Journal of Immunological Methods, 25 (1979) 365--374
365
© Elsevier/North-Holland Biomedical Press
DETECTION OF ANTIBODY-FORMING CELLS AND HUMORAL ANTIBODY TO HORSERADISH PEROXIDASE *
MICHAEL BURKE, DOROTHY HARRIS, B. DAVID STOLLAR, HALINA BOREL and YVES BOREL Division of Immunology, Department of Pediatrics, ChiMren's Hospital Medical Center and Harvard Medical School, Boston, MA 02115, and Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, U.S.A.
(Received 19 June 1978, accepted 12 September 1978)
Two new simple methods for detecting antibody-forming cells by hemolytic plaque assay and hemagglutinating antibody to horseradish peroxidase have been developed in mice. Both techniques utilize as target, sheep erythrocytes coupled directly with horseradish peroxidase. These assays are sensitive, antigen-specific and are useful to quantitate both direct and indirect antibody-forming cells and humoral antibodies.
INTRODUCTION Since the introduction of horseradish peroxidase (HRP) as a marker for antibodies (Avrameas and Uriel, 1960; Nakane and Pierce, 1966), the enzyme has been extensively used in immunocytochemistry. It is a hemecontaining glycoprotein of relatively low molecular weight (about 40,000) and its intracellular localization may be detected by light, electron (Avrameas, 1972) and fluorescent microscopy (Papadimitrou et al., 1976). HRP is itself highly immunogenic and has been shown to elicit the formation of IgG (Avrameas, 1975) and IgE (Spitzer et al., 1973) antibodies. Anti-HRP antibody-forming cells (AFC) have been demonstrated by immunocytochemical staining methods in lymph nodes of rabbits and rodents (Avrameas and Leduc, 1970; Kuhlmann and Avrameas, 1972; Miller et al., 1974) and by hemolytic plaque assay in the rabbit (Lemieux et al., 1974} and mouse (Tern y n c k et al., 1977). Circulating antibody has been detected in mice (Hess et al., 1971), various rodents and sheep (Avrameas, 1975). Here we report simple techniques to detect AFC and hemagglutinating antibody to HRP in the mouse, using as a target sheep erythrocytes coated directly with HRP. The methods are sensitive, antigen-specific and provide rapid quantitative assays of anti-HRP AFC and antibody.
* These studies were supported by NIH Grant No. AI 13867.
366 MATERIALS AND METHODS
Animals C57BL/6, BALB/c and (C57BL/6 × DBA/2)F1 referred to as BDF,, 6--10week-old male mice and an outbred rabbit were obtained from Jackson Laboratories, Bar Harbor, ME. Antigens HRP grade I (RZ = 3) and HRP grade II (RZ = 1.3) were purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN. Dinitrophenyl conjugated keyhole limpet hemocyanin (DNPs0KLH) was prepared as previously described (Golan and Borel, 1971). (The subscript refers to the number of moles of hapten per mole of carrier.) Conjugation o f HRP to sheep red blood cells (SRBC) SRBC obtained from Colorado Serum Co. Laboratories were washed extensively (4 or 5 times) with 0.15 M NaHCO3 before coupling with HRP. Target HRP-conjugated SRBC were prepared by a modification of the periodate oxidation technique from Erlanger and Beiser (1964) and Nakane and Kawaoi (1974). 5 mg of HRP grade I or 10 mg of HRP grade II were dissolved in 0.5 ml of 0.15 M NaHCO3. An equal volume of 0.12 M NaIO4 was added and the mixture was gently stirred for 30 min at room temperature. The mixture was then immediately added to a Sephadex G-25 column and developed against an 0.15 M NaHCO3 (pH 9.5) buffer. The eluate from the colored peak containing the oxidized HI%P was collected and then added dropwise to 2.5 ml of a 20% cell suspension of washed SRBC in M NaHCO3 inside a 50 ml centrifuge tube. The mixture was gently stirred at room temperature for 15 min after which time 100 mg of t-butylamine borane (Aldrich Chemical Co., Milwaukee, WI) in 5 ml of bicarbonate solution were added and the mixture stirred for another 3 min. The tube was then quickly filled with isotonic bicarbonate and centrifuged at 2000 rev/min for 10 min. The cells were washed extensively in bicarbonate (about 4--6 times) and either used immediately or stored overnight at 4 °C. HRP-coated SRBC were tested in microtitration V-bottom plates with rabbit anti-HRP serum prior to their use in the plaque assay. Hemolytic plaque assay AFC were determined by a modification of the hemolytic plaque assay (Jerne and Nordin, 1963) using HRP-SRBC as target cells. Spleen cells were suspended in tissue culture medium TCM 858 from Difco. Agar purified from Difco and DEAE dextran 10 mg/ml pH 7.0 were used. Guinea pig complement was o b t a i n e d from Biological Associates, MD and used in a 1/30 dilution. 1.5--3.0 × 106 nucleated cells were assayed per plate. Direct (19S) plaques were counted 90--120 min after the addition of complement. The indirect plaque-forming cells were revealed by use of a rabbit anti-mouse IgG
367 serum shown to react with IgG1, IgG2a, IgG2b and IgG3 on immunodiffusion. The serum was used at a dilution of 1/200. IgG (7S) PFC were obtained by subtracting the direct PFC from the total number of PFC. In the inhibition study, free HRP was added to the agar plate prior to incubation, as described in Results. In one experiment, trinitrophenyl-substituted sheep erythrocytes (TNPSRBC), prepared by the m e t h o d of Rittenberg and Pratt (1969), were used in the plaque assay. Statistical analysis was performed according to Student's t test. For PFC, the geometric mean (+S.E.) for each group was calculated.
Microhemagglu tination assay for circulating an tibody Circulating antibody was determined by a modification of the hemagglutination assay of Wegmann and Smithies (1966). Briefly, properly absorbed antisera were serially diluted in phosphate-buffered saline (PBS) pH 7.4, containing 0.5% of bovine serum albumin (BSA). SRBC coated with HRP and uncoated were suspended at a concentration of 1/6% in PBS, containing 0.25% of BSA. For hemagglutination inhibition, one drop of free antigen, HRP and ovalbumin, respectively at a concentration of 0.5 mg/ml in PBS alone was added to one drop of the respective serum dilution in the wells of microtiter V-plates. For agglutination, one drop of PBS diluent alone replaced the antigen solution. The plates were then covered with another microtiter plate, wrapped with plastic film and incubated 30--45 min at 37°C. After this time, one drop of the red cell suspension was added to each well, the microtiter plates covered as above and incubated overnight at room temperature. The plates were then spun at 1500 rev/min for 90 sec and the direct agglutination titer read. For the indirect titer, the plates were treated in the following manner. The supernatant was removed by suction using a 19-gauge needle, 3 drops of PBS alone were delivered into each well and the contents were mixed by agitation with a thin jet of air. After the plates were spun at 2000 rev/min for 2 min, the supernatant was removed and 2 drops of rabbit anti-mouse IgG serum diluted 1 : 200 in PBS containing 0.25% of BSA was added to each well. The contents were mixed, the plates covered as before and incubated at 37°C for 2 h. They were spun again at 1500 rev/min for 90 sec and the indirect agglutination titer was read. As controls, uncoated SRBC were tested in a parallel manner with the respective antisera. In addition, HRP-coated cells were added to PBS diluent and to absorbed normal mouse serum dilutions, with and w i t h o u t inhibiting antigen. Static electrical effects, 'spread', were minimized by spraying the undersurface of the plates before use with an anti-static aerosol. Although the spread could not be entirely prevented at high antibody concentrations in the indirect test, the titration end-point was evident.
368
Hyperimmune antiserum to HRP This was obtained by bleeding a rabbit which had been immunized with 3 weekly injections of 1 mg HRP grade I in complete Freund's adjuvant. Precipitating antibody to HRP was demonstrated in the serum (up to a dilution of 1 : 8) by immunodiffusion in an Ouchterlony plate. RESULTS
Demonstration of the presence o f coupled HRP-SRBC by passive hemagglutination Initially, to demonstrate that HRP was indeed bound to the sheep erythrocytes by the conjugation procedure outlined above, direct passive hemagglutination was performed, using SRBC both uncoated and coated with HRP grade I or II and h y p e r i m m u n e rabbit anti-HRP antiserum and, as a control, normal (non-immunized) rabbit serum. With HRP-SRBC and anti-Hl~P serum a direct titer of 1 : 2560 was found. There was no hemagglutination when either normal serum or uncoated SRBC was used. Development o f plaque-forming cells (PFC) in mice immunized with HRP Groups of 6 C57BL/6 mice were immunized with a single intraperitoneal injection 0.2 mg HRP (grade II) in complete Freund's adjuvant (CFA). The mice were sacrificed and their spleen cells assayed for direct and indirect PFC on various days after antigen challenge. The peak of anti-HRP response was obtained on day 5 for direct PFC and day 6 for indirect PFC (Fig. 1). The background responses with uncoated SRBC was minimal in mice immunized
20,000
uJZ15,000 (D
~
I
10,00C 5,000 I
0
I
L
I
4 5 6 7 DAYSAFTERIMMUNIZATION
8
Fig. 1. D i r e c t ( ) and indirect ( ...... ) P F C / s p l e e n ( g e o m e t r i c m e a n ± S . E . ) to c o n j u g a t e d H R P - S R B C f o r g r o u p s o f 6 m i c e i m m u n i z e d w i t h 0 . 2 m g H R P , g r a d e II, in C F A . Number of plaques counted per plate, direct
day day day day day
4 5 6 7 8
60 -175 -66-104-56--
250 336 380 236 232
indirect 76 -- 122 38 - - 2 5 0 30 -- 120 2-36
369 with HRP. As an additional control, 5 unimmunized mice produced virtually no PFC to HRP-coated SRBC.
Effect o f using different grades of HRP on target cells In order to compare the effect of coupling different grades of HRP to SRBC, 5 C57BL/6 mice were immunized with 0.1 mg HRP grade I, in CFA i.p. and PFC (direct and indirect) were measured on day 6 using SRBC coupled to both grades I and II o f HRP, (HRP (I)-SRBC and HRP (II)-SRBC respectively). No significant difference was observed between the two types o f conjugated target cells (Table 1) for direct as well as indirect PFC, although indirect PFC obtained with HRP (I)-SRBC were higher than those using HRP (II)-SRBC. Thus, grade II HRP may be readily used to detect direct AFC to HRP. Inhibition study 5 C57BL/6 mice were immunized with 0.1 mg HRP grade I in CFA and PFC determined on day 6. Direct and indirect anti-HRP PFC response were estimated using SRBC coupled to both grades I and II HRP. 1.25, 2.5 and 5.0 X 10 -9 moles of grade I HRP respectively were added to agar plates. As shown in Fig. 2, the direct PFC responses with both HRP (I)-SRBC and HRP (II)-SRBC were inhibited by the addition of free HRP, and this was dosedependent. Similar results (not shown here) were obtained for indirect PFC responses in both cases. Immune response to an unrelated antigen (DNP-KLH) As a further study for the specificity o f the plaque-forming cell to HRP, 5 C57BL/6 mice were immunized i.p. with 0.2 mg o f DNPs0KLH in CFA. On day 5, the mice were sacrificed and PFC were assessed using 3 types of target cells: (a) trinitrophenyl-substituted sheep erythrocytes (TNP-SRBC), (b) HRP-SRBC and (c) uncoated SRBC. At the time of pouring the agar, HRP grade I, in a dose of 5.0 X 10 -9 moles, was added to a second group of plates containing TNP-SRBC. Fig. 3 shows that animals immunized with DNP-KLH do not produce AFC to HRP and the addition of HRP in a con-
TABLE 1 PFC PER SPLEEN USING SRC COUPLED TO HRP GRADES I AND II The number of plaques counted per plate was HRP grade I direct 54--194, grade II direct 50--164, grade I indirect 6--22, grade II indirect 1--8.
Direct Indirect
HRP ( I ) - S R C
HRP(II)-SRC
P
13,847 (4222) 1,510 (408)
12,049 (3798) 720 (146)
NS NS
370
TARGET CELLS HRP[I)-SRBC
IT
T A R G E T ( E L L S HRP ( ] I ) S R B C
8000
(1) -H v
6000
Z
LU W --/ (1. u') (D U(1.
4000
\ I
2000
i
1
I
I
L
0
t,25
2.5
5,0
tl
I
0
~
~,25 2.5
I
5.0
Number of moles xIO-9of HRP odded to A(~or
Fig. 2. I n h i b i t i o n of d i r e c t P F C b y free HRP. S h o w n are P F C / s p l e e n ( g e o m e t r i c m e a n ± S.E.) for 5 mice i m m u n i z e d w i t h 0.1 mg HRP, grade I, in C F A 6 days previously. T h e target cells are S R B C b o u n d to HRP, grades I a n d II, respectively. A d o s e - d e p e n d e n t inhibit i o n of a n t i - H R P PFC o c c u r s o n t h e a d d i t i o n o f free H R P t o t h e agar.
centration response.
that
inhibited
anti-HRP
P F C has n o e f f e c t o n the a n t i - T N P
Immunization o f different strains o f mice T o d e t e r m i n e w h e t h e r o t h e r strains o f m i c e r e s p o n d t o H R P , g r o u p s o f
371 35,000 TNP-SRBC 30,000
25,°°° /
1
~ 2o,oooi (O 15,000 -
10,000
5,000 HRP-SRBC SRBC &lone HRP added (5 x lO-gmoles)
Fig. 3. D i r e c t P F C / s p l e e n ( g e o m e t r i c mean-+ S.E.) for mice i m m u n i z e d w i t h 0.2 m g D N P s 0 K L H in CFA. T h e r e is a high r e s p o n s e w h e n S R B C b o u n d t o TNP are used as target cells, b u t n o t for u n c o a t e d S R C o r for HRP-SRC. T h e a d d i t i o n o f H R P t o t h e agar does n o t i n h i b i t P F C t o TNP-SRC.
5 mice -- C57BL/6, BDF~ and BALB/c -- were each immunized with 0.1 mg HRP grade I in CFA i.p. Direct PFC were measured on day 5. BDF1 mice appear to respond m or e than the BALB/c in terms o f direct AFC (Table 2).
Detection of circulating antibody by passive hemagglutination 6 C57BL/6 mice were immunized with an i.p. injection o f 0.1 mg HRP grade I in CFA and then bled on days 7 and 21. T h e y were then given a b o o s ter injection with 0.05 mg HRP in CFA and bled one week later (day 28). The sera was pooled, absorbed with SRBC, and the anti-HRP hemagglutinating a n t i b o d y was determined as described in Methods. As d e m o n s t r a t e d in Table 3, a specific anti-HRP titer was obtained on days 7 and 21 after primary immunization, as well as one week after secondary immu n izatio n with HRP. These titers were enhanced by the addition of anti-mouse IgG. F u r t h e r m o r e , the hemagglutination was completely inhibTABLE 2 D I R E C T P F C P E R S P L E E N IN V A R I O U S S T R A I N S O F MICE I M M U N I Z E D WITH HRP Strain
N u m b e r o f mice
P F C / s p l e e n (_+S.E.)
P
BDF1 BALB/c C57BL/6
5 5 5
1 7 , 2 9 9 4 (_+2044) 3 , 6 5 7 (_+1584) 1 2 , 0 4 9 (-+3798)
NS
365
© Elsevier/North-Holland Biomedical Press
DETECTION OF ANTIBODY-FORMING CELLS AND HUMORAL ANTIBODY TO HORSERADISH PEROXIDASE *
MICHAEL BURKE, DOROTHY HARRIS, B. DAVID STOLLAR, HALINA BOREL and YVES BOREL Division of Immunology, Department of Pediatrics, ChiMren's Hospital Medical Center and Harvard Medical School, Boston, MA 02115, and Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, U.S.A.
(Received 19 June 1978, accepted 12 September 1978)
Two new simple methods for detecting antibody-forming cells by hemolytic plaque assay and hemagglutinating antibody to horseradish peroxidase have been developed in mice. Both techniques utilize as target, sheep erythrocytes coupled directly with horseradish peroxidase. These assays are sensitive, antigen-specific and are useful to quantitate both direct and indirect antibody-forming cells and humoral antibodies.
INTRODUCTION Since the introduction of horseradish peroxidase (HRP) as a marker for antibodies (Avrameas and Uriel, 1960; Nakane and Pierce, 1966), the enzyme has been extensively used in immunocytochemistry. It is a hemecontaining glycoprotein of relatively low molecular weight (about 40,000) and its intracellular localization may be detected by light, electron (Avrameas, 1972) and fluorescent microscopy (Papadimitrou et al., 1976). HRP is itself highly immunogenic and has been shown to elicit the formation of IgG (Avrameas, 1975) and IgE (Spitzer et al., 1973) antibodies. Anti-HRP antibody-forming cells (AFC) have been demonstrated by immunocytochemical staining methods in lymph nodes of rabbits and rodents (Avrameas and Leduc, 1970; Kuhlmann and Avrameas, 1972; Miller et al., 1974) and by hemolytic plaque assay in the rabbit (Lemieux et al., 1974} and mouse (Tern y n c k et al., 1977). Circulating antibody has been detected in mice (Hess et al., 1971), various rodents and sheep (Avrameas, 1975). Here we report simple techniques to detect AFC and hemagglutinating antibody to HRP in the mouse, using as a target sheep erythrocytes coated directly with HRP. The methods are sensitive, antigen-specific and provide rapid quantitative assays of anti-HRP AFC and antibody.
* These studies were supported by NIH Grant No. AI 13867.
366 MATERIALS AND METHODS
Animals C57BL/6, BALB/c and (C57BL/6 × DBA/2)F1 referred to as BDF,, 6--10week-old male mice and an outbred rabbit were obtained from Jackson Laboratories, Bar Harbor, ME. Antigens HRP grade I (RZ = 3) and HRP grade II (RZ = 1.3) were purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN. Dinitrophenyl conjugated keyhole limpet hemocyanin (DNPs0KLH) was prepared as previously described (Golan and Borel, 1971). (The subscript refers to the number of moles of hapten per mole of carrier.) Conjugation o f HRP to sheep red blood cells (SRBC) SRBC obtained from Colorado Serum Co. Laboratories were washed extensively (4 or 5 times) with 0.15 M NaHCO3 before coupling with HRP. Target HRP-conjugated SRBC were prepared by a modification of the periodate oxidation technique from Erlanger and Beiser (1964) and Nakane and Kawaoi (1974). 5 mg of HRP grade I or 10 mg of HRP grade II were dissolved in 0.5 ml of 0.15 M NaHCO3. An equal volume of 0.12 M NaIO4 was added and the mixture was gently stirred for 30 min at room temperature. The mixture was then immediately added to a Sephadex G-25 column and developed against an 0.15 M NaHCO3 (pH 9.5) buffer. The eluate from the colored peak containing the oxidized HI%P was collected and then added dropwise to 2.5 ml of a 20% cell suspension of washed SRBC in M NaHCO3 inside a 50 ml centrifuge tube. The mixture was gently stirred at room temperature for 15 min after which time 100 mg of t-butylamine borane (Aldrich Chemical Co., Milwaukee, WI) in 5 ml of bicarbonate solution were added and the mixture stirred for another 3 min. The tube was then quickly filled with isotonic bicarbonate and centrifuged at 2000 rev/min for 10 min. The cells were washed extensively in bicarbonate (about 4--6 times) and either used immediately or stored overnight at 4 °C. HRP-coated SRBC were tested in microtitration V-bottom plates with rabbit anti-HRP serum prior to their use in the plaque assay. Hemolytic plaque assay AFC were determined by a modification of the hemolytic plaque assay (Jerne and Nordin, 1963) using HRP-SRBC as target cells. Spleen cells were suspended in tissue culture medium TCM 858 from Difco. Agar purified from Difco and DEAE dextran 10 mg/ml pH 7.0 were used. Guinea pig complement was o b t a i n e d from Biological Associates, MD and used in a 1/30 dilution. 1.5--3.0 × 106 nucleated cells were assayed per plate. Direct (19S) plaques were counted 90--120 min after the addition of complement. The indirect plaque-forming cells were revealed by use of a rabbit anti-mouse IgG
367 serum shown to react with IgG1, IgG2a, IgG2b and IgG3 on immunodiffusion. The serum was used at a dilution of 1/200. IgG (7S) PFC were obtained by subtracting the direct PFC from the total number of PFC. In the inhibition study, free HRP was added to the agar plate prior to incubation, as described in Results. In one experiment, trinitrophenyl-substituted sheep erythrocytes (TNPSRBC), prepared by the m e t h o d of Rittenberg and Pratt (1969), were used in the plaque assay. Statistical analysis was performed according to Student's t test. For PFC, the geometric mean (+S.E.) for each group was calculated.
Microhemagglu tination assay for circulating an tibody Circulating antibody was determined by a modification of the hemagglutination assay of Wegmann and Smithies (1966). Briefly, properly absorbed antisera were serially diluted in phosphate-buffered saline (PBS) pH 7.4, containing 0.5% of bovine serum albumin (BSA). SRBC coated with HRP and uncoated were suspended at a concentration of 1/6% in PBS, containing 0.25% of BSA. For hemagglutination inhibition, one drop of free antigen, HRP and ovalbumin, respectively at a concentration of 0.5 mg/ml in PBS alone was added to one drop of the respective serum dilution in the wells of microtiter V-plates. For agglutination, one drop of PBS diluent alone replaced the antigen solution. The plates were then covered with another microtiter plate, wrapped with plastic film and incubated 30--45 min at 37°C. After this time, one drop of the red cell suspension was added to each well, the microtiter plates covered as above and incubated overnight at room temperature. The plates were then spun at 1500 rev/min for 90 sec and the direct agglutination titer read. For the indirect titer, the plates were treated in the following manner. The supernatant was removed by suction using a 19-gauge needle, 3 drops of PBS alone were delivered into each well and the contents were mixed by agitation with a thin jet of air. After the plates were spun at 2000 rev/min for 2 min, the supernatant was removed and 2 drops of rabbit anti-mouse IgG serum diluted 1 : 200 in PBS containing 0.25% of BSA was added to each well. The contents were mixed, the plates covered as before and incubated at 37°C for 2 h. They were spun again at 1500 rev/min for 90 sec and the indirect agglutination titer was read. As controls, uncoated SRBC were tested in a parallel manner with the respective antisera. In addition, HRP-coated cells were added to PBS diluent and to absorbed normal mouse serum dilutions, with and w i t h o u t inhibiting antigen. Static electrical effects, 'spread', were minimized by spraying the undersurface of the plates before use with an anti-static aerosol. Although the spread could not be entirely prevented at high antibody concentrations in the indirect test, the titration end-point was evident.
368
Hyperimmune antiserum to HRP This was obtained by bleeding a rabbit which had been immunized with 3 weekly injections of 1 mg HRP grade I in complete Freund's adjuvant. Precipitating antibody to HRP was demonstrated in the serum (up to a dilution of 1 : 8) by immunodiffusion in an Ouchterlony plate. RESULTS
Demonstration of the presence o f coupled HRP-SRBC by passive hemagglutination Initially, to demonstrate that HRP was indeed bound to the sheep erythrocytes by the conjugation procedure outlined above, direct passive hemagglutination was performed, using SRBC both uncoated and coated with HRP grade I or II and h y p e r i m m u n e rabbit anti-HRP antiserum and, as a control, normal (non-immunized) rabbit serum. With HRP-SRBC and anti-Hl~P serum a direct titer of 1 : 2560 was found. There was no hemagglutination when either normal serum or uncoated SRBC was used. Development o f plaque-forming cells (PFC) in mice immunized with HRP Groups of 6 C57BL/6 mice were immunized with a single intraperitoneal injection 0.2 mg HRP (grade II) in complete Freund's adjuvant (CFA). The mice were sacrificed and their spleen cells assayed for direct and indirect PFC on various days after antigen challenge. The peak of anti-HRP response was obtained on day 5 for direct PFC and day 6 for indirect PFC (Fig. 1). The background responses with uncoated SRBC was minimal in mice immunized
20,000
uJZ15,000 (D
~
I
10,00C 5,000 I
0
I
L
I
4 5 6 7 DAYSAFTERIMMUNIZATION
8
Fig. 1. D i r e c t ( ) and indirect ( ...... ) P F C / s p l e e n ( g e o m e t r i c m e a n ± S . E . ) to c o n j u g a t e d H R P - S R B C f o r g r o u p s o f 6 m i c e i m m u n i z e d w i t h 0 . 2 m g H R P , g r a d e II, in C F A . Number of plaques counted per plate, direct
day day day day day
4 5 6 7 8
60 -175 -66-104-56--
250 336 380 236 232
indirect 76 -- 122 38 - - 2 5 0 30 -- 120 2-36
369 with HRP. As an additional control, 5 unimmunized mice produced virtually no PFC to HRP-coated SRBC.
Effect o f using different grades of HRP on target cells In order to compare the effect of coupling different grades of HRP to SRBC, 5 C57BL/6 mice were immunized with 0.1 mg HRP grade I, in CFA i.p. and PFC (direct and indirect) were measured on day 6 using SRBC coupled to both grades I and II o f HRP, (HRP (I)-SRBC and HRP (II)-SRBC respectively). No significant difference was observed between the two types o f conjugated target cells (Table 1) for direct as well as indirect PFC, although indirect PFC obtained with HRP (I)-SRBC were higher than those using HRP (II)-SRBC. Thus, grade II HRP may be readily used to detect direct AFC to HRP. Inhibition study 5 C57BL/6 mice were immunized with 0.1 mg HRP grade I in CFA and PFC determined on day 6. Direct and indirect anti-HRP PFC response were estimated using SRBC coupled to both grades I and II HRP. 1.25, 2.5 and 5.0 X 10 -9 moles of grade I HRP respectively were added to agar plates. As shown in Fig. 2, the direct PFC responses with both HRP (I)-SRBC and HRP (II)-SRBC were inhibited by the addition of free HRP, and this was dosedependent. Similar results (not shown here) were obtained for indirect PFC responses in both cases. Immune response to an unrelated antigen (DNP-KLH) As a further study for the specificity o f the plaque-forming cell to HRP, 5 C57BL/6 mice were immunized i.p. with 0.2 mg o f DNPs0KLH in CFA. On day 5, the mice were sacrificed and PFC were assessed using 3 types of target cells: (a) trinitrophenyl-substituted sheep erythrocytes (TNP-SRBC), (b) HRP-SRBC and (c) uncoated SRBC. At the time of pouring the agar, HRP grade I, in a dose of 5.0 X 10 -9 moles, was added to a second group of plates containing TNP-SRBC. Fig. 3 shows that animals immunized with DNP-KLH do not produce AFC to HRP and the addition of HRP in a con-
TABLE 1 PFC PER SPLEEN USING SRC COUPLED TO HRP GRADES I AND II The number of plaques counted per plate was HRP grade I direct 54--194, grade II direct 50--164, grade I indirect 6--22, grade II indirect 1--8.
Direct Indirect
HRP ( I ) - S R C
HRP(II)-SRC
P
13,847 (4222) 1,510 (408)
12,049 (3798) 720 (146)
NS NS
370
TARGET CELLS HRP[I)-SRBC
IT
T A R G E T ( E L L S HRP ( ] I ) S R B C
8000
(1) -H v
6000
Z
LU W --/ (1. u') (D U(1.
4000
\ I
2000
i
1
I
I
L
0
t,25
2.5
5,0
tl
I
0
~
~,25 2.5
I
5.0
Number of moles xIO-9of HRP odded to A(~or
Fig. 2. I n h i b i t i o n of d i r e c t P F C b y free HRP. S h o w n are P F C / s p l e e n ( g e o m e t r i c m e a n ± S.E.) for 5 mice i m m u n i z e d w i t h 0.1 mg HRP, grade I, in C F A 6 days previously. T h e target cells are S R B C b o u n d to HRP, grades I a n d II, respectively. A d o s e - d e p e n d e n t inhibit i o n of a n t i - H R P PFC o c c u r s o n t h e a d d i t i o n o f free H R P t o t h e agar.
centration response.
that
inhibited
anti-HRP
P F C has n o e f f e c t o n the a n t i - T N P
Immunization o f different strains o f mice T o d e t e r m i n e w h e t h e r o t h e r strains o f m i c e r e s p o n d t o H R P , g r o u p s o f
371 35,000 TNP-SRBC 30,000
25,°°° /
1
~ 2o,oooi (O 15,000 -
10,000
5,000 HRP-SRBC SRBC &lone HRP added (5 x lO-gmoles)
Fig. 3. D i r e c t P F C / s p l e e n ( g e o m e t r i c mean-+ S.E.) for mice i m m u n i z e d w i t h 0.2 m g D N P s 0 K L H in CFA. T h e r e is a high r e s p o n s e w h e n S R B C b o u n d t o TNP are used as target cells, b u t n o t for u n c o a t e d S R C o r for HRP-SRC. T h e a d d i t i o n o f H R P t o t h e agar does n o t i n h i b i t P F C t o TNP-SRC.
5 mice -- C57BL/6, BDF~ and BALB/c -- were each immunized with 0.1 mg HRP grade I in CFA i.p. Direct PFC were measured on day 5. BDF1 mice appear to respond m or e than the BALB/c in terms o f direct AFC (Table 2).
Detection of circulating antibody by passive hemagglutination 6 C57BL/6 mice were immunized with an i.p. injection o f 0.1 mg HRP grade I in CFA and then bled on days 7 and 21. T h e y were then given a b o o s ter injection with 0.05 mg HRP in CFA and bled one week later (day 28). The sera was pooled, absorbed with SRBC, and the anti-HRP hemagglutinating a n t i b o d y was determined as described in Methods. As d e m o n s t r a t e d in Table 3, a specific anti-HRP titer was obtained on days 7 and 21 after primary immunization, as well as one week after secondary immu n izatio n with HRP. These titers were enhanced by the addition of anti-mouse IgG. F u r t h e r m o r e , the hemagglutination was completely inhibTABLE 2 D I R E C T P F C P E R S P L E E N IN V A R I O U S S T R A I N S O F MICE I M M U N I Z E D WITH HRP Strain
N u m b e r o f mice
P F C / s p l e e n (_+S.E.)
P
BDF1 BALB/c C57BL/6
5 5 5
1 7 , 2 9 9 4 (_+2044) 3 , 6 5 7 (_+1584) 1 2 , 0 4 9 (-+3798)
NS
