Journal oflmmunological Methods, 14 (1977) 111--122 © Elsevier/North-Holland Biomedical Press
111
RADIOIMMUNOASSAY OF CLASS-SPECIFIC ANTIBODIES (RIACA): CHICKEN ANTIBODIES TO DNP
MATTI K. VILJANEN, KAISA GRANFORS and PAAVO TOIVANEN Department of Medical Microbiology, Turku University, SF-20520 Turhu, Finland
(Received 27 February 1976, accepted 8 September 1976)
A radioimmunological method for the quantitation of class-specific antibodies has been developed. The method allows the quantitation of nanogram per ml concentrations of IgG- and IgM-anti-DNP antibodies without any physical or chemical pretreatment of the sample. The principle of the method is the same as in the radioimmunoassay of classspecific antibodies (RIACA) against BSA (Viljanen et al., 1975) developed recently by us. DNP was coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies were allowed to react with the coupled DNP and then quantitated by their capacity to bind 12SI-labeled anti-chicken-p or anti-chicken-% The inter-assay variation coefficients ranged from 8.1 to 14.7% and the mean standard deviations of duplicate determinations were about 11%. The combination of this method with the exact immunoradiometric quantitation of the total serum IgM and IgG, and with an immunoabsorption technique, makes possible to quantitate classspecific antibodies in weight units.
INTRODUCTION Most of the techniques used for the quantitation of anti-hapten antibodies are b a s e d o n t h e i n h i b i t i o n o f a serological r e a c t i o n b y free h a p t e n . T h e react i o n t o be i n h i b i t e d c a n be t h e p r e c i p i t a t i o n (Pauling et al., 1 9 4 4 ; K a p l a n a n d K a b a t , 1 9 6 6 ; J a t o n et al., 1 9 6 7 ; M o r e n o a n d K a b a t , 1 9 6 9 ) , h a e m a g g l u t i n a t i o n ( G r o f f et al., 1 9 6 7 ) or t h e lysis o f h a p t e n c o a t e d e r y t h r o c y t e s ( P a s a n e n a n d M~ikel~i, 1969). T h e i n a c t i v a t i o n o f a h a p t e n a t e d b a c t e r i o p h a g e b y a n t i - h a p t e n a n t i b o d i e s , a n d t h e i n h i b i t i o n o f this r e a c t i o n b y free h a p t e n has f o r m e d t h e basis o f a v e r y sensitive m e t h o d f o r t h e assay o f a n t i - h a p t e n a n t i b o d i e s (Sarvas a n d M~ikel~i, 1 9 7 0 ; J o r m a l a i n e n a n d M~ikel~i, 1971). Since the affinity of the antibodies affects both the inhibition of precipitation and t h e i n a c t i v a t i o n o f t h e h a p t e n a t e d phage, b o t h o f t h e s e t e c h n i q u e s p r o v i d e an o p p e r t u n i t y t o e v a l u a t e t h e q u a l i t y o f t h e a n t i b o d i e s a n a l y z e d . In absol u t e q u a n t i t a t i o n , h o w e v e r , this p r e s e n t s a d r a w b a c k : the m a t u r i t y o f t h e i m m u n e r e s p o n s e has t o be t a k e n i n t o c o n s i d e r a t i o n w h e n titers are c o m pared. T h e class-specific q u a n t i t a t i o n o f a n t i - h a p t e n a n t i b o d i e s has h i t h e r t o b e e n carried o u t b y r a t h e r c o m p l i c a t e d a n d o n l y s e m i q u a n t i t a t i v e t e c h n i q u e s . T h e
112
immunoglobulin class of the antibody can be determined by a combined use of the inhibition techniques and of physical separation of different immunoglobulins, or by using 2-mercapto ethanol. The haptenated phage techniques can also be used to evaluate the contribution of IgM and IgG to the antibody titer w i t h o u t further treatment of the sample (Sarvas and M~kel~i, 1970). The comparison of haemagglutinating and haemolytic antibody titers has also been used in the direct estimation of IgM and IgG antibodies (Neveu and Borduas, 1975). We have recently developed a radioimmunoassay of class-specific antibodies (RIACA) against BSA in the chicken (Viljanen et al., 1975). In the present work, the same principle is applied for the assay of anti-DNP antibod{es. DNP is coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies are allowed to react with the coupled DNP and then quantitated by their capacity to bind '2SI-labeled anti-p or anti-% The combination of this m e t h o d with immunoradiometric quantitation of the total immunoglobulin concentration (Viljanen, 1975) and an immunoabsorption technique made it possible to quantitate class-specific antibodies in weight units instead of titers. The accuracy and sensitivity of the m e t h o d allows exact quantitation of low antibody concentrations w i t h o u t any pretreatment of the sample. The technique also permits simultaneous quantitation of class-specific antibodies against both hapten and carrier. Thus, it makes possible detailed studies about the mechanisms involved in the immune responses against hapten-carrier conjugates. MATERIALS AND METHODS
Principle of the method The principle of the m e t h o d is the same as in the anti-BSA RIACA (Viljanen et al., 1975). DNP is conjugated to a cyanogen bromide activated paper disk employing lysine as a connection link. One of the amino groups in lysine is covalently coupled to an activated disk. DNP is then conjugated to the other amino group in the lysine molecule. For the reaction of anti-DNP antibodies with the coupled DNP, a DNP-disk is put into the sample to be analyzed. After an incubation, the disk is washed free of u n b o u n d antibodies, and antibodies on the disk are quantitated by their capacity to bind ,2 s I - l a b e l e d anti-chicken-p or anti-chicken-~.
Buffers As a general buffer 0.1 M sodium phosphate buffer, pH 7.5, was used (buffer A). For a n t i g e n ~ a n t i b o d y reactions this buffer was supplemented with 1.0% normal sheep plasma (buffer B). Paper disks with bound antibodies were washed with physiological saline containing 0.5% Tween 20.
113
Preparation of anti-chicken-p and anti-chicken-7 Chicken serum immunoglobulins were precipitated with sodium sulphate according to Benedict (1967). The precipitate was dissolved in borate buffer, pH 8.2 (T/2 = 0.16) and dialysed for 20 h against the same buffer. The dialysate was applied into a Sephadex G-200 column (2.5 X 90 cm) with the aid of a peristaltic pump from down to up. The column was equilibrated with the borate buffer, pH 8.2; the same buffer was used also for the elution. The two first elution peaks containing mostly IgM or IgG, respectively, were concentrated with 30% polyethylenglycol. The IgM fraction was recycled twice with the same Sephadex G-200 column. The IgG fraction was dialysed against a phosphate buffer, 0.1 M, pH 6.4, and further purified with the aid of a DEAE-cellulose chromatography; phosphate buffers of 0.1 M, pH 6.4, and of 0.2 M, pH 5.8, were used. In immunoelectrophoresis and in immunodiffusion against anti-chicken serum, both IgM and IgG preparations gave only one precipitation arc. These preparations were used to immunize rabbits and sheep. I m m u n e sera were absorbed by using solid immunoabsorbents (cyanogen bromide activated Sepharose 4B, Pharmacia, Uppsala, Sweden). Both antiIgG and anti-IgM sera were absorbed with agammaglobulinemic chicken serum. In addition, anti-IgG serum was absorbed with a purified chicken IgM, and anti-IgM serum was absorbed with newly-hatched chicken serum. The columns with serum-conjugated immunoabsorbents were equilibrated and eluted with borate buffer, pH 8.2 (T/2 = 0.16) and 0.5 M NaC1. Regeneration of the columns was carried out with 3 M NaSCN, pH 8.5 (Leslie and Martin, 1973). The absorbed anti-7 and anti-p antisera were further purified by absorption into and from Sepharose 4B conjugated chicken IgG and chicken IgM, respectively. The final preparations of anti-chicken-p and antichicken-3, were free of any other antibodies and serum proteins.
Iodination of anti-immunoglobulins A modified chloramine-T m e t h o d of Hunter and Greenwood (1962) was applied in the iodination of the anti-chicken-p and anti-chicken-% After the iodination the preparations were supplemented with 10% normal sheep plasma and stored at +4°C. Specific activities ranged from 10 to 30 pCi/pg.
Preparation of conjugates for immunization DNP-albumin conjugates were prepared according to the m e t h o d of Eisen (1953) using sodium 2,4,-dinitrobenzene sulphonate (Eastman Kodak Co., Rochester, N.Y. 14650, U.S.A.) and crystalline bovine serum albumin (Fraction V, Armour Pharmaceutical Co., Ltd., Eastbourne, England}. The hapten per carrier ratio used in this study was 24/1.
114
Activation of paper disks R o u n d filter paper disks (diameter 6 mm, weight 1.3 mg) were activated by cyanogen bromide in pH 10.5--11.0 for 5 min as described earlier (Viljanen et al., 1975).
Coupling o f DNP into the disks A b o u t 1 g o f activated disks and 1.5 g of L-lysine-monohydrochlorid were added in 40 ml of 0.1 M NaHCO3. The m i xt ure was enrolled overnight at r o o m t e m p e r a t u r e and u n b o u n d lysine was washed away three times with 40 ml of 0.1 NaHCO3. Any remaining active groups were reacted with 40 ml of 1.0 M ethanolamine at pH 8 for 2 h. The disks were then washed thrice with 40 ml o f 0.1 M acetate buf f e r at pH 4.0 and twice with 40 ml of assay buffer A. The disks were decanted relative free of buffer and added to 40 ml o f 0.01 M K2CO3 with 1.5 g of sodium 2,4,-dinitrobenzene sulphonate. This mix tu r e was enrolled overnight and the disks were then washed four times with 40 ml of buffer A. The disks were dried at r o o m t e m p e r a t u r e and stored at +4 °C.
Preparation of DNP-Sepharose 4B column A b o u t 200 mg of L-lysine-monohydrochlorid and an equal a m o u n t of sodium 2,4,-dinitrobenzene sulphonate were mixed with 5 g of swollen cyanogen b r o mi de activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) in 25 ml o f 0.01 M borate buffer containing 0.1 M NaC1 at pH 8.0. The conjugation procedure was carried out modifying slightly the instructions supplied by the manufacturer. Ca. 15 × 200 m m column filled with this conjugate was used for the i m m u n o a b s o r p t i o n procedures.
Preparation o f diluent plasma Normal chicken plasma was used for the preparation of anti-DNP antib o d y free plasma. Natural antibodies were eliminated by absorption to DNPSepharose 4B column avoiding dilution of the plasma during the procedure. T h e absorbed preparation (abs-NCP) was used as a diluent in the preparation o f standard curves.
Quantitation of IgM- and IgG-anti-DNP antibodies Phase 1. Sample plasma (0.5 pl or 100 pl), 1.0 ml of buffer B and a DNPdisk were mixed in a p o l y s t y r e n e tube (12 × 70 mm). The tubes were closed tightly and mixed for 1--3 h in a roller (30 rpm) at r o o m temperature. T h e r e a f t e r the disks were washed thrice with 2.5 ml of a mixture containing physiological saline and 0.5% Tween 20.
115
Phase 2. Ca. 15,000 cpm of ~2SI-labeled anti-chicken-p or anti-chicken-7 in 1.0 ml of buffer B was added to each tube. The tubes were enrolled overnight at room temperature, and the disks were washed as in the phase 1. The radioactivity on the disks was counted using the original tubes in a gammacounter (Wallac, Turku, Finland) for 10 min or until 10,000 cpm. All determinations were carried out in triplicate. Standardization As a standard, pooled plasma of DNP-BSA immunized chickens was used. The chickens had been immunized s.c. with 4 injections of 0.1 mg of DNPBSA in complete Freund's adjuvant, given at three week's intervals. The pool was stored at --20 ° C, divided in small aliquots. For a standard curve, serial dilutions were made for each daily assay. To eliminate the variations of protein concentration, abs-NCP was used as a diluent. For screening assays the standard plasma was diluted 1 : 1, 1 : 2, 1 : 10 and 1 • 100, and for the detection of low antibody concentrations (with 100 pl of the sample) the 10-fold dilutions were continued up to 1 : 10 000. The abs-NCP served as a negative control in both assays.
Quantitation of total IgM and IgG An immunoradiometric technique was applied for the quantitation of the total concentrations of IgM and IgG (Viljanen, 1975). The protein components of a plasma to be tested, including the immunoglobulins, were coupled covalently into an activated paper disk. After the coupling, the disk was washed free of u n b o u n d proteins, and the immunoglobulins on the disk were quantitated by their capacity to bind ~2SI-labeled anti-p or anti-%
Quantitation of anti-DNP antibodies in weight units The following procedure was applied to quantitate the anti-DNP antibodies in weight units per ml instead of percent of the standard plasma. First, concentrations of IgM and IgG in the standard plasma were quantitated as described above, and then, ca. 40 ml of the standard plasma was applied into the DNP-Sepharose 4B column. When about 75% of the total plasma volume had been eluted out of the column, a 1.0 ml sample was taken for further testing. In this way any dilution of the plasma during the absorption was avoided. In the sample, the concentrations of IgM- and IgG-anti-DNP were quantitated by RIACA. In addition, the total concentrations of IgM and IgG in the sample plasma were analyzed by the immunoradiometric technique. The assess the a m o u n t of immunoglobulins unspecifically bound to the column, the normal chicken plasma was absorbed exactly in the same way as the standard plasma. The weight unit correspondence of
116 one percent of the standard plasma was calculated from the following equation: A b -- Ababs R=
l g - - Igabs - - Iguabs
where, R = percent/weight unit ratio (percent × ml/pg); A b = concentration of DNP-specific IgM or IgG before absorption (%); Ababs = concentration of DNP-specific IgM or IgG after absorption (%); Ig = concentration of total IgM or IgG before absorption (pg/ml); Igab~ = concentration of total igM or IgG in the absorbed plasma (pg/ml); Iguab~ = a m o u n t of unspecifically absorbed IgM or IgG (pg/ml). Accuracy and sensitivity
The variation coefficients of the dose metameter and the detection limits were calculated according to Ekins (1974). The inter-assay variances were determined using two reference plasmas (A and B) included in each daily assay. Collection of samples
The blood samples from the experimental animals were collected from a wing vein into heparinized syringes. The plasmas were separated by immediate centrifugation after collection. The plasmas were stored at --20°C. RESULTS Q u a n t i t a t i o n o f a n t i b o d i e s in w e i g h t u n i t s
The DNP-Sepharose 4B column was able to absorb all of the anti-DNP antibodies, both of IgM and IgG classes, from the standard plasma applied. The decrease in the total concentration of IgG was 1730 pg/ml and t h a t of IgM 491 pg/ml. When the same procedure was applied for the normal chicken plasma, no unspecific absorption of IgG was observed. In contrast, concentration of IgM in the normal chicken plasma was decreased slightly by the absorption to the DNP-Sepharose 4B column. This decrease must be due to the unspecific binding of IgM, or to the binding of natural IgM-anti-DNP to the column. We prefer the latter alternative, since, when the abs-NCP was recycled through the DNP-Sepharose 4B column, no absorption of IgM occurred. On the basis of these findings, we conclude that the total concentration of IgG-anti-DNP in the standard plasma was 1730 pg/ml and that of IgM-anti-DNP 491 pg/ml.
117 Standard curves
F o r screening purposes the t e c hni que was used in a less sensitive form. Only 0.5 pl o f the sample plasma was used for the assay and the lowest IgGanti-DNP and IgM-anti-DNP concent r a t i ons used for the standard curve were 17.3 pg/ml and 4.9 pg/ml, respectively (fig. 1). The det ect i on limit of this screening system f or the antibodies of IgG class was 8.2 pg/ml and t hat for the antibodies o f IgM class 1.3 pg/ml. All samples with low a n t i b o d y concent rat i ons were also measured by a more sensitive version o f the m e t hod. Using 100 pl o f the sample plasma per an assay tube the detection limits were highly improved. With 100 pl volumes, the d etec t i on limit for the antibodies o f lgG class was 35 ng/ml and t h at for the antibodies of IgM class 4.9 ng/ml (fig. 2). The sensitivity of the m e t h o d can be increased even from this by applying larger sample volumes and longer incubation times.
IgM
4
x
IgG
~3
o
.
m
J
0
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,~
"
10
.
.
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10
.
.
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.
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5 x 1 0 3 pg ml I g G - a n t i - D N P
.
110
3
pg ml
IgM-anti-DNP
Fig. 1. Standard curves for screening of IgG-anti-DNP and IgM-anti-DNP concentrations. 0.5 gl of the sample plasma was used for each determination. Detection limits for IgG and IgM antibodies were 8.2 pg/ml and 1,3 pg/ml, respectively.
118
/
4
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=E a.
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"
.
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. 1 0. 2 .
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. 1 0.3
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10 4
' 1=0s
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5 x"1 0 3 ng rnl I g M - a n t i - D N P
ng rnl
Fig. 2. S t a n d a r d curves for d e t e c t i o n o f low c o n c e n t r a t i o n s o f a n t i - D N P a n t i b o d i e s . 100 pl o f t h e s a m p l e p l a s m a was used for e a c h d e t e r m i n a t i o n . D e t e c t i o n limit for IgGa n t i - D N P was 35 n g / m l a n d t h a t o f IgM-anti-DNP 4.9 ng/ml.
Variation coefficients o f duplicate determinations The variation coefficients of duplicate determinations were calculated from 30 r a n d o m l y selected duplicate determinations. T h e y represented IgGanti-DNP concentrations from 1.0 pg/ml upwards and IgM-anti-DNP concentrations from 0.2 pg/ml upwards. With duplicate determinations the mean variation coefficients of response m e t a m e t e r were
111
RADIOIMMUNOASSAY OF CLASS-SPECIFIC ANTIBODIES (RIACA): CHICKEN ANTIBODIES TO DNP
MATTI K. VILJANEN, KAISA GRANFORS and PAAVO TOIVANEN Department of Medical Microbiology, Turku University, SF-20520 Turhu, Finland
(Received 27 February 1976, accepted 8 September 1976)
A radioimmunological method for the quantitation of class-specific antibodies has been developed. The method allows the quantitation of nanogram per ml concentrations of IgG- and IgM-anti-DNP antibodies without any physical or chemical pretreatment of the sample. The principle of the method is the same as in the radioimmunoassay of classspecific antibodies (RIACA) against BSA (Viljanen et al., 1975) developed recently by us. DNP was coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies were allowed to react with the coupled DNP and then quantitated by their capacity to bind 12SI-labeled anti-chicken-p or anti-chicken-% The inter-assay variation coefficients ranged from 8.1 to 14.7% and the mean standard deviations of duplicate determinations were about 11%. The combination of this method with the exact immunoradiometric quantitation of the total serum IgM and IgG, and with an immunoabsorption technique, makes possible to quantitate classspecific antibodies in weight units.
INTRODUCTION Most of the techniques used for the quantitation of anti-hapten antibodies are b a s e d o n t h e i n h i b i t i o n o f a serological r e a c t i o n b y free h a p t e n . T h e react i o n t o be i n h i b i t e d c a n be t h e p r e c i p i t a t i o n (Pauling et al., 1 9 4 4 ; K a p l a n a n d K a b a t , 1 9 6 6 ; J a t o n et al., 1 9 6 7 ; M o r e n o a n d K a b a t , 1 9 6 9 ) , h a e m a g g l u t i n a t i o n ( G r o f f et al., 1 9 6 7 ) or t h e lysis o f h a p t e n c o a t e d e r y t h r o c y t e s ( P a s a n e n a n d M~ikel~i, 1969). T h e i n a c t i v a t i o n o f a h a p t e n a t e d b a c t e r i o p h a g e b y a n t i - h a p t e n a n t i b o d i e s , a n d t h e i n h i b i t i o n o f this r e a c t i o n b y free h a p t e n has f o r m e d t h e basis o f a v e r y sensitive m e t h o d f o r t h e assay o f a n t i - h a p t e n a n t i b o d i e s (Sarvas a n d M~ikel~i, 1 9 7 0 ; J o r m a l a i n e n a n d M~ikel~i, 1971). Since the affinity of the antibodies affects both the inhibition of precipitation and t h e i n a c t i v a t i o n o f t h e h a p t e n a t e d phage, b o t h o f t h e s e t e c h n i q u e s p r o v i d e an o p p e r t u n i t y t o e v a l u a t e t h e q u a l i t y o f t h e a n t i b o d i e s a n a l y z e d . In absol u t e q u a n t i t a t i o n , h o w e v e r , this p r e s e n t s a d r a w b a c k : the m a t u r i t y o f t h e i m m u n e r e s p o n s e has t o be t a k e n i n t o c o n s i d e r a t i o n w h e n titers are c o m pared. T h e class-specific q u a n t i t a t i o n o f a n t i - h a p t e n a n t i b o d i e s has h i t h e r t o b e e n carried o u t b y r a t h e r c o m p l i c a t e d a n d o n l y s e m i q u a n t i t a t i v e t e c h n i q u e s . T h e
112
immunoglobulin class of the antibody can be determined by a combined use of the inhibition techniques and of physical separation of different immunoglobulins, or by using 2-mercapto ethanol. The haptenated phage techniques can also be used to evaluate the contribution of IgM and IgG to the antibody titer w i t h o u t further treatment of the sample (Sarvas and M~kel~i, 1970). The comparison of haemagglutinating and haemolytic antibody titers has also been used in the direct estimation of IgM and IgG antibodies (Neveu and Borduas, 1975). We have recently developed a radioimmunoassay of class-specific antibodies (RIACA) against BSA in the chicken (Viljanen et al., 1975). In the present work, the same principle is applied for the assay of anti-DNP antibod{es. DNP is coupled covalently to a cyanogen bromide activated paper disk with the augmentation of lysine molecule. Anti-DNP antibodies are allowed to react with the coupled DNP and then quantitated by their capacity to bind '2SI-labeled anti-p or anti-% The combination of this m e t h o d with immunoradiometric quantitation of the total immunoglobulin concentration (Viljanen, 1975) and an immunoabsorption technique made it possible to quantitate class-specific antibodies in weight units instead of titers. The accuracy and sensitivity of the m e t h o d allows exact quantitation of low antibody concentrations w i t h o u t any pretreatment of the sample. The technique also permits simultaneous quantitation of class-specific antibodies against both hapten and carrier. Thus, it makes possible detailed studies about the mechanisms involved in the immune responses against hapten-carrier conjugates. MATERIALS AND METHODS
Principle of the method The principle of the m e t h o d is the same as in the anti-BSA RIACA (Viljanen et al., 1975). DNP is conjugated to a cyanogen bromide activated paper disk employing lysine as a connection link. One of the amino groups in lysine is covalently coupled to an activated disk. DNP is then conjugated to the other amino group in the lysine molecule. For the reaction of anti-DNP antibodies with the coupled DNP, a DNP-disk is put into the sample to be analyzed. After an incubation, the disk is washed free of u n b o u n d antibodies, and antibodies on the disk are quantitated by their capacity to bind ,2 s I - l a b e l e d anti-chicken-p or anti-chicken-~.
Buffers As a general buffer 0.1 M sodium phosphate buffer, pH 7.5, was used (buffer A). For a n t i g e n ~ a n t i b o d y reactions this buffer was supplemented with 1.0% normal sheep plasma (buffer B). Paper disks with bound antibodies were washed with physiological saline containing 0.5% Tween 20.
113
Preparation of anti-chicken-p and anti-chicken-7 Chicken serum immunoglobulins were precipitated with sodium sulphate according to Benedict (1967). The precipitate was dissolved in borate buffer, pH 8.2 (T/2 = 0.16) and dialysed for 20 h against the same buffer. The dialysate was applied into a Sephadex G-200 column (2.5 X 90 cm) with the aid of a peristaltic pump from down to up. The column was equilibrated with the borate buffer, pH 8.2; the same buffer was used also for the elution. The two first elution peaks containing mostly IgM or IgG, respectively, were concentrated with 30% polyethylenglycol. The IgM fraction was recycled twice with the same Sephadex G-200 column. The IgG fraction was dialysed against a phosphate buffer, 0.1 M, pH 6.4, and further purified with the aid of a DEAE-cellulose chromatography; phosphate buffers of 0.1 M, pH 6.4, and of 0.2 M, pH 5.8, were used. In immunoelectrophoresis and in immunodiffusion against anti-chicken serum, both IgM and IgG preparations gave only one precipitation arc. These preparations were used to immunize rabbits and sheep. I m m u n e sera were absorbed by using solid immunoabsorbents (cyanogen bromide activated Sepharose 4B, Pharmacia, Uppsala, Sweden). Both antiIgG and anti-IgM sera were absorbed with agammaglobulinemic chicken serum. In addition, anti-IgG serum was absorbed with a purified chicken IgM, and anti-IgM serum was absorbed with newly-hatched chicken serum. The columns with serum-conjugated immunoabsorbents were equilibrated and eluted with borate buffer, pH 8.2 (T/2 = 0.16) and 0.5 M NaC1. Regeneration of the columns was carried out with 3 M NaSCN, pH 8.5 (Leslie and Martin, 1973). The absorbed anti-7 and anti-p antisera were further purified by absorption into and from Sepharose 4B conjugated chicken IgG and chicken IgM, respectively. The final preparations of anti-chicken-p and antichicken-3, were free of any other antibodies and serum proteins.
Iodination of anti-immunoglobulins A modified chloramine-T m e t h o d of Hunter and Greenwood (1962) was applied in the iodination of the anti-chicken-p and anti-chicken-% After the iodination the preparations were supplemented with 10% normal sheep plasma and stored at +4°C. Specific activities ranged from 10 to 30 pCi/pg.
Preparation of conjugates for immunization DNP-albumin conjugates were prepared according to the m e t h o d of Eisen (1953) using sodium 2,4,-dinitrobenzene sulphonate (Eastman Kodak Co., Rochester, N.Y. 14650, U.S.A.) and crystalline bovine serum albumin (Fraction V, Armour Pharmaceutical Co., Ltd., Eastbourne, England}. The hapten per carrier ratio used in this study was 24/1.
114
Activation of paper disks R o u n d filter paper disks (diameter 6 mm, weight 1.3 mg) were activated by cyanogen bromide in pH 10.5--11.0 for 5 min as described earlier (Viljanen et al., 1975).
Coupling o f DNP into the disks A b o u t 1 g o f activated disks and 1.5 g of L-lysine-monohydrochlorid were added in 40 ml of 0.1 M NaHCO3. The m i xt ure was enrolled overnight at r o o m t e m p e r a t u r e and u n b o u n d lysine was washed away three times with 40 ml of 0.1 NaHCO3. Any remaining active groups were reacted with 40 ml of 1.0 M ethanolamine at pH 8 for 2 h. The disks were then washed thrice with 40 ml o f 0.1 M acetate buf f e r at pH 4.0 and twice with 40 ml of assay buffer A. The disks were decanted relative free of buffer and added to 40 ml o f 0.01 M K2CO3 with 1.5 g of sodium 2,4,-dinitrobenzene sulphonate. This mix tu r e was enrolled overnight and the disks were then washed four times with 40 ml of buffer A. The disks were dried at r o o m t e m p e r a t u r e and stored at +4 °C.
Preparation of DNP-Sepharose 4B column A b o u t 200 mg of L-lysine-monohydrochlorid and an equal a m o u n t of sodium 2,4,-dinitrobenzene sulphonate were mixed with 5 g of swollen cyanogen b r o mi de activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) in 25 ml o f 0.01 M borate buffer containing 0.1 M NaC1 at pH 8.0. The conjugation procedure was carried out modifying slightly the instructions supplied by the manufacturer. Ca. 15 × 200 m m column filled with this conjugate was used for the i m m u n o a b s o r p t i o n procedures.
Preparation o f diluent plasma Normal chicken plasma was used for the preparation of anti-DNP antib o d y free plasma. Natural antibodies were eliminated by absorption to DNPSepharose 4B column avoiding dilution of the plasma during the procedure. T h e absorbed preparation (abs-NCP) was used as a diluent in the preparation o f standard curves.
Quantitation of IgM- and IgG-anti-DNP antibodies Phase 1. Sample plasma (0.5 pl or 100 pl), 1.0 ml of buffer B and a DNPdisk were mixed in a p o l y s t y r e n e tube (12 × 70 mm). The tubes were closed tightly and mixed for 1--3 h in a roller (30 rpm) at r o o m temperature. T h e r e a f t e r the disks were washed thrice with 2.5 ml of a mixture containing physiological saline and 0.5% Tween 20.
115
Phase 2. Ca. 15,000 cpm of ~2SI-labeled anti-chicken-p or anti-chicken-7 in 1.0 ml of buffer B was added to each tube. The tubes were enrolled overnight at room temperature, and the disks were washed as in the phase 1. The radioactivity on the disks was counted using the original tubes in a gammacounter (Wallac, Turku, Finland) for 10 min or until 10,000 cpm. All determinations were carried out in triplicate. Standardization As a standard, pooled plasma of DNP-BSA immunized chickens was used. The chickens had been immunized s.c. with 4 injections of 0.1 mg of DNPBSA in complete Freund's adjuvant, given at three week's intervals. The pool was stored at --20 ° C, divided in small aliquots. For a standard curve, serial dilutions were made for each daily assay. To eliminate the variations of protein concentration, abs-NCP was used as a diluent. For screening assays the standard plasma was diluted 1 : 1, 1 : 2, 1 : 10 and 1 • 100, and for the detection of low antibody concentrations (with 100 pl of the sample) the 10-fold dilutions were continued up to 1 : 10 000. The abs-NCP served as a negative control in both assays.
Quantitation of total IgM and IgG An immunoradiometric technique was applied for the quantitation of the total concentrations of IgM and IgG (Viljanen, 1975). The protein components of a plasma to be tested, including the immunoglobulins, were coupled covalently into an activated paper disk. After the coupling, the disk was washed free of u n b o u n d proteins, and the immunoglobulins on the disk were quantitated by their capacity to bind ~2SI-labeled anti-p or anti-%
Quantitation of anti-DNP antibodies in weight units The following procedure was applied to quantitate the anti-DNP antibodies in weight units per ml instead of percent of the standard plasma. First, concentrations of IgM and IgG in the standard plasma were quantitated as described above, and then, ca. 40 ml of the standard plasma was applied into the DNP-Sepharose 4B column. When about 75% of the total plasma volume had been eluted out of the column, a 1.0 ml sample was taken for further testing. In this way any dilution of the plasma during the absorption was avoided. In the sample, the concentrations of IgM- and IgG-anti-DNP were quantitated by RIACA. In addition, the total concentrations of IgM and IgG in the sample plasma were analyzed by the immunoradiometric technique. The assess the a m o u n t of immunoglobulins unspecifically bound to the column, the normal chicken plasma was absorbed exactly in the same way as the standard plasma. The weight unit correspondence of
116 one percent of the standard plasma was calculated from the following equation: A b -- Ababs R=
l g - - Igabs - - Iguabs
where, R = percent/weight unit ratio (percent × ml/pg); A b = concentration of DNP-specific IgM or IgG before absorption (%); Ababs = concentration of DNP-specific IgM or IgG after absorption (%); Ig = concentration of total IgM or IgG before absorption (pg/ml); Igab~ = concentration of total igM or IgG in the absorbed plasma (pg/ml); Iguab~ = a m o u n t of unspecifically absorbed IgM or IgG (pg/ml). Accuracy and sensitivity
The variation coefficients of the dose metameter and the detection limits were calculated according to Ekins (1974). The inter-assay variances were determined using two reference plasmas (A and B) included in each daily assay. Collection of samples
The blood samples from the experimental animals were collected from a wing vein into heparinized syringes. The plasmas were separated by immediate centrifugation after collection. The plasmas were stored at --20°C. RESULTS Q u a n t i t a t i o n o f a n t i b o d i e s in w e i g h t u n i t s
The DNP-Sepharose 4B column was able to absorb all of the anti-DNP antibodies, both of IgM and IgG classes, from the standard plasma applied. The decrease in the total concentration of IgG was 1730 pg/ml and t h a t of IgM 491 pg/ml. When the same procedure was applied for the normal chicken plasma, no unspecific absorption of IgG was observed. In contrast, concentration of IgM in the normal chicken plasma was decreased slightly by the absorption to the DNP-Sepharose 4B column. This decrease must be due to the unspecific binding of IgM, or to the binding of natural IgM-anti-DNP to the column. We prefer the latter alternative, since, when the abs-NCP was recycled through the DNP-Sepharose 4B column, no absorption of IgM occurred. On the basis of these findings, we conclude that the total concentration of IgG-anti-DNP in the standard plasma was 1730 pg/ml and that of IgM-anti-DNP 491 pg/ml.
117 Standard curves
F o r screening purposes the t e c hni que was used in a less sensitive form. Only 0.5 pl o f the sample plasma was used for the assay and the lowest IgGanti-DNP and IgM-anti-DNP concent r a t i ons used for the standard curve were 17.3 pg/ml and 4.9 pg/ml, respectively (fig. 1). The det ect i on limit of this screening system f or the antibodies of IgG class was 8.2 pg/ml and t hat for the antibodies o f IgM class 1.3 pg/ml. All samples with low a n t i b o d y concent rat i ons were also measured by a more sensitive version o f the m e t hod. Using 100 pl o f the sample plasma per an assay tube the detection limits were highly improved. With 100 pl volumes, the d etec t i on limit for the antibodies o f lgG class was 35 ng/ml and t h at for the antibodies of IgM class 4.9 ng/ml (fig. 2). The sensitivity of the m e t h o d can be increased even from this by applying larger sample volumes and longer incubation times.
IgM
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IgM-anti-DNP
Fig. 1. Standard curves for screening of IgG-anti-DNP and IgM-anti-DNP concentrations. 0.5 gl of the sample plasma was used for each determination. Detection limits for IgG and IgM antibodies were 8.2 pg/ml and 1,3 pg/ml, respectively.
118
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Fig. 2. S t a n d a r d curves for d e t e c t i o n o f low c o n c e n t r a t i o n s o f a n t i - D N P a n t i b o d i e s . 100 pl o f t h e s a m p l e p l a s m a was used for e a c h d e t e r m i n a t i o n . D e t e c t i o n limit for IgGa n t i - D N P was 35 n g / m l a n d t h a t o f IgM-anti-DNP 4.9 ng/ml.
Variation coefficients o f duplicate determinations The variation coefficients of duplicate determinations were calculated from 30 r a n d o m l y selected duplicate determinations. T h e y represented IgGanti-DNP concentrations from 1.0 pg/ml upwards and IgM-anti-DNP concentrations from 0.2 pg/ml upwards. With duplicate determinations the mean variation coefficients of response m e t a m e t e r were
