a t i v e i n all o t h e r t e c h n i q u e s . Sample number 139 (titre <16) was positive in the three immunoassays, but negative in Hepanosticon. Sample number 133, which had the highest anticomplementary titre (<64), w a s p o s i t i v e b y all t h e o t h e r m e t h o d s .
TABLE7 DetectionofHBsAginfouranticomplementarysera. R e f e r e n c e no. of serum
CF *
Hepanosticon
AusRIA II .
FRCRIA
.
EIA
44
<16
.
76
<16
.
139
<16
--
+
+
+
133
<64
+
+
+
+
.
.
.
.
.
* T h e figure in t h e CF test i n d i c a t e s t h e first negative d i l u t i o n o f t h e s a m p l e w h e n tit r a t e d in t h e a b s e n c e o f anti-HB s.
352 DISCUSSION The new enzyme-immunoassay Hepanostika Microelisa System proves promising. Its sensitivity is of at least the same order as that of the radioimmunological methods. Handling of radioactive material is avoided and expensive equipment is not needed. According to the instructions, three readers were used in this study. The reading agreed by two of them was taken as the final result, and by this criterion most of the discrepant readings turned out to be negative. The confirmatory test is esssential for all samples showing positivity or discrepant results in the EIA screening test. Most of those samples weakly positive or reading discrepantly on screening were negative after confirmation. In order to avoid such false positives, the strength of the negative control should be adjusted upwards, but this may, of course, diminish the sensitivity of the test. Reading with the naked eye is laborious, and differences in subjective interpretation are c o m m o n with weakly positive samples. A potential improvement may be the special multichannelled photometer (Finnpipette® Analyzer System) used by Leinikki and P~issil~ (1976). This measures absorbance vertically through the bottoms of the cuvettes, and is equipped with a programmable desk calculator. None of the test systems studied gave ideal results. In the FRC-RIA, a sheep anti-HBs against subtype ad was used, and this m e t h o d was the most sensitive for detection of the ad specimen. EIA gave the highest titre for the a~ specimen; similar findings have been reported by Wolters et al. (1976a,b). Most of the serum samples in this study were of subtype ay, and this may explain why EIA gave more positives than the other methods. It is possible that an immunoassay using a mixture of subtype specific antibodies is less sensitive to a particular subtype than a m e t h o d having only the subtype specific antibody against the antigen being tested. The optimal result in screening would be obtained with a test system which uses separate procedures for each antigen subtype. Many hepatitis patients have anti-complementary sera, which is partly due to the high frequency of immune complexes among these patients (Lambert et al., 1976). These anti-complementary samples cannot be resolved with the CF test. The standardization of the reagents and the m a n y controls needed in this technique make it unsatisfactory as compared, for instance, with Hepanosticon. The latter is simple, has about the same degree of sensitivity as CF, and anticomplementarity does n o t interfere with the outcome. Acknowledgements This study was partly supported by grants from The Finnish Medical Society Duodecim and The Sigrid Juselius Foundation. Equipment for the Hepanostika Microelisa System and Hepanosticon was supplied by Organon Teknika.
353 L a b s y s t e m s O y division o f F i n n p i p e t t e K y p r o v i d e d us with the new multichannelled Labpipettes. T h e t e c h n i c a l assistance o f Miss Ulla Viitanen, Miss H e l e n a Kainulainen, Mrs. Anja KallionpS,~i, Miss Satu Kangas and Miss Kirsi-Marja Pylkk~inen is gratefully a c k n o w l e d g e d . REFERENCES Catt, K. and G.W. Tregear, 1967, Science 158, 1570. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Hunter, W.M. and F.C. Greenwood, 1962, Nature (London) 194,495. Krause, U. and V. Raunio, 1967, Acta Pathol. Microbio|. Scand. 71,328. Lambert, P-H., E. Tribollet, A. Celada, K. Madalinski, P.C. Frei and P-A. Miescher, 1976, J. Clin. Invest. (submitted). Leinikki, P. and Suvi P~ssil~, 1976, J. Clin. Pathol., 29 (in press). Reesink, H.W., W.J. Duimel and H.G.J. Brummelhuis, 1973, Lancet 2, 1351. Salmi, A., 1969, Acta Pathol. Microbiol. Scand. 76, 271. Steinbuch, M. and R. Audran, 1969, Arch. Biochem. Biophys. 134,279. Wadsworth, C., 1957, Int. Arch. All. 10,350. Wolters, G., L. Kuijpers, J. Ka~aki and A. Schuurs, 1976a, J. Inf. Dis. (in press). Wolters, G., L. Kuijpers, J. KaSaki and A. Schuurs, 1976b, J. Clin. Patho|. 29,873.