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Phylogeny of immunoglobulin structure and function—VIII
0161-5890.80
PHYLOGENY
OF IMMUNOGLOBULIN FUNCTION-VIII.
STRUCTURE
0301-036s
10200~0
AND
INTERMOLECULAR HETEROGENEITY OF SHARK 19s IgM ANTIBODlES TO PNEUMOCOCCAL POLYSACCHARIDE* T. VINCENT SHANKEYt and L. WILLIAM CLEMS Department
of Immunology Laboratory.
and Medical Microbiology, College of Medicine. and Whitney University of Florida, Gainesville, FL 32610, U.S.A.
(Received 2 Mu!
Marine
1979)
Abstract-Significant IgM antibody responses to the pneumococcal capsular polysaccharidc were demonstrated in nurse sharks immunized with pneumococcal vaccines. The antibodies isolated from immune sera by affinity chromatography were exclusively of the 19s variety; no 7s antibodies were isolated from any of six animals studied for periods up to twelve months. Equilibrium dialysis studies of the isolated antibodies demonstrated several important points: (I) the IgM antibodies contained ten combining sites per 19s molecule. (2) in all cases, the isolated antibodies showed marked heterogeneity of combining sites, (3) the antibodies were all of low average affinity, and (4) there was no increase in the average affinity of the antibodies isolated from any single animal for periods of up to twelve months after the start of immunization. In order IO determine if a single IgM molecule contains ten equivalent combining sites. the antibodies isolated from several animals were fractionated by liquid isoelectric focusing. Equilibrium dialysis experiments using focused fractions showed the presence of ten functionally identical combining sites per IgM molecule. As a proof of the structural homogeneity of focused fractions, antibodies were separated into H and L chains.9 recombined and tested for the regeneration of the original combining site by equilibrium dialysis. The results indicated that recombinonts of focused antibody fractions contained binding sites identical to those of the intact antibody, whereas heterogeneous (unfocused) recombinants and isolated H and L chains failed to show any binding activity. The conclusion from this study is that the heterogeneity of ligand binding exhibited by nurse shark 19s antibodies to the capsular polysaccharide of the Type III pneumc-coccus can be attributed to intermolecular heterogeneity.
INTRODUCTION
1969; Young et al., 1971). numerous other studies with conventional IgM antibodies have indicated considerable heterogeneity of hapten binding with valences of less than ten. In certain instances valences of less than ten were attributable to steric factors due to antigen size (Stone & Metzger, 1968; Edberg ef al., 1972) whereas in others, haptens were employed which would not likely impose steric limitations on the antibody combining sites. In this latter context an average of five high and five low affiinity sites per 19s antibody molecule has been frequently observed (Onoue er al., 1968; Oriol et al.. 1971; Voss & Sigel, 1972; Oriol & Rousset, 1974). In terms of resolving this issue there would seem to be two different, but not necessarily exclusive. possibilities. The first is that ligand binding heterogeneity is a consequence of differences in affinities between antibodies in a population, i.e. intermolecular differences due presumably to differences in antibody primary structure. The second possibility is that ligand binding
Mammalian IgM antibodies contain ten heavylight chain pairs and thus, by analogy with IgG antibodies, should contain ten equivalent ligand binding sites per molecule (reviewed by Metzer, 1970). Although studies employing structurally homogeneous IgM monoclonal proteins have demonstrated the presence of ten homogeneous binding sites per molecule (Ashman & Metzer. l This work was supported by NSF grant PCM 75-16479, NIH grant 1 ROI Al 14137andspecialfundsfrom thecenter for Environmental Programs. IFAS, University of Florida. +Present address: Department of Pathology. School of Medicine. University of Pennsylvania. Philadelphia, PA. U.S.A.
:Address correspondence to: L. W. Clem. Department of Microbiology, University Medical Center, Jackson, MS 39216. U.S.A. S;Abbreviations used: Tris buffer. 0.01 M Tris. 0.14 M NaCI. 0.01 M EDTA. O.l”,, NaN (pH 7.4); S3. capsular carbohydrate of Type III pneumococci; S8. capsular carbohydrate of Type VIII pncumococci: H and L chains: component heavy and light polypeptidc chains of immunoglobulins: DNP. 2.4-dinitrophcnyl. 365
366
T. VINCENT
SHANKEY
reflects differences within heterogeneity individual antibody molecules, i.e. intramolecular differences. The solution to this question has, to a large part. been elusive due to difficulties in obtaining sufficient amounts of structurally homogeneous IgM antibodies for study. The physicochemical properties of immunoglobulins from lower vertebrates indicate that such animals are restricted to the synthesis of but one immunoglobulin isotype and that this isotype is analogous to IgM (reviewed by Clem & Leslie, 1969; Carton, 1973; Grey, 1969). Furthermore, studies using sharks indicate that certain antigens, especially A-variant streptococci, can elicit the production of very large amounts of 19s antibodies (up to 10 mg ah/ml serum, Clem & Leslie, 1971). Although it has not been possible to isolate a suitable hapten for studying affinities and valences with this antigen (Shankey, 1977), the finding in sharks of reasonably good 19s antibody responses to both the DNP hapten covalently coupled to streptococcal cells and the capsular polysaccharide of pneumococcal cells, has now provided sufficient antibody to defined ligands for study. This paper describes the binding properties of shark 19s antibodies to the S3 polysaccharide and the following paper in this series (V. Shankey and L. W. Clem, in preparation) describes similar studies with shark antibodies to the DNP moiety. A summation of these findings suggests the intriguing possibility that both inter- and intramolecular factors may be involved in 19s IgM ligand binding heterogeneity.
MATERIALS
AND METHODS
Vaccines and immunization
Fully encapsulated strains of Types III and VIII Streptococcus (Diplococcus) pneumoniae were a generous gift of Dr. A. M. Pappenheimer, Jr. (Biological Laboratories, Harvard University, Cambridge, MA). Vaccines were prepared from both types of pneumococci as described by Kimball et al. (1971) and were stored at 4°C in 0.1% formalized saline until used. Nurse sharks (Ginglymyostoma cirratum) of both sexes, weighing lo&200 kg, were maintained in tidal sea water at the Lerner Marine Laboratories, Bimini, Bahamas. Immunizations, performed by intravenous injection of 30 ml of either Type III or Type VIII
and L. WILLIAM
CLEM
vaccine (1 O9 cells/ml), and bleedings. were accomplished as described elsewhere (Clem & Leslie, 1971). Sera were obtained by centrifugation after allowing the blood to clot overnight at room temperature and were subsequently stored at -20°C until used. Isolation of capsular carbohydrate
Type III and Type VIII pneumococci were first passed through DBA mice to select highly encapsulated strains. Fresh isolates were used to inoculate 2-liter broth cultures of Brain Heart Infusion (Difco Labs) supplemented with 2% sterile horse serum (Pel-Freeze). Cell lysis and extraction of the capsular carbohydrates were performed as described by Campbell & Pappenheimer (1966). The purified carbohydrates were then treated sequentially with DNase, RNase and pronase as described by Katz & Pappenheimer (1969). isolation and labelling of the S3 hexasaccharide hapten
Purified S3 was partially acid hydrolyzed by the procedure of Campbell & Pappenheimer (1966), and the hydrolysis products were gel filtered at 4°C on a 2.5 x 120 cm column of Sephadex G-25 (fine mesh) equilibrated with 0.01 M NH,HCO,. The hexasaccharide peak was identified by determining the ratio of reducing sugar (Nelson, 1944) to total sugar (Dubois et al., 1956) of eluted products. Fractions containing the hexasaccharide were pooled and concentrated by flash evaporation. was Labelling of the hexasaccharide accomplished by reduction with tritiated sodium borohydride. Three milligrams of hexasaccharide (in 1 ml) were mixed at 4°C with 250 mCi tritiated sodium borohydride (Amersham/ Searle, 8 Ci/mmole) dissolved in 0.1 ml distilled water. After incubation at room temperature, the reaction mixture was gel filtered on a 2.5 x 120 cm Sephadex G-25 (fine mesh) column with 0.01 M NH,HCO,. equilibrated Radioactivity eluted from the column in two peaks: the first at the position of the unlabelled hexasaccharide and the second coincident with the totally included volume of the column. The activity of the labelled hexasaccharide was 3.8 x 1OScounts/min/~mole. Isolation of antibodies
Antibodies to the capsular carbohydrate of Type III pneumococci were isolated from nurse shark sera by use of immunoadsorbent colums
Phylogeny of lmmunoglobulin
prepared by linking S3 to Sepharose by a modification of the method of Axen et al. (1967). Briefly, 5 g L-lysine dissolved in 500 ml 0.5 M NaHCO, was added to 100 ml (packed volume) Sepharose 4B (Pharmacia) previously activated with 4 g CNBr (Eastman Organic). The pH was adjusted to 8.5 and the mixture was stirred at 4°C for 12 hr. After washing, 100 mg S3 previously activated with 400 mg CNBr was added to the lysine-Sepharose, the pH was adjusted to 8.5 and the mixture was stirred at 4°C for 12 hr. Before use, the immunoadsorbent was washed with 0.1 M glycine (pH 9.0) to block unreacted sites on the activated Sepharose. Antibodies were eluted from immunoadsorbant columns with 0.5 M glycine/l.O M NaCl, pH 2.3, neutralized, concentrated by positive pressure dialysis and gel filtered at 4°C on a 2.5 x 100 cm Sephadex G-200 column equilibrated with Tris buffer. Eluted fractions were monitored for protein by absorbance at 280 nm and antibody containing fractions were pooled and concentrated by positive pressure dialysis. Equilibrium dialysis
Equilibrium dialysis was performed essentially as described by Clem & Small (1970). One side of the cell was filled with 250 ~1purified antibody (from 0.5 x lO-‘j to 5 x 10e6 M depending upon the experiment) in 0.05 M phosphate buffer (pH 7.2), while the other side of the cell was filled with an equal volume of the tritiated hexasaccharide hapten (from 0.1 x 10m6to 50 x 10m6 M) in the same buffer. Dialysis cells were rotated (- 1 rev/min) at 4°C for 72 hr, a period sufficient for the equilibration of hapten with normal nurse shark Ig or buffer. Duplicate 50 ~1 samples from each side of the cell were counted in 4.5 ml Aquasol- (New England Nuclear) using a Beckman LS- 1OOC liquid scintillation spectrometer. Samples were counted for 10 min or until 0.57$, counting error was reached. Data were calculated and plotted by the method of Scatchard (1949). Each data point represents duplicate counts of duplicate samples from duplicate cells. Antibody concentrations for equilibrium dialysis experiments were determined by differential refractometry as described by Babul & Stellwagen (1969). In order to assure that the protein concentration determined by this method was an accurate assessment of active antibody, only freshly isolated samples from immunoadsorbants were used for equilibrium dialysis.
Structure and Function-VIII
367
Preparative liquid isoelectric focusing
Isoelectric focusing was performed in an LKB 110 ml isoelectric focusing column (model 8 10 1) using a final concentration of 1.2% carrier Ampholines (LKB Instruments) stabilized by a linear 5-45x sucrose gradient (Ultrapure sucrose, Schwartz/Mann). The cathode used was 1% ethylene diamine, while the anode was 1% phosphoric acid in 50% sucrose. Antibodies isolated from a single bleeding (25-40 mg in 5 ml) were mixed with an equal volume of dense ampholyte solution, and immediately layered to its isobuoyant density using a length of 20 gauge polyethylene tubing inserted into the column before loading the ampholyte solution. Focusing was initiated at 400 V, and was increased to 1200 V as rapidly as possible without exceeding 6 mA or 9 W. Focusing was not continued for more than 24 hr since noticeable loss of cathodal ampholytes was observed after that time. After focusing, 1 ml fractions were collected, the pH was determined immediately and the protein was monitored by absorbance at 280 nm. Fractions containing a visible precipitate were clarified by the addition of an equal volume of Tris buffer before measuring absorbance. Recombination of antibody H and L chains
Heterogeneous or homogeneous nurse shark anti-S3 antibody was mildly reduced with 2mercaptoethanol (Eastman Organic) and alkylated with iodoacetamide (Sigma) in Tris buffer as previously described (Clem & Small, 1967). After extensive dialysis against Tris buffer, the antibody was dialyzed against 5 M guanidine-HCl containing 0.01 M iodoacetamide (pH 5.5) and subsequently gel filtered on a 1.5 x 90 cm Bio Gel A5M column equilibrated with 5 M guanidine_HCl. Eluted fractions were monitored for protein by absorbance at 280 nm. Subsequent recombination of the separated H and L chains was accomplished by mixing equimolar amounts of H and L chains, using J?$&= 11.7 for isolated H chains and Ei& = 13.1 for isolated L chains (Clem & Small, 1967). Mixtures of H and L chains in 5 M guanidineHCl were concentrated by positive pressure (7.5 lb max) to a concentration of 5 mg protein per ml. They were then dialyzed at 4°C against three changes of Tris buffer (100 volumes each). The size of recombinant antibody molecules was determined by density gradient ultracentrifugation using a linear 8-267; sucrose gradient. Samples were spun for 24 hr at 160,000 g in an
T. VINCENT SHANKEY and L. WILLIAM CLEM
368
Table 1. Precipitable
serum antibody to S3 of nurse sharks immunized pneumococcal vaccine
Shark No.
4 Months
23 50 51
0.20 0.25 0.30
with Type III
Precipitable antibody (me/ml) Tie after primary immunization’ 8 Months 6 Months
12 Months 0.30 0.30 0.50
0.50 0.40 0.80
0.40 0.30 0.80
‘Each animal received vaccine on days 1, 2, 50 and 120.
SW-40 Ti rotor and fractions of 0.25 ml were subsequently eluted by pumping 50% sucrose into the bottom of each tube. Fractions were monitored for protein by absorbance at 280 nm. Normal nurse shark 19s and 7s IgM were used as molecular-weight markers. The composition of the -7s recombinant peak obtained from density gradient ultracentrifugation was determined by elecgels in SDS-polyacrylamide trophoresis (Laemmli, 1970). Gels stained with 0.025% Buffalo Black were scanned at 600 nm using a Beckman spectrophotometer equipped with a linear transporter, and areas under peaks were measured with a planimeter. RESULTS
Level of anti-carbohydrate
antibody
The amount of precipitable anti-capsular carbohydrate antibody in each serum sample was determined by quantitative precipitation. The sera obtained from each of three nurse sharks immunized with Type III vaccine were tested for precipitable antibody using the S3 polysaccharide as antigen. The results, shown in Table 1, indicated that each of these animals produced relatively low levels of precipitable anti-S3 antibody. Due to the relatively poor response to the Type
III vaccine, four additional animals were immunized with Type VIII vaccine with the intention of eliciting cross-reactive antibodies to S3, a phenomena frequently observed with rabbits and horses immunized with the Type VIII vaccine (Speyer et al., 1973). The results of quantitative precipitation tests with these sera, using the S3 and S8 antigens (Table 2) indicated that all four animals produced significant antibody responses. In most cases, the amount of precipitable anti-S8 antibody did not increase during the period of vaccination. These results also showed that sharks immunized with Type VIII vaccine produced antibodies which precipitated with S3. In order to determine the extent of cross-reaction, the sera from animals receiving Type VIII vaccine were tested both by sequential and by simultaneous precipitation with S3 and S8. The results (Table 2) indicated that sera from two of these animals (numbers 59 and 61) contained the same amount of precipitable antibody with S8 alone as with S3 and S8. This indicated that these animals produced some antibodies to S8 which completely cross-reacted with S3, and thus were probably recognizing the common (cellobiuronic acid) residues found in both S3 and S8. On the other hand, the results of quantitative precipitation tests with the sera from animals 58 and 64 showed increased amounts of precipitable
Table 2. Precipitable serum antibody to S3 and S8 of nurse sharks immunized with Type VIII pneumococcal vaccine
Shark No.
s3b
Precipitable antibody (mg/ml) Time after primary immunization’ 5 Months 1 Month S8 S8’ S3 S3 + S8 S3 + S8“
58 59 61 64
0.40 0.20 0.25 0.50
1.20 0.60 0.25 0.70
a Each animal received b Antibody precipitable ‘Antibody precipitable ‘Antibody precipitable ’ Not done.
1.60 0.63 0.30 1.20
0.93 0.15 0.32 0.60
vaccine on days 1, 2, 3, 32 and 64. with S3 alone. with S8 alone. with S3 and S8 together.
1.00 1.40 1.40 0.70
1.90 1.35 1.40 1.24
s3 0.40
10 Months S8 S3 + S8 1.10 N.D.’ N.D. N.D.
1.58
Phylogeny of lmmunoglobulin
Structure
369
and Function-VIII
In all cases, the isolated anti-S3 antibody eluted from a Sephadex G-200 gel filtration column at the void volume and analysis of this material in the analytical ultracentrifuge indicated a single homogeneous boundary with Isolation of anti-S3 antibodies + 17s (at 3-5 mg/ml). an S,,, of analysis of the purified The sera of two animals (numbers 23 and 51) Immunoelectrophoretic anti-S3 antibody demonstrated a single immunized with Type III and from all four animals immunized with Type VIII vaccine were component when reacted with rabbit anti-whole nurse shark serum and developed an identical used as a source of antibodies to S3. Antibodies were isolated by elution from S3-Sepharose precipitin band with rabbit antisera to nurse immunoadsorbant columns using 0.5 A4 shark immunoglobins (Clem er al., 1967). glycine/l.O M NaCl (pH 2.3). In all cases, the isolated antibody represented from 25 to 50% of Afjnity of anti-S3 anribodies The affinity of the isolated nurse shark the precipitable serum antibody to S3 and was >90% active as determined by quantitative antibodies to S3 was measured by equilibrium precipitation or affinity column recycling. dialysis using the tritiated hexasaccharide hapten
antibody with S3 and S8 compared to S8 alone. This would indicate the presence of at least two different populations of antibodies, one capable of reacting with S8, the other with S3.
04 03 :
-0 I -10
N.S.
23
CK+O 4 Months
N.S
04
03
I2 Months
02
02
51
-
4 Months
-
I2
Months
+z+_
0 I
f
5
N.S Ooo m
58 I Month 5 Months IO Months
N.S.
04
03 F
IO
N.S @DO -
Fig.
I.
Scatchard
IO
61 I Month
I-
59
-
I Month
-
5 Months
N.S. 64 0-0-0
-
I
Month
5 Months
5 Months
plots for isolated 19s IgM antibodies isolated from each of six different Times indicated refer to the time after primary immunization.
nurse sharks.
370
T. VINCENT
SHANKEY
and L. WILLIAM
CLEM
obtained from S3. This hapten was used since obtain homogeneous IgM antibodies to address preliminary equilibrium dialysis experiments the question of whether homogeneous antibody indicated that nurse shark anti-S3 antibodies exhibited homogeneous hapten binding. During failed to react appreciably with the isoelectric focusing, all nurse shark IgM tetrasaccharide and formed precipitates with the antibodies developed significant bands of visible larger octasaccharide hapten obtained from S3. precipitate in the focusing column. In order to Speyer et al. (1973) have reported a similar demonstrate that these precipitates were precipitation with rabbit anti-S3 antibodies with homogeneous antibody at its isoelectric point the octasaccharide hapten. and not heterogeneous antibody precipitated at The results of equilibrium dialysis studies with its isobuoyant density, several experiments were isolated anti-S3 antibodies are shown in Fig. 1 conducted. First, an isolated nurse shark anti-S3 and illustrate several points. In each case, the antibody was divided into two unequal aliquots; isolated exhibited considerable antibody both focused in an identical manner even though heterogeneity of binding sites of relatively low different amounts of antibody were applied to each column. Second, an anti-S3 antibody peak affinity (K, < 5 x IO4 L/M). The antibody isolated from any single animal showed no which focused at pI 4.5 was subsequently significant increase in affinity for periods up to refocused in a narrower ampholyte gradient, and was shown to elute from this second focusing twelve months after the start of immunization. Similarly, in all cases five or more binding sites column at the same isoelectric point ( f 0.1 pH were measured and in two cases (animals 58 and unit). Finally, a focused anti-S3 antibody peak 64) the Scatchard plots readily extrapolated to a was refocused on an identical ampholyte valence of ten. gradient with the polarity of the column reversed, and again the peak eluted at the same Binding of homogeneous antibody obtained by isoelectric point ( f. 0.1 pH unit). Since focusing isoelectric focusing experiments conducted with higher ampholyte Liquid isoelectric focusing was utilized to concentrations failed to prevent precipitation of
4 Months 30
0
I Month
50
100
Fraction
100
number
Fig. 2. Liquid isoelectric focusing elution profiles of 19s anti-S3 primary immunization. Top: Shark No. 51, antibodies isolated Middle: Shark No. 58, antibodies isolated at 5 months (left) and antibodies isolated at 1 month (left) and
antibodies isolated at different times after at 4 months (left) and 12 months (right). 10 months (right). Bottom: Shark No. 64, 5 months (right).
Phylogeny of lmmunoglobulin
the antibody, a final ampholyte concentration of 1.2’;; was routinely used. Comparison of the liquid isoelectric focusing elution profiles of 19s anti-S3 antibodies isolated from three animals at different times after primary immunization (Fig. 2) showed a typical pattern of six to ten major peaks, generally focusing at isoelectric points between 4 and 7. In all cases, there was no significant change in the number of isoelectric points of the major antibody peaks during periods of up to eight months of immunization. Liquid isoelectric focusing analysis of the isolated 19s anti-S8 antibodies from two animals (not shown) showed a similar heterogeneous elution profile with seven or nine major peaks. Since it was considered crucial to obtain homogeneous antibodies for subsequent studies, only the first peaks eluting from focusing columns were utilized. This was done in order to minimize possible contamination from other peaks during elution from the focusing column. Furthermore, the eluted peaks were repurified on an S3-Sepharose immunoadsorbant before use in equilibrium dialysis experiments, since as 40
N.S. 58
r
000 Prc -focus 0..
pl 4.6
NS. 64
000
Prc -focus
r
Fig. 3. Scatchard plots showing the S3 hapten binding by focused and non-focused 19s shark anti-S3 antibodies. Results expressed as r/900,000 daltons. MlMM 11’3-F
Structure and Function--VIII
371
much as 50% of the focused nurse shark antibody was found to be inactivated by the focusing procedure. This effect was shown to be particular to 1gM antibodies, since less than 10% of focused rabbit IgG antibodies to S3 were inactivated by similar isoelectric focusing procedures. Kim & Karush (1974) have reported a similar inactivation of equine IgM antibodies after isoelectric focusing. The results of equilibrium dialysis experiments using these focused nurse shark antibodies, shown in Fig. 3, indicated that focused antibody shows homogeneous binding as contrasted with heterogeneous binding seen with the unfocused anti-S3 antibody obtained from these same animals. Since the technique used to determine antibody concentration for these equilibrium dialysis experiments is an accurate assessment of the active antibody concentrations, these results clearly indicate that the isoelectrically focused 19s IgM antibodies exhibit- ten functionally identical binding sites per molecule. Antibody recombination studies In order to verify the homogeneity of focused nurse shark anti-S3 antibodies, focused fractions were mildly reduced and alkylated, separated into H and L chains and subsequently recombined and examined for the regeneration of the original combining site. Since recombinant anti-S3 antibodies could not be repurified by affinity chromatography (acid elution from the immunoadsorbant would separate the recombined chains), an alternative approach was employed to assess the active antibody concentration for equilibrium dialysis experiments. The recombined H and L chains from focused antibody were first subjected to preparative density gradient ultracentrifugation wherein a majority (270%) of recombinant molecules sedimented only slightly slower than the 7S nurse shark immunoglobulin marker (Fig. 4). An aliquot of each -7s protein was subjected to electrophoresis in SDSpolyacrylamide gels. In each case m 70% of the stained material migrated in the gel region expected to contain H chains whereas the remainder was in the L chain region (inserts, Fig. 4). It therefore seems reasonable to suggest that the -7s recombinants were composed of 2 H and 2 L chains and thus a molecular weight of 180,000 was assumed. Another aliquot of _ 7S material was applied to an S3 immunoadsorbant. The results indicated that 70:/, of the recombined (PI 4.60) antibody from animal
372
T. VINCENT SHANKEY and L. WILLIAM CLEM N.S 000
58 pl 46 19s
Antibody
0 l l Recombi non&
64
pl 4.4
19 S Antibody Recombinants
.’
0’
l...’ ,
IO
.
\ I
20
Fraction
%. +._.--*: 30
40
number
Fig. 4. Sucrose density gradient ultracentrifugation profile of H and L chain recombinants. Top: focused (~14.6) anti-S3 antibody from shark 58. Bottom: focused (PI 4.4) anti-S3 antibody from shark 64. Arrows indicate the positions of labeled shark 7S and 19s immunoglobulin markers. Inserts show the SDS-polyacrylamide gel electrophoresis of the 7S recombinants compared to labeled shark immunogiobulin H and L chains.
number 58 was active while 60% of the recombined (PI 4.40) antibody from animal number &4was active. The remainder of the u 7S samples were subjected to equilibrium dialysis against the S3 hexasaccharide. The antibody concentration used for calculating the results of these equilibrium dialysis experiments represented the total protein concentration, as determined by differential refractometry, corrected for inactive antibody using the results of immunoadsorbant passage. The equilibrium dialysis data (Fig.5) was analyzed by linear regression analysis which indicated that the plot for animal 58 (p1 4.60) both before and after recombination extrapolated to 2.0 sites per 7s sub-unit (regression coefficients of 0.98 and 0.92, respectively), while the plot for animal 64 (PI 4.40) extrapolated to 2.02 sites before (regression coefficient 0.98) and 1.95 sites after recombination (regression coefficient 0.93). It should also be pointed out that recombinant m 7s molecules obtained from heterogeneous (unfocused) anti-S3 antibodies from these same two nurse sharks (similarly containing equimolar H and L chains as judged by electrophoresis in
IO r
2.0
Fig. 5. Scatchard plots showing the S3 hapten binding by focused intact anti-S3 19s molecules and 7S H and L chain recombinants. Results are expressed as r/180,000 daltons.
SDS-polyacrylamide gels) were incapable of binding to S3Ammunoadsorbant (< lOO,‘,>and showed no detectable hapten binding by equilibrium dialysis. Similarly, isolated H and L chains from unfocused antibody failed to exhibit binding of the S3 hapten in equilibrium dialysis experiments.
DISCUSSION
Previous attempts to induce antibody formation to a variety of antigens have given sharks the reputation of being immunologically poor responders (reviewed by Carton, 1973). However, the responses to pneumococcal vaccines reported here, and the previously reported responses to streptococcal vaccines (Clem & Leslie, 1971) indicate that sharks are capable of synthesizing significant quantities of antibodies in response to bacterial vaccines. Similar vaccination procedures in mammals and birds (Leslie & Clem, 1973; Krause, 1970; Haber, 1971) have been shown to result in the frequent production of large quantities of antibody. While
Phylogeny of Immunoglobulin Structure and Function-VIII
quantitatively quite good, the shark response is necessarily limited to the production of only IgM antibodies. Thus, the utilization of similar procedures with these and other bacterial vaccines could provide large quantities of shark IgM antibodies to different haptens with which to address a number of questions about the characteristics of the immune response and antibodies from lower vertebrates. The failure to detect 7S IgM antibodies in response to pneumococcal vaccines is in contrast to reports of shark antibody responses to a variety of protein, bacterial and viral antigens (Clem & Small, 1967; Clem et al., 1967; Suran et al., 1967; Schulkind et al., 1971) and to hapten-protein conjugates (Voss & Sigel, 197 1). Although 7S IgM antibodies generally were observed late in the immune response in these studies, 7S anti-S3 or S8 antibodies were not isolated from any of the animals studied here, even after twelve months of immunization. Similarly, 7S IgM antibodies have not been detected in any of the sera of twelve nurse sharks immunized with streptococcal antigens (Clem & Leslie, 1971; L. W. Clem, unpublished observations). Since the anti-carbohydrate antibody response in sharks immunized with pneumococcal or streptococcal vaccines also differs quantitatively, it is possible that these results reflect differences in the control of antibody synthesis and/or release in sharks presented with different antigens. The alternate possibilitity, that these animals synthesized 7S IgM anti-carbohydrate antibodies which could not be recovered using immunoadsorbants, is unlikely. The 7S sub-units obtained by mild reduction of purified 19s anti-S3 (and antistreptococcal carbohydrate, L. W Clem, unpublished observations) antibodies have been isolated using these same immunoadsorbants, suggesting then, that naturally occurring 7S IgM antibodies of similar or higher affinities should be recoverable by this technique. In contrast to the usual pattern of maturation seen in the immune response of higher vertebrates (Eisen & Siskind, 1964; Kimball, 1972), the nurse shark response to prolonged administration of pneumococcal vaccines failed to exhibit maturation. This was evidenced by the lack of any significant increase in the amount of precipitable serum antibody with prolonged immunization, the lack of changes in the isoelectric focusing elution profiles of the isolated antibody, and finally by a lack of significant increases in the affinities of the
373
isolated antibodies from any of the six animals studied here. This lack of maturation appears to be a common finding for IgM antibody responses -for lower vertebrates (Voss & Sigel, 1972; Clem & Small, 1970; Fiebig ef al., 1977) and for mammals (Voss & Eisen, 1968; Sarvas & Mlkelii, 1970; Kim & Karush, 1974; Chua et al., 1975). The presence of a ten heavy-light chain structure predicts that shark 19s IgM antibodies should contain ten equivalent hapten combining sites. However, previous studies of shark antibodies have measured a valence of less than one for each heavy-light chain pair (Voss & Sigel, 1972). In contrast, the results of the study reported here clearly demonstrate that isoelectrically focused shark 19s antibodies to S3 contain ten equivalent, low affinity combining sites. Since it was previously shown that functionally homogeneous antibodies are not necessarily structurally homogeneous (Hoffman & Campbell, 1969), it was considered insufficient to conclude that isoelectrically focused shark anti-S3 antibodies were structurally homogeneous based on homogeneous binding of the S3 hapten. In view of studies on the regeneration of original antibody combining sites following separation and recombination of H and L chains from homogeneous mammalian IgG antibodies (Unman, 1971), recombination of focused and unfocused shark anti-S3 antibodies was undertaken. The demonstration that isoelectritally focused shark IgM antibodies contain an identical combining site after H and L chain separation and subsequent recombination, while recombinants of heterogeneous (unfocused) shark antibodies lacked any binding activity, thus indicates that isoelectrically focused fractions are structurally homogeneous. Therefore, a reasonable conclusion is that heterogeneity of hapten binding with shark antibodies to this ligand is an intermolecular phenomenon. This finding is seemingly in conflict with the results obtained with shark antibodies to the DNP moiety reported in the following paper in this series. Studies of recombinants of IgM molecules have generally been limited to an assessment of physiochemical or antigenic properties or recombinants of myeloma proteins (Grey & Mannik, 1965; Gordon & Cohen, 1966; Miller & Terry, 1968; Harboe et al., 1969; Carson & Weigert, 1973). These studies have usually shown that IgM myeloma proteins recombine to form N 7S sub-units composed of both H and L
374
T. VINCENT SHANKEY and L. WILLIAM CLEM
chains. A single report which studied the binding characteristics of IgM antibodies after the recombination of L chains and Fdp-chain fragments of isoelectrically focused Fabp suggested that recombinants of heterogeneous Fabp produce an active combining site identical to the site on the Fabp fragments before chain dissociation (Mitchell et al., 1977). It was concluded by these authors that the light chains of intact IgM antibodies do not contribute significantly to the binding of hapten. It should be noted, however, that no direct evidence was presented in that study to indicate the Fabp fragments actually dissociated into free L and Fdp chains allowing subsequent recombination of heterogeneous molecules. studies of recombination The results performed with shark IgM antibodies to S3 suggest that both H and L chains contribute to the formation of the shark antibody combining site. The lack of significant hapten binding by either isolated chain and the failure of m 7S recombinants of heterogeneous H and L chains to exhibit significant binding thus suggests that the antibody combining site of these lower vertebrate antibodies is formed by the juxtaposition of the appropriate H and L chains. Furthermore, since both the H and L chains apparently contribute to the formation of the combining site, it is reasonable that the affinity of the intact site should equal the product of individual affinity contributions of the isolated H and L chains, as previously shown for mammalian IgG antibodies (Painter er al., 1972). Unfortunately, the lack of significant binding by isola$d H and L chains of shark anti-S3 antibodies makes this determination impossible. Finally, the results of this study give some insight into the complexity of the immune response of lower vertebrates. The anti-S3 antibodies of the animals used in this study all showed considerable heterogeneity by liquid isoelectric focusing. This implies that within a single shark there is the capacity to generate a number of different combining sites to any single ligand. However, detailed analysis of each focused fraction would be necessary to show that each combining site is unique. Previously reported studies on the molecular heterogeneity of the shark antibody response to DNP and streptococcal vaccines (Clem et af., 1975) support the concept that there exist a number of different combining sites in the ‘library’ of sites available to any individual shark. It would be of considerable interest to determine whether the
‘libraries’ available to the entire species contain common combining sites, or whether each ‘library’ is unique. REFERENCES
Ashman R.F. & Metzger H. (1969) A Waldenstrom microglobulin which binds nitrophenyl ligands. J. biol. Chem. 244, 3405-3414.
Axen R., Porath J. & Ernback S. (1967) Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature, Lond. 214. 1302-1304. Babul J. & Stellwagen E. (1969) Measurement of protein concentration with interference optics. Analyr. E&hem. 28,216221.
Campbell J. H. & Pappenheimer A. M. (1966) Quantitative studies of the specificity of anti-pneumococcal polysaccharide antibodies, types III and VIII. I. Isolation of oligosaccharides from acid and from enzymatic hydrolysates of S3 and S8. Immunochemistry 3, 195-212. Carson D. & Weigert M. (1973) Immunochemical analysis of the cross-reacting idiotypes of mouse myeloma proteins with anti-dextran activity and normal anti-dextran antibody. Proc. natn. Acad. Sci. U.S.A. 70, 235-239. Carton Y. (1973) La riponse immunitaire chez les agnathes et les poissons. Structuredes immunoglobulins. Ann. Biol. 12, 139-187.
Clem L. W., deBoutaud F. & Sigel M. M. (1967) Phylogeny and function. II. structure of immunoglobulin lmmunoglobulins of the nurse shark. J. Immun. 99, 12261235. Clem L. W. & Small P. A. (1967) Phylogeny of immunoglobulin structure and function. I. Immunoglobulins of the lemon shark. J. exp. Med. 125, 893-920. Clem L. W. & Small P. A. (1970) Phylogeny of immunoglobuhn structure and function. V. Valences and association constants of teleost antibodies to a haptemc determinant. J. exp. Med. 132, 385-400. Clem L. W. & Leslie G. A. (1971) Production of 19s IaM . antibodies with restricted heterogeneity from sharks. Proc. natn. Acad. Sci. U.S.A. 68, 139-141. _ Clem L. W.. McLean W. E. & Shankev V. (1975) Oualitative
and quantitative aspects of the antibody.library-of
sharks.
Adv. exp. Med. Biol. 64, 231-239.
Chua M-M., Morgan D. A. & Karush F. (1973) Equine antihapten antibody. IX. IgM anti-lactose antibodies. J. Immun. 114,99-101.
Dubois M., Giles K. A., Hamilton J. K., Roberts P. A & Smith F. (1956) Calorimetric method for determination of sugars and related substances. Analyt. Chem. 28,350-357. Edberg S. C., Bronson P. M. & Van Oss C. J. (1972) The valency of IgM and IgG rabbit anti-dextran antibody as a function of the size of the dextran molecule. Immunochemistry 9, 273-288.
Eisen H. N. & Siskind G. W. (1964) Variations in affinities of antibodies during the immune response. Biochemisrry 3, 996-1008.
Fiebig H., Gruhn R. & Ambrosius H. (1977) Studies on the control of IgM antibody synthesis. III. Preferential formation of anti-DNP antibodies in the course of the immune response of carp. Immunochemistry 9, 721-726. Gordon S. & Cohn S. (1966) Recombination of heavy and light chains from human immunoglobulins. Immunology 10, 549-557.
Grey H. M. & recombination of myeloma proteins. Grey H. M. (1969) Immun. 10, 51-104.
Mannik M. (1965) Specificity of H and L chains from human yGJ. exp. Med. 122, 619-632. Phylogeny of immunoglobulins. Adv.
Haber E. (1971) Homogeneous elicited antibodies: induction, characterization, isolation. and structure. Ann. N. Y. Acod. Sci. 190,285-305.
Phylogeny of lmmunoglobulin Harboe M.. Solheim B. Cr. & Deverill J. (1969) Individual macroglobulins. of human antigenic specilicity Localization of antigenic determinants and their use to demonstrate formation of hybrid molecules after reduction and alkylation. J. exp. Med. 129, 1217-1281. Hoffman D. R. & Campbell D. H. (1969) Model systems for the study of drug hypersensitivity. I. The specificity of rabbit anti-aspiryl system. J. Immun. 103, 655-661. Katz M. & Pappenheimer A. M. (1969) Quantitative studies of the specificity of anti-pneumococcal antibodies, types III and VIII. 1V. Binding of labeled hexasaccharides derived from S3 by anti-S3 antibodies and their Fab fragments. J. fmmun. 103, 491-495. Kim Y. D. & Karush F. (1974) Equine anti-hapten antibody. VIII. isoelectric fractions of IgM and 7s anti-lactose antibody. Immunochemistry 11, 147-152. Kimball J. W.. Pappenheimer A. M. & Jaton J-C. (1971) The response in rabbits to prolonged immunization with type III pneumococci. J. Immun. 106, 1177-l 184. Kimball J. W. (1972) Maturation of the immune response to type Ill pneumococcal polysaccharide. Immunochemistry 9, 1169-l 184. Klinman N. R. (1971) Region of homogeneous binding activity after recombination of chains of “monofocal” antibody. J. Immun. 106, 1330-1337. Krause R. M. (1970) The search for antibodies with molecular uniformity. Adv. Immun. 12, I-56. Laemmli U. K. (1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685.
Leslie G. A & Clem L. W. (I 973) Chicken antibodies to group A streptococcal carbohydrate. J. Immun. 110, 191-196. Metzger H. (1970) Structure and function of 7 M macroglobulins. Adv. Immun. 12, 57-l 16. Miller E. J. & Terry W. D. (1968) Antigenic determinants of human IgM requiring interaction of 1, and K chains. J. Immun. 101, 1300-I 307. Mitchell K. F., Karush F. & Morgan D. 0. (1977) IgM antibody. III. The role of light chains in equine anti-lactose Fablr. Immunochemistry 14, 781-786. Nelson N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. biol Chem. 153, 375-380. Onoue K., Grossberg A. L., Yagi Y. & Pressman D. (1968) lmmunoglobulin M antibodies with ten combining sites. Science 162, 574-576. Oriol R., Binaghi R. & Coltorti E. (1971) Valence and
Structure and Function-VIII
315
association constant of rat macroglobulin antibody. J. 104, 932-937. Oriol R. & Rousset M. (1974) The IgM antibody site. I. Increase in binding affinity of rat 19s and 7s antidinitrophenyl antibodies during the course of the immune response. J. Immun. 112, 2227-2234. Painter R. G., Sage H. J. & Tanford C. (1972) Contributions of heavy and light chains of rabbit immunoglobulin to antibody activity. I. Binding studies on isolated heavy and light chains. Biochemistry 11, 1327-l 345. Sarvas H. & MHkelH,0. (1970) Haptenated bacteriophage in the assay of antibody quantity and affinity: Maturation of an immune response. Immunochemistry. 7,933-943. Scatchard G. (1949) The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660-672. Schulkind M. L., Robbins J. B. & Clem L. W. (1971) Reactivities of shark 19s and 7s 1gM antibodies to Salmonella typhimurium. Nature, Land. 14, 182-l 83. Shankey V. (1977)Heterogeneity of IgM ligand binding: an interor intramolecular phenomenon? Doctoral dissertation, University of Florida. Gainesville. FL. Speyer J. L. Emans J. B:, Kimball J. W., & Pappenheimer A. M. (1973) Quantitative studies of the specificity of antipneumococcal polysaccharide antibodies, types III and VIII. V. Cross-reacting antibodies in rabbbit antisera. Immunochemistry 10,257-263. Stone M. J. & Metzger H. (1968) Binding properties of a Waldenstrom macroglobulin antibody. J. biol. Chem. 243, Immun.
5977-5984.
Suran A. A., Tarail M. H. & Papermaster B. W. (1967) lmmunoglobulins of the leopard shark. 1. Isolation and chacterization of 17s and 7s immunoglobulins with precipitating activity. J. Immun. 99, 679-686. Voss E. W. & Eisen H. N. (1968) Valences and affinity of IgM antibodies to the 2.4-dinitrophenyl (DNP) group. Fedn. Proc. Fe&.
Am. Sot. exp. Biol. 27, 684.
Voss E. W. & Sigel M. M. (1971) Distribution of 19s and 7s IgM antibodies during the immune response in the nurSe shark. J. Immun. 106, 1323-1329. Voss E. W. & Sigel M. M. (1972) Valence and temporal change in affinity of purified 7s and 18s nurse shark anti2,4_dinitrophenyl antibodies. J. Immun. 109, 665-673. Young N. M., Jocius I. B. & Leon M. A. (1971) Binding properties of a mouse immunoglobulin M myeloma protein with carbohydrate specificity. Biochemistry 10, 3457-3460.
PHYLOGENY
OF IMMUNOGLOBULIN FUNCTION-VIII.
STRUCTURE
0301-036s
10200~0
AND
INTERMOLECULAR HETEROGENEITY OF SHARK 19s IgM ANTIBODlES TO PNEUMOCOCCAL POLYSACCHARIDE* T. VINCENT SHANKEYt and L. WILLIAM CLEMS Department
of Immunology Laboratory.
and Medical Microbiology, College of Medicine. and Whitney University of Florida, Gainesville, FL 32610, U.S.A.
(Received 2 Mu!
Marine
1979)
Abstract-Significant IgM antibody responses to the pneumococcal capsular polysaccharidc were demonstrated in nurse sharks immunized with pneumococcal vaccines. The antibodies isolated from immune sera by affinity chromatography were exclusively of the 19s variety; no 7s antibodies were isolated from any of six animals studied for periods up to twelve months. Equilibrium dialysis studies of the isolated antibodies demonstrated several important points: (I) the IgM antibodies contained ten combining sites per 19s molecule. (2) in all cases, the isolated antibodies showed marked heterogeneity of combining sites, (3) the antibodies were all of low average affinity, and (4) there was no increase in the average affinity of the antibodies isolated from any single animal for periods of up to twelve months after the start of immunization. In order IO determine if a single IgM molecule contains ten equivalent combining sites. the antibodies isolated from several animals were fractionated by liquid isoelectric focusing. Equilibrium dialysis experiments using focused fractions showed the presence of ten functionally identical combining sites per IgM molecule. As a proof of the structural homogeneity of focused fractions, antibodies were separated into H and L chains.9 recombined and tested for the regeneration of the original combining site by equilibrium dialysis. The results indicated that recombinonts of focused antibody fractions contained binding sites identical to those of the intact antibody, whereas heterogeneous (unfocused) recombinants and isolated H and L chains failed to show any binding activity. The conclusion from this study is that the heterogeneity of ligand binding exhibited by nurse shark 19s antibodies to the capsular polysaccharide of the Type III pneumc-coccus can be attributed to intermolecular heterogeneity.
INTRODUCTION
1969; Young et al., 1971). numerous other studies with conventional IgM antibodies have indicated considerable heterogeneity of hapten binding with valences of less than ten. In certain instances valences of less than ten were attributable to steric factors due to antigen size (Stone & Metzger, 1968; Edberg ef al., 1972) whereas in others, haptens were employed which would not likely impose steric limitations on the antibody combining sites. In this latter context an average of five high and five low affiinity sites per 19s antibody molecule has been frequently observed (Onoue er al., 1968; Oriol et al.. 1971; Voss & Sigel, 1972; Oriol & Rousset, 1974). In terms of resolving this issue there would seem to be two different, but not necessarily exclusive. possibilities. The first is that ligand binding heterogeneity is a consequence of differences in affinities between antibodies in a population, i.e. intermolecular differences due presumably to differences in antibody primary structure. The second possibility is that ligand binding
Mammalian IgM antibodies contain ten heavylight chain pairs and thus, by analogy with IgG antibodies, should contain ten equivalent ligand binding sites per molecule (reviewed by Metzer, 1970). Although studies employing structurally homogeneous IgM monoclonal proteins have demonstrated the presence of ten homogeneous binding sites per molecule (Ashman & Metzer. l This work was supported by NSF grant PCM 75-16479, NIH grant 1 ROI Al 14137andspecialfundsfrom thecenter for Environmental Programs. IFAS, University of Florida. +Present address: Department of Pathology. School of Medicine. University of Pennsylvania. Philadelphia, PA. U.S.A.
:Address correspondence to: L. W. Clem. Department of Microbiology, University Medical Center, Jackson, MS 39216. U.S.A. S;Abbreviations used: Tris buffer. 0.01 M Tris. 0.14 M NaCI. 0.01 M EDTA. O.l”,, NaN (pH 7.4); S3. capsular carbohydrate of Type III pneumococci; S8. capsular carbohydrate of Type VIII pncumococci: H and L chains: component heavy and light polypeptidc chains of immunoglobulins: DNP. 2.4-dinitrophcnyl. 365
366
T. VINCENT
SHANKEY
reflects differences within heterogeneity individual antibody molecules, i.e. intramolecular differences. The solution to this question has, to a large part. been elusive due to difficulties in obtaining sufficient amounts of structurally homogeneous IgM antibodies for study. The physicochemical properties of immunoglobulins from lower vertebrates indicate that such animals are restricted to the synthesis of but one immunoglobulin isotype and that this isotype is analogous to IgM (reviewed by Clem & Leslie, 1969; Carton, 1973; Grey, 1969). Furthermore, studies using sharks indicate that certain antigens, especially A-variant streptococci, can elicit the production of very large amounts of 19s antibodies (up to 10 mg ah/ml serum, Clem & Leslie, 1971). Although it has not been possible to isolate a suitable hapten for studying affinities and valences with this antigen (Shankey, 1977), the finding in sharks of reasonably good 19s antibody responses to both the DNP hapten covalently coupled to streptococcal cells and the capsular polysaccharide of pneumococcal cells, has now provided sufficient antibody to defined ligands for study. This paper describes the binding properties of shark 19s antibodies to the S3 polysaccharide and the following paper in this series (V. Shankey and L. W. Clem, in preparation) describes similar studies with shark antibodies to the DNP moiety. A summation of these findings suggests the intriguing possibility that both inter- and intramolecular factors may be involved in 19s IgM ligand binding heterogeneity.
MATERIALS
AND METHODS
Vaccines and immunization
Fully encapsulated strains of Types III and VIII Streptococcus (Diplococcus) pneumoniae were a generous gift of Dr. A. M. Pappenheimer, Jr. (Biological Laboratories, Harvard University, Cambridge, MA). Vaccines were prepared from both types of pneumococci as described by Kimball et al. (1971) and were stored at 4°C in 0.1% formalized saline until used. Nurse sharks (Ginglymyostoma cirratum) of both sexes, weighing lo&200 kg, were maintained in tidal sea water at the Lerner Marine Laboratories, Bimini, Bahamas. Immunizations, performed by intravenous injection of 30 ml of either Type III or Type VIII
and L. WILLIAM
CLEM
vaccine (1 O9 cells/ml), and bleedings. were accomplished as described elsewhere (Clem & Leslie, 1971). Sera were obtained by centrifugation after allowing the blood to clot overnight at room temperature and were subsequently stored at -20°C until used. Isolation of capsular carbohydrate
Type III and Type VIII pneumococci were first passed through DBA mice to select highly encapsulated strains. Fresh isolates were used to inoculate 2-liter broth cultures of Brain Heart Infusion (Difco Labs) supplemented with 2% sterile horse serum (Pel-Freeze). Cell lysis and extraction of the capsular carbohydrates were performed as described by Campbell & Pappenheimer (1966). The purified carbohydrates were then treated sequentially with DNase, RNase and pronase as described by Katz & Pappenheimer (1969). isolation and labelling of the S3 hexasaccharide hapten
Purified S3 was partially acid hydrolyzed by the procedure of Campbell & Pappenheimer (1966), and the hydrolysis products were gel filtered at 4°C on a 2.5 x 120 cm column of Sephadex G-25 (fine mesh) equilibrated with 0.01 M NH,HCO,. The hexasaccharide peak was identified by determining the ratio of reducing sugar (Nelson, 1944) to total sugar (Dubois et al., 1956) of eluted products. Fractions containing the hexasaccharide were pooled and concentrated by flash evaporation. was Labelling of the hexasaccharide accomplished by reduction with tritiated sodium borohydride. Three milligrams of hexasaccharide (in 1 ml) were mixed at 4°C with 250 mCi tritiated sodium borohydride (Amersham/ Searle, 8 Ci/mmole) dissolved in 0.1 ml distilled water. After incubation at room temperature, the reaction mixture was gel filtered on a 2.5 x 120 cm Sephadex G-25 (fine mesh) column with 0.01 M NH,HCO,. equilibrated Radioactivity eluted from the column in two peaks: the first at the position of the unlabelled hexasaccharide and the second coincident with the totally included volume of the column. The activity of the labelled hexasaccharide was 3.8 x 1OScounts/min/~mole. Isolation of antibodies
Antibodies to the capsular carbohydrate of Type III pneumococci were isolated from nurse shark sera by use of immunoadsorbent colums
Phylogeny of lmmunoglobulin
prepared by linking S3 to Sepharose by a modification of the method of Axen et al. (1967). Briefly, 5 g L-lysine dissolved in 500 ml 0.5 M NaHCO, was added to 100 ml (packed volume) Sepharose 4B (Pharmacia) previously activated with 4 g CNBr (Eastman Organic). The pH was adjusted to 8.5 and the mixture was stirred at 4°C for 12 hr. After washing, 100 mg S3 previously activated with 400 mg CNBr was added to the lysine-Sepharose, the pH was adjusted to 8.5 and the mixture was stirred at 4°C for 12 hr. Before use, the immunoadsorbent was washed with 0.1 M glycine (pH 9.0) to block unreacted sites on the activated Sepharose. Antibodies were eluted from immunoadsorbant columns with 0.5 M glycine/l.O M NaCl, pH 2.3, neutralized, concentrated by positive pressure dialysis and gel filtered at 4°C on a 2.5 x 100 cm Sephadex G-200 column equilibrated with Tris buffer. Eluted fractions were monitored for protein by absorbance at 280 nm and antibody containing fractions were pooled and concentrated by positive pressure dialysis. Equilibrium dialysis
Equilibrium dialysis was performed essentially as described by Clem & Small (1970). One side of the cell was filled with 250 ~1purified antibody (from 0.5 x lO-‘j to 5 x 10e6 M depending upon the experiment) in 0.05 M phosphate buffer (pH 7.2), while the other side of the cell was filled with an equal volume of the tritiated hexasaccharide hapten (from 0.1 x 10m6to 50 x 10m6 M) in the same buffer. Dialysis cells were rotated (- 1 rev/min) at 4°C for 72 hr, a period sufficient for the equilibration of hapten with normal nurse shark Ig or buffer. Duplicate 50 ~1 samples from each side of the cell were counted in 4.5 ml Aquasol- (New England Nuclear) using a Beckman LS- 1OOC liquid scintillation spectrometer. Samples were counted for 10 min or until 0.57$, counting error was reached. Data were calculated and plotted by the method of Scatchard (1949). Each data point represents duplicate counts of duplicate samples from duplicate cells. Antibody concentrations for equilibrium dialysis experiments were determined by differential refractometry as described by Babul & Stellwagen (1969). In order to assure that the protein concentration determined by this method was an accurate assessment of active antibody, only freshly isolated samples from immunoadsorbants were used for equilibrium dialysis.
Structure and Function-VIII
367
Preparative liquid isoelectric focusing
Isoelectric focusing was performed in an LKB 110 ml isoelectric focusing column (model 8 10 1) using a final concentration of 1.2% carrier Ampholines (LKB Instruments) stabilized by a linear 5-45x sucrose gradient (Ultrapure sucrose, Schwartz/Mann). The cathode used was 1% ethylene diamine, while the anode was 1% phosphoric acid in 50% sucrose. Antibodies isolated from a single bleeding (25-40 mg in 5 ml) were mixed with an equal volume of dense ampholyte solution, and immediately layered to its isobuoyant density using a length of 20 gauge polyethylene tubing inserted into the column before loading the ampholyte solution. Focusing was initiated at 400 V, and was increased to 1200 V as rapidly as possible without exceeding 6 mA or 9 W. Focusing was not continued for more than 24 hr since noticeable loss of cathodal ampholytes was observed after that time. After focusing, 1 ml fractions were collected, the pH was determined immediately and the protein was monitored by absorbance at 280 nm. Fractions containing a visible precipitate were clarified by the addition of an equal volume of Tris buffer before measuring absorbance. Recombination of antibody H and L chains
Heterogeneous or homogeneous nurse shark anti-S3 antibody was mildly reduced with 2mercaptoethanol (Eastman Organic) and alkylated with iodoacetamide (Sigma) in Tris buffer as previously described (Clem & Small, 1967). After extensive dialysis against Tris buffer, the antibody was dialyzed against 5 M guanidine-HCl containing 0.01 M iodoacetamide (pH 5.5) and subsequently gel filtered on a 1.5 x 90 cm Bio Gel A5M column equilibrated with 5 M guanidine_HCl. Eluted fractions were monitored for protein by absorbance at 280 nm. Subsequent recombination of the separated H and L chains was accomplished by mixing equimolar amounts of H and L chains, using J?$&= 11.7 for isolated H chains and Ei& = 13.1 for isolated L chains (Clem & Small, 1967). Mixtures of H and L chains in 5 M guanidineHCl were concentrated by positive pressure (7.5 lb max) to a concentration of 5 mg protein per ml. They were then dialyzed at 4°C against three changes of Tris buffer (100 volumes each). The size of recombinant antibody molecules was determined by density gradient ultracentrifugation using a linear 8-267; sucrose gradient. Samples were spun for 24 hr at 160,000 g in an
T. VINCENT SHANKEY and L. WILLIAM CLEM
368
Table 1. Precipitable
serum antibody to S3 of nurse sharks immunized pneumococcal vaccine
Shark No.
4 Months
23 50 51
0.20 0.25 0.30
with Type III
Precipitable antibody (me/ml) Tie after primary immunization’ 8 Months 6 Months
12 Months 0.30 0.30 0.50
0.50 0.40 0.80
0.40 0.30 0.80
‘Each animal received vaccine on days 1, 2, 50 and 120.
SW-40 Ti rotor and fractions of 0.25 ml were subsequently eluted by pumping 50% sucrose into the bottom of each tube. Fractions were monitored for protein by absorbance at 280 nm. Normal nurse shark 19s and 7s IgM were used as molecular-weight markers. The composition of the -7s recombinant peak obtained from density gradient ultracentrifugation was determined by elecgels in SDS-polyacrylamide trophoresis (Laemmli, 1970). Gels stained with 0.025% Buffalo Black were scanned at 600 nm using a Beckman spectrophotometer equipped with a linear transporter, and areas under peaks were measured with a planimeter. RESULTS
Level of anti-carbohydrate
antibody
The amount of precipitable anti-capsular carbohydrate antibody in each serum sample was determined by quantitative precipitation. The sera obtained from each of three nurse sharks immunized with Type III vaccine were tested for precipitable antibody using the S3 polysaccharide as antigen. The results, shown in Table 1, indicated that each of these animals produced relatively low levels of precipitable anti-S3 antibody. Due to the relatively poor response to the Type
III vaccine, four additional animals were immunized with Type VIII vaccine with the intention of eliciting cross-reactive antibodies to S3, a phenomena frequently observed with rabbits and horses immunized with the Type VIII vaccine (Speyer et al., 1973). The results of quantitative precipitation tests with these sera, using the S3 and S8 antigens (Table 2) indicated that all four animals produced significant antibody responses. In most cases, the amount of precipitable anti-S8 antibody did not increase during the period of vaccination. These results also showed that sharks immunized with Type VIII vaccine produced antibodies which precipitated with S3. In order to determine the extent of cross-reaction, the sera from animals receiving Type VIII vaccine were tested both by sequential and by simultaneous precipitation with S3 and S8. The results (Table 2) indicated that sera from two of these animals (numbers 59 and 61) contained the same amount of precipitable antibody with S8 alone as with S3 and S8. This indicated that these animals produced some antibodies to S8 which completely cross-reacted with S3, and thus were probably recognizing the common (cellobiuronic acid) residues found in both S3 and S8. On the other hand, the results of quantitative precipitation tests with the sera from animals 58 and 64 showed increased amounts of precipitable
Table 2. Precipitable serum antibody to S3 and S8 of nurse sharks immunized with Type VIII pneumococcal vaccine
Shark No.
s3b
Precipitable antibody (mg/ml) Time after primary immunization’ 5 Months 1 Month S8 S8’ S3 S3 + S8 S3 + S8“
58 59 61 64
0.40 0.20 0.25 0.50
1.20 0.60 0.25 0.70
a Each animal received b Antibody precipitable ‘Antibody precipitable ‘Antibody precipitable ’ Not done.
1.60 0.63 0.30 1.20
0.93 0.15 0.32 0.60
vaccine on days 1, 2, 3, 32 and 64. with S3 alone. with S8 alone. with S3 and S8 together.
1.00 1.40 1.40 0.70
1.90 1.35 1.40 1.24
s3 0.40
10 Months S8 S3 + S8 1.10 N.D.’ N.D. N.D.
1.58
Phylogeny of lmmunoglobulin
Structure
369
and Function-VIII
In all cases, the isolated anti-S3 antibody eluted from a Sephadex G-200 gel filtration column at the void volume and analysis of this material in the analytical ultracentrifuge indicated a single homogeneous boundary with Isolation of anti-S3 antibodies + 17s (at 3-5 mg/ml). an S,,, of analysis of the purified The sera of two animals (numbers 23 and 51) Immunoelectrophoretic anti-S3 antibody demonstrated a single immunized with Type III and from all four animals immunized with Type VIII vaccine were component when reacted with rabbit anti-whole nurse shark serum and developed an identical used as a source of antibodies to S3. Antibodies were isolated by elution from S3-Sepharose precipitin band with rabbit antisera to nurse immunoadsorbant columns using 0.5 A4 shark immunoglobins (Clem er al., 1967). glycine/l.O M NaCl (pH 2.3). In all cases, the isolated antibody represented from 25 to 50% of Afjnity of anti-S3 anribodies The affinity of the isolated nurse shark the precipitable serum antibody to S3 and was >90% active as determined by quantitative antibodies to S3 was measured by equilibrium precipitation or affinity column recycling. dialysis using the tritiated hexasaccharide hapten
antibody with S3 and S8 compared to S8 alone. This would indicate the presence of at least two different populations of antibodies, one capable of reacting with S8, the other with S3.
04 03 :
-0 I -10
N.S.
23
CK+O 4 Months
N.S
04
03
I2 Months
02
02
51
-
4 Months
-
I2
Months
+z+_
0 I
f
5
N.S Ooo m
58 I Month 5 Months IO Months
N.S.
04
03 F
IO
N.S @DO -
Fig.
I.
Scatchard
IO
61 I Month
I-
59
-
I Month
-
5 Months
N.S. 64 0-0-0
-
I
Month
5 Months
5 Months
plots for isolated 19s IgM antibodies isolated from each of six different Times indicated refer to the time after primary immunization.
nurse sharks.
370
T. VINCENT
SHANKEY
and L. WILLIAM
CLEM
obtained from S3. This hapten was used since obtain homogeneous IgM antibodies to address preliminary equilibrium dialysis experiments the question of whether homogeneous antibody indicated that nurse shark anti-S3 antibodies exhibited homogeneous hapten binding. During failed to react appreciably with the isoelectric focusing, all nurse shark IgM tetrasaccharide and formed precipitates with the antibodies developed significant bands of visible larger octasaccharide hapten obtained from S3. precipitate in the focusing column. In order to Speyer et al. (1973) have reported a similar demonstrate that these precipitates were precipitation with rabbit anti-S3 antibodies with homogeneous antibody at its isoelectric point the octasaccharide hapten. and not heterogeneous antibody precipitated at The results of equilibrium dialysis studies with its isobuoyant density, several experiments were isolated anti-S3 antibodies are shown in Fig. 1 conducted. First, an isolated nurse shark anti-S3 and illustrate several points. In each case, the antibody was divided into two unequal aliquots; isolated exhibited considerable antibody both focused in an identical manner even though heterogeneity of binding sites of relatively low different amounts of antibody were applied to each column. Second, an anti-S3 antibody peak affinity (K, < 5 x IO4 L/M). The antibody isolated from any single animal showed no which focused at pI 4.5 was subsequently significant increase in affinity for periods up to refocused in a narrower ampholyte gradient, and was shown to elute from this second focusing twelve months after the start of immunization. Similarly, in all cases five or more binding sites column at the same isoelectric point ( f 0.1 pH were measured and in two cases (animals 58 and unit). Finally, a focused anti-S3 antibody peak 64) the Scatchard plots readily extrapolated to a was refocused on an identical ampholyte valence of ten. gradient with the polarity of the column reversed, and again the peak eluted at the same Binding of homogeneous antibody obtained by isoelectric point ( f. 0.1 pH unit). Since focusing isoelectric focusing experiments conducted with higher ampholyte Liquid isoelectric focusing was utilized to concentrations failed to prevent precipitation of
4 Months 30
0
I Month
50
100
Fraction
100
number
Fig. 2. Liquid isoelectric focusing elution profiles of 19s anti-S3 primary immunization. Top: Shark No. 51, antibodies isolated Middle: Shark No. 58, antibodies isolated at 5 months (left) and antibodies isolated at 1 month (left) and
antibodies isolated at different times after at 4 months (left) and 12 months (right). 10 months (right). Bottom: Shark No. 64, 5 months (right).
Phylogeny of lmmunoglobulin
the antibody, a final ampholyte concentration of 1.2’;; was routinely used. Comparison of the liquid isoelectric focusing elution profiles of 19s anti-S3 antibodies isolated from three animals at different times after primary immunization (Fig. 2) showed a typical pattern of six to ten major peaks, generally focusing at isoelectric points between 4 and 7. In all cases, there was no significant change in the number of isoelectric points of the major antibody peaks during periods of up to eight months of immunization. Liquid isoelectric focusing analysis of the isolated 19s anti-S8 antibodies from two animals (not shown) showed a similar heterogeneous elution profile with seven or nine major peaks. Since it was considered crucial to obtain homogeneous antibodies for subsequent studies, only the first peaks eluting from focusing columns were utilized. This was done in order to minimize possible contamination from other peaks during elution from the focusing column. Furthermore, the eluted peaks were repurified on an S3-Sepharose immunoadsorbant before use in equilibrium dialysis experiments, since as 40
N.S. 58
r
000 Prc -focus 0..
pl 4.6
NS. 64
000
Prc -focus
r
Fig. 3. Scatchard plots showing the S3 hapten binding by focused and non-focused 19s shark anti-S3 antibodies. Results expressed as r/900,000 daltons. MlMM 11’3-F
Structure and Function--VIII
371
much as 50% of the focused nurse shark antibody was found to be inactivated by the focusing procedure. This effect was shown to be particular to 1gM antibodies, since less than 10% of focused rabbit IgG antibodies to S3 were inactivated by similar isoelectric focusing procedures. Kim & Karush (1974) have reported a similar inactivation of equine IgM antibodies after isoelectric focusing. The results of equilibrium dialysis experiments using these focused nurse shark antibodies, shown in Fig. 3, indicated that focused antibody shows homogeneous binding as contrasted with heterogeneous binding seen with the unfocused anti-S3 antibody obtained from these same animals. Since the technique used to determine antibody concentration for these equilibrium dialysis experiments is an accurate assessment of the active antibody concentrations, these results clearly indicate that the isoelectrically focused 19s IgM antibodies exhibit- ten functionally identical binding sites per molecule. Antibody recombination studies In order to verify the homogeneity of focused nurse shark anti-S3 antibodies, focused fractions were mildly reduced and alkylated, separated into H and L chains and subsequently recombined and examined for the regeneration of the original combining site. Since recombinant anti-S3 antibodies could not be repurified by affinity chromatography (acid elution from the immunoadsorbant would separate the recombined chains), an alternative approach was employed to assess the active antibody concentration for equilibrium dialysis experiments. The recombined H and L chains from focused antibody were first subjected to preparative density gradient ultracentrifugation wherein a majority (270%) of recombinant molecules sedimented only slightly slower than the 7S nurse shark immunoglobulin marker (Fig. 4). An aliquot of each -7s protein was subjected to electrophoresis in SDSpolyacrylamide gels. In each case m 70% of the stained material migrated in the gel region expected to contain H chains whereas the remainder was in the L chain region (inserts, Fig. 4). It therefore seems reasonable to suggest that the -7s recombinants were composed of 2 H and 2 L chains and thus a molecular weight of 180,000 was assumed. Another aliquot of _ 7S material was applied to an S3 immunoadsorbant. The results indicated that 70:/, of the recombined (PI 4.60) antibody from animal
372
T. VINCENT SHANKEY and L. WILLIAM CLEM N.S 000
58 pl 46 19s
Antibody
0 l l Recombi non&
64
pl 4.4
19 S Antibody Recombinants
.’
0’
l...’ ,
IO
.
\ I
20
Fraction
%. +._.--*: 30
40
number
Fig. 4. Sucrose density gradient ultracentrifugation profile of H and L chain recombinants. Top: focused (~14.6) anti-S3 antibody from shark 58. Bottom: focused (PI 4.4) anti-S3 antibody from shark 64. Arrows indicate the positions of labeled shark 7S and 19s immunoglobulin markers. Inserts show the SDS-polyacrylamide gel electrophoresis of the 7S recombinants compared to labeled shark immunogiobulin H and L chains.
number 58 was active while 60% of the recombined (PI 4.40) antibody from animal number &4was active. The remainder of the u 7S samples were subjected to equilibrium dialysis against the S3 hexasaccharide. The antibody concentration used for calculating the results of these equilibrium dialysis experiments represented the total protein concentration, as determined by differential refractometry, corrected for inactive antibody using the results of immunoadsorbant passage. The equilibrium dialysis data (Fig.5) was analyzed by linear regression analysis which indicated that the plot for animal 58 (p1 4.60) both before and after recombination extrapolated to 2.0 sites per 7s sub-unit (regression coefficients of 0.98 and 0.92, respectively), while the plot for animal 64 (PI 4.40) extrapolated to 2.02 sites before (regression coefficient 0.98) and 1.95 sites after recombination (regression coefficient 0.93). It should also be pointed out that recombinant m 7s molecules obtained from heterogeneous (unfocused) anti-S3 antibodies from these same two nurse sharks (similarly containing equimolar H and L chains as judged by electrophoresis in
IO r
2.0
Fig. 5. Scatchard plots showing the S3 hapten binding by focused intact anti-S3 19s molecules and 7S H and L chain recombinants. Results are expressed as r/180,000 daltons.
SDS-polyacrylamide gels) were incapable of binding to S3Ammunoadsorbant (< lOO,‘,>and showed no detectable hapten binding by equilibrium dialysis. Similarly, isolated H and L chains from unfocused antibody failed to exhibit binding of the S3 hapten in equilibrium dialysis experiments.
DISCUSSION
Previous attempts to induce antibody formation to a variety of antigens have given sharks the reputation of being immunologically poor responders (reviewed by Carton, 1973). However, the responses to pneumococcal vaccines reported here, and the previously reported responses to streptococcal vaccines (Clem & Leslie, 1971) indicate that sharks are capable of synthesizing significant quantities of antibodies in response to bacterial vaccines. Similar vaccination procedures in mammals and birds (Leslie & Clem, 1973; Krause, 1970; Haber, 1971) have been shown to result in the frequent production of large quantities of antibody. While
Phylogeny of Immunoglobulin Structure and Function-VIII
quantitatively quite good, the shark response is necessarily limited to the production of only IgM antibodies. Thus, the utilization of similar procedures with these and other bacterial vaccines could provide large quantities of shark IgM antibodies to different haptens with which to address a number of questions about the characteristics of the immune response and antibodies from lower vertebrates. The failure to detect 7S IgM antibodies in response to pneumococcal vaccines is in contrast to reports of shark antibody responses to a variety of protein, bacterial and viral antigens (Clem & Small, 1967; Clem et al., 1967; Suran et al., 1967; Schulkind et al., 1971) and to hapten-protein conjugates (Voss & Sigel, 197 1). Although 7S IgM antibodies generally were observed late in the immune response in these studies, 7S anti-S3 or S8 antibodies were not isolated from any of the animals studied here, even after twelve months of immunization. Similarly, 7S IgM antibodies have not been detected in any of the sera of twelve nurse sharks immunized with streptococcal antigens (Clem & Leslie, 1971; L. W. Clem, unpublished observations). Since the anti-carbohydrate antibody response in sharks immunized with pneumococcal or streptococcal vaccines also differs quantitatively, it is possible that these results reflect differences in the control of antibody synthesis and/or release in sharks presented with different antigens. The alternate possibilitity, that these animals synthesized 7S IgM anti-carbohydrate antibodies which could not be recovered using immunoadsorbants, is unlikely. The 7S sub-units obtained by mild reduction of purified 19s anti-S3 (and antistreptococcal carbohydrate, L. W Clem, unpublished observations) antibodies have been isolated using these same immunoadsorbants, suggesting then, that naturally occurring 7S IgM antibodies of similar or higher affinities should be recoverable by this technique. In contrast to the usual pattern of maturation seen in the immune response of higher vertebrates (Eisen & Siskind, 1964; Kimball, 1972), the nurse shark response to prolonged administration of pneumococcal vaccines failed to exhibit maturation. This was evidenced by the lack of any significant increase in the amount of precipitable serum antibody with prolonged immunization, the lack of changes in the isoelectric focusing elution profiles of the isolated antibody, and finally by a lack of significant increases in the affinities of the
373
isolated antibodies from any of the six animals studied here. This lack of maturation appears to be a common finding for IgM antibody responses -for lower vertebrates (Voss & Sigel, 1972; Clem & Small, 1970; Fiebig ef al., 1977) and for mammals (Voss & Eisen, 1968; Sarvas & Mlkelii, 1970; Kim & Karush, 1974; Chua et al., 1975). The presence of a ten heavy-light chain structure predicts that shark 19s IgM antibodies should contain ten equivalent hapten combining sites. However, previous studies of shark antibodies have measured a valence of less than one for each heavy-light chain pair (Voss & Sigel, 1972). In contrast, the results of the study reported here clearly demonstrate that isoelectrically focused shark 19s antibodies to S3 contain ten equivalent, low affinity combining sites. Since it was previously shown that functionally homogeneous antibodies are not necessarily structurally homogeneous (Hoffman & Campbell, 1969), it was considered insufficient to conclude that isoelectrically focused shark anti-S3 antibodies were structurally homogeneous based on homogeneous binding of the S3 hapten. In view of studies on the regeneration of original antibody combining sites following separation and recombination of H and L chains from homogeneous mammalian IgG antibodies (Unman, 1971), recombination of focused and unfocused shark anti-S3 antibodies was undertaken. The demonstration that isoelectritally focused shark IgM antibodies contain an identical combining site after H and L chain separation and subsequent recombination, while recombinants of heterogeneous (unfocused) shark antibodies lacked any binding activity, thus indicates that isoelectrically focused fractions are structurally homogeneous. Therefore, a reasonable conclusion is that heterogeneity of hapten binding with shark antibodies to this ligand is an intermolecular phenomenon. This finding is seemingly in conflict with the results obtained with shark antibodies to the DNP moiety reported in the following paper in this series. Studies of recombinants of IgM molecules have generally been limited to an assessment of physiochemical or antigenic properties or recombinants of myeloma proteins (Grey & Mannik, 1965; Gordon & Cohen, 1966; Miller & Terry, 1968; Harboe et al., 1969; Carson & Weigert, 1973). These studies have usually shown that IgM myeloma proteins recombine to form N 7S sub-units composed of both H and L
374
T. VINCENT SHANKEY and L. WILLIAM CLEM
chains. A single report which studied the binding characteristics of IgM antibodies after the recombination of L chains and Fdp-chain fragments of isoelectrically focused Fabp suggested that recombinants of heterogeneous Fabp produce an active combining site identical to the site on the Fabp fragments before chain dissociation (Mitchell et al., 1977). It was concluded by these authors that the light chains of intact IgM antibodies do not contribute significantly to the binding of hapten. It should be noted, however, that no direct evidence was presented in that study to indicate the Fabp fragments actually dissociated into free L and Fdp chains allowing subsequent recombination of heterogeneous molecules. studies of recombination The results performed with shark IgM antibodies to S3 suggest that both H and L chains contribute to the formation of the shark antibody combining site. The lack of significant hapten binding by either isolated chain and the failure of m 7S recombinants of heterogeneous H and L chains to exhibit significant binding thus suggests that the antibody combining site of these lower vertebrate antibodies is formed by the juxtaposition of the appropriate H and L chains. Furthermore, since both the H and L chains apparently contribute to the formation of the combining site, it is reasonable that the affinity of the intact site should equal the product of individual affinity contributions of the isolated H and L chains, as previously shown for mammalian IgG antibodies (Painter er al., 1972). Unfortunately, the lack of significant binding by isola$d H and L chains of shark anti-S3 antibodies makes this determination impossible. Finally, the results of this study give some insight into the complexity of the immune response of lower vertebrates. The anti-S3 antibodies of the animals used in this study all showed considerable heterogeneity by liquid isoelectric focusing. This implies that within a single shark there is the capacity to generate a number of different combining sites to any single ligand. However, detailed analysis of each focused fraction would be necessary to show that each combining site is unique. Previously reported studies on the molecular heterogeneity of the shark antibody response to DNP and streptococcal vaccines (Clem et af., 1975) support the concept that there exist a number of different combining sites in the ‘library’ of sites available to any individual shark. It would be of considerable interest to determine whether the
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