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INHERITANCE OF ANTIBODY SPECIFICITY II. Anti -(4-Hydroxy-5-Bromo-3-Nitrophenyl)Acetyl in the Mouse*

(From the Department of Serology and Bacteriology, University of Helsinki, 00290 Helsinki 29, Finland)

genes code for the variable polypeptide sequence of immunoglobulin molecules . Available evidence indicates that there are three pools of V genes, one shared by all kappa chain subtypes, another by lambda chain subtypes, and the third by all heavy chain classes and subclasses . In the case of kappa and lambda V genes the evidence is mainly sequence data of myeloma proteins whereas several additional pieces of evidence suggest that all heavy chains share a common pool of variable polypeptide sequences . The structural genes for these sequences are called V H genes . V  genes of the rabbit and the mouse have been defined on Mendelian terms. In the rabbit the a-locus allotypes, a  a Z, and a,, have helped in gaining the important information that V and constant (C) genes of the heavy chain remain close but not absolute linkage (1) . Meiotic recombinations occur at a frequency of approximately 0 .3% (2, 3) . Different Mendelian markers of V H genes have been observed in the mouse. Contrary to a,, a,, and a, allotype markers of the rabbit, each of the mouse V .-gene markers is present in only a small proportion of immunoglobulin molecules sharing an antibody specificity . Both this and the mapping data suggest that each of the mouse markers represents a small proportion of all V genes .
Variable (V) 1

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At least six Mendelian V H -gene markers have been published : the gene con-

trolling the idiotype found in antiarsonate antibodies of A/J and AL/N mice (4), the gene controlling the idiotype in the antistreptococcal A carbohydrate antibody in A/J mice (5), the gene controlling the anti-alpha-1-3-dextran response in BALB/c mice (6), the gene controlling the T15 idiotype found in the antiphosphorylcholine antibody in BALB/c mice (7), the gene controlling the fine specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies in C57BL/6 mice (8), and the gene controlling the antiphosphorylcholine antibody with the 5107 idiotype in the BALB/c mice (9) . The six markers are based on three different
This study was supported by the Medical Research Council, Academy of Finland. $ Fellow of Funda(~ao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil . Present Address: Institute for Genetics, Cologne University, D-5000 Cologne 41, Weyertal 121, Germany. ' Abbreviations used in this paper: aminocap, N, e-amino-n-caproic acid ; BSA, bovine serum albumin; CG, chicken globulin ; DIP, (4-hydroxy-3-5-diiodophenyl)acetyl ; FSI, fine specificity index (see Results section) ; HPI, haptenated phage inactivation ; HPII, haptenated phage inactivation inhibition ; I, o, concentration of hapten causing 50% inhibition of a serological reaction ; IgCH, immunoglobulin heavy chain constant region ; 2-ME, 2-mercaptoethanol ; NBrP, (4-hydroxy5-bromo-3-nitrophenyl)acetyl ; NCIP, (4-hydroxy-5-cloro-3-nitrophenyl)acetyl ; NIP, (4-hydroxy-5iodo-3-nitrophenyl)acetyl ; NNP, (4-hydroxy-3,5-dinitrophenyl)acetyl ; NP, (4-hydroxy-3-nitrophenyl)acetyl ; V, variable ; V  gene, coding for the heavy chain V region .



141, 1975

Bethesda. Eastbourne. antistreptococcal A carbohydrate (5) . and IAH strains were from our colony. This extinction was pH dependent. Michael Potter (National Cancer Institute.1-3-dextran (6).) . Bar Harbor. England. Alum-precipitated NBrP  CG (100. The haptens used are shown in Table I. Md . The method for preparing further derivatives of NCIP was analogous to the method for preparing NBrP derivatives .g . Immunization of Mice . e.800. The flask was filled with chlorine under a hood. The second injection was administered 40 days after the first. and shaken overnight at room temperature under the hood . stoppered quickly. The resulting yellow crystals were washed with acetic acid and on October 25.. NBrP CG contained 14 mol of NBrP/150. Ltd. and NBrP BSA contained 14 mol of NBrP/mol of BSA. This was indistinguishable from the corresponding values for NIP and NBrP but different from the NP value (pH 7.rupress. Br I CI NO Z H I RZ NO.. Maine. Most of them were prepared according to the method of Brownstone et al. and (c) mice of the same genotype have a characteristic fine specificity in their antibody to an antigenic determinant (8) . BSA (Cohn fraction V) was obtained from Armour and Company. The serum antibodies from mice were obtained by bleeding 12 days after the second injection if not otherwise mentioned . NBrP BSA (100 jug) was injected subcutaneously (four sites) in complete Freund's adjuvant .4 . CG was obtained from blood serum by 45% ammonium sulphate saturation . In this paper we report a V gene that manifests itself by causing a high response to hapten (4-hydroxy-5-bromo-3-nitrophenyl)acetyl (NBrP) and a characteristic fine specificity in this antibody . Strain C57BL/Ka and CB20 were kindly given us by Dr .. Antibody Assays . NO. and the coefficient of extinction was 4. 2009 OH CHZ COOH Compound NBrP NIP NCIP NNP NP DIP R.Published THEREZA IMANISHI AND O . and the pH midpoint for the conversion from colored to noncolored was was injected intraperitoneally with (first injection) or without (second injection) 10 1 Bordetella pertussis bacteria as adjuvant .2). MAKELA 841 technical principles : (a) most mice of the same genotype share an idiotype or an isoelectric-focusing pattern in specific antibody. NBrP and its derivatives and (4-hydroxy-5TABLE I Haptens Used in This Study Materials and Methods Downloaded from jem. NIH.g . I iodo-3-nitrophenyl)acetyl (NIP) azide were prepared as previously described (11) . DBA/2N. C3H. e. (10) . NO Z NO Z NO. (b) mice of the same genotype are either high or low responders to an antigenic determinant. The immunogens were NBrP coupled to chicken globulin (CG) or bovine serum albumin (BSA) . CBA. The next day it was refilled with chlorine and incubated over another night. When dissolved they had an extinction maximum at a of 430. alpha. Most of our mouse strains were obtained from the Jackson Laboratories. (4-hydroxy-5-cloro3-nitrophenyl)acetyl (NCIP) was prepared by adding 2 g of (4-hydroxy-3-nitrophenyl)acetyl (NP) into 25 ml of glacial acetic acid in a 1 liter stoppered flask.000 g of CG. Haptens and Their Conjugates . The amount of antibody was measured by inactivation of haptenated (NBrP) bacteriophage (HPI test) which has been described earlier (11) . C57BL/6. . BALB/c. Mice .

and NNP while their affinity for both NP and NCIP was much lower . 80% inhibition corresponded to the phage survivor count of fivefold diluted antibody.Published 842 INHERITANCE OF ANTIBODY SPECIFICITY. Reasonably constant and accurate Iso values could be obtained by using half-log steps in the concentration series . (4-hydroxy-3 . NIP aminocap. had a high affinity for NBrP. Antiallotype Sera .5-dinitrophenyl)acetyl (NNP) aminocap. It manifested itself 100r Results Downloaded from jem. 10'10 10-9 10-8 10-7 10-6 10-5 10-4 hapten concentration 1. . The general method for studying fine specificity was hapten inhibition of NBrP-phage inactivation (HPII) . C57BL/6 antibodies. 1 (individual mice) and Table II (mean Iso values) .rupress. 2009 BALB/c 50 a x 0 C 0 a L c d U d Q 100 C57BL/6 " N&P-aminocap NIP -aminocap " NCIP-aminocap NNP-aminocap " NP -aminocap 50 Of 10-11 FIG. pertussis bacteria coated with C57BL/6 antibodies according to Dresser and Wortis (12) . A hapten concentration was said to cause 50% inhibition of NBrP-T4 inactivation (HPI) if it increased the number of phage survivors to the survival value of twice diluted antibody. Affinity of the antibodies was estimated by inhibiting the HPI reaction with varying concentrations of the above on October 25. NP aminocap. Affinity and Fine Specificity . Inhibition of anti-NBrP by NBrP-aminocap and related haptens. and (4-hydroxy-3-5-diiodophenyl)acetyl (DIP) aminocap . on the other hand. BALB/c antibodies had the highest affinity for the immunogenic NBrP but both NIP and NCIP followed closely thereafter . The main characteristics of anti-NBrP produced by the BALB/c and C57BL/6 mice emerge from Fig. Antibodies were obtained from a BALB/c or a C57BL/6 mouse 12 days after the second injection of NBrP-CG. II Affinity Assays. Anti-Ig-1° was obtained by immunizing BALB/c mice with B. Affinity for NNP was lower and for NP lower still . NIP. Anti-Ig-1a was prepared by immunizing C57BL/6 mice with B. Five related haptens including the immunogenic NBrP were used for the inhibition . Six free haptens in the form of N. NCIP aminocap. ISo values were interpolated from the crossing of the inhibition curves and the 50°10 horizontal line . The typing was done by the double-diffusion method . e-amino-n-caproic acid (aminocap) derivatives were used : NBrP aminocap. Maturation of the response was seen in both strains of mice . etc. 1. The relationship of hapten concentration to HPI inhibition is illustrated in Fig. Anti-NBrP Antibodies in Mouse Sera. pertussis bacteria coated with BALB/c antibodies .

of mice Time after immunization (days) days BALB/c 7 8 8 21 7 7 7 14 17 29 12 sect 14 17 130 21 6 5.2 1. but the relative affinities were again the same as those for the anti-NBrP  CG response (Table III) . In spite of this maturation the strain characteristics remained clearly visible at all stages . It differed from category 1 by having lower affinity for NP than for NCIP.4 5. IAH. and RF/J) belonged to this category.4 2.8 46 11 610 250 69 67 77 34 21. MA/J. NIP. All five tested allotype Ig-l b strains belonged to this group .400 1. but low affinity for NCIP.8 63 20 110 45 9. and NNP .7 9.000 4.9 4. Category 2 is characterized by very low affinity for DIP and NP.Published THEREZA IMANISHI AND O. Inheritance of the Strain Characteristics . and by high affinity for NCIP (higher than for NNP) . Category 1 is characterized by low I SO values (high relative affinity) for NIP and NNP. The low number of AKR mice makes the classification of this strain unreliable . Other inbred strains of mice were tested for the fine specificity of their anti-NBrP .4 2. CBA. DBA/2N. antibodies grew more "specific" for the immunogen .600 2. A/J. The anti-NBrP of this category differed from that of category 2 by its high affinity for NBrP. By this criterion. and (6) relative affinities for haptens other than the immunogenic one decreased . relatively high affinity for DIP and NP.100 620 100 60 87 18 16 15 1. We wanted to present the data of individual mice in a two-dimensional form. 2009 18 29 12 sec 3 . and for this purpose the fine . C57L. DIP. $12 days after the secondary immunization (sec) . MAKELA TABLE 11 84 3 Affinity of Anti-NBrP for Various Haptens at Different Stages of Immunization No . ISa values are geometric means of the nanomolar hapten concentrations causing 50% inhibition of NBrP-T4 phage inactivation (8). Affinity of the anti-NBrP produced by BALB/c and C57BL/6 was lower than when NBrP-CG was used.200 I* s values for: NBrPNIPNNPNPNC1Paminocapaminocapammocapammocapaminocap Strain C57BL/6 Downloaded from jem. including several allotype Ig-la strains . CG was the carrier molecule for NBrP in most of these experiments . Category 3 had only two on October 25. Most tested strains (BALB/c. We did a limited experiment using NBrP-BSA as the immunogen . in two ways : (a) ISO values for all haptens decreased.rupress.600 1.5 320 100 140 * As primary response sera were tested in the presence of 2-ME the data are representative of 7S antibodies only . and C3H of allotype Ig-1" (and perhaps the only tested allotype Ig-1° strain AKR) . ST/bJ. The results indicate that mouse strains can be divided into at least three categories on the basis of their anti-NBrP (Table IV) .

of mice 10 7 ME-resistent titer (log)* 6 .400 180 ND ND ND ND 880 9.6 7 . 2009 Affinity of Anti-NBrPAntibody for NBrP and Related Haptens in Different Inbred Strains of Mice Strain CBA C3H BALB/c MA/J C57L ST/bJ IAH C5713L/6 C57BL/Ka C57BL/Ks LP/J SJL/J RF/J DBA/2N AKR A/J CE/J H-2 k k d k b k TABLE IV IgC  allotype Ig-1° Ig-1° Ig-1" No. All values are geometric means of the nanomolar hapten concentrations causing 50% reduction of the phage inactivation .800 60 68 110 93 270 600 1.800 2.900 800 2.3 26 2 . of mice 10 12 21 4 6 6 8 18 14 5 14 10 11 h o* values for : NBrPNIPNNPNPNCIPDIPaminocap aminocap aminocap aminocap aminocap aminocap 2 .300 1.3 67 49 51 27 74 4 .4 t 0 .4 8. Titers are given as log mean t standard error .14 Isu$ values for : NBrPNIPNNPNPNCIPaminocapaminocapaminocapaminocapaminocap 18 8 .4 9 . however.2 f 0 .8 5 . that the scatter between individual .900 65 180 15 9 . Ig-1 .3 5 .200 760 2.8 6 .3 1 . This suggested an index where the I so value for NCIP was divided by the mean of corresponding NNP and NP values .org on October 25.7 7 .4 2 . and NCIP . NP.4 2 .7 8 . with NBrP-T4 phage .100 3.6 1 . We found.7 23 33 12 66 66 530 340 1.5 10 7 .1 5 .6 5 . specificity index (FSI) was calculated for each mouse.98 1 .4 1 . Ig-1" 6 5 6 a k Ig-1^' Ig-F 10 * All the animals were bled 12 days after secondary immunization and were tested with the HPII method.Published 84 4 INHERITANCE OF ANTIBODY SPECIFICITY .7 13 1 .6 5 .9 1 .0 4.100 1.000 105 13 180 Strain BALB/c C57BL/6 * Animals were bled 12 days after the second injection .9 5 . $ Values are geometric means of nanomolar hapten concentrations causing 50% reduction of the phage inactivation .400 ND ND ND b d d b Ig-1 ° s k d k Ig-1 .5 5 .9 2 .9 3 .8 46 61 19 50 27 10.2 0 .5 13 140 200 110 120 340 14 8 . II TABLE III Amount and Affinity of Anti-NBrPAntibodies in the Secondary Response to NBrP-BSA No . High affinity for NCIP had a negative correlation to high affinity for NNP and for NP.1 6 . We found a high positive correlation between the affinities for NP and for NNP.91 0 . All the tests were performed using 2-ME to inactivate IgM antibodies .9 4. Downloaded from jem.6 2 .7 7 .600 2.7 2 .8 1 . For the index we selected the most informative haptens NNP.700 1.5 160 12 5.400 1.09 5 .0 2 . NBrP-T4 phage was used for HPI and HPII tests .rupress.0 4.4 2 .9 4 .8 8 .8 2.

C x BI. genes in the fine specificity of anti-NBrP antibodies. C x BH. and adopted this index: FSI = ISO (NBrP) x ISU (N CIP) ISa (NNP) x ISO (NP) . 2) . we tested CB20 mice that have the Ig heavy chain constant region (IgC. Our data do not suggest that H-2-linked genes have a role in determining the specificity of the anti-NBrP antibodies .) genes of C57BL/Ka in a BALB/c background genome (7). MAKELA 845 mice was reduced by using the mean of NCIP and NBrP values as the denominator. Since there was a correlation between the allotype and the specificity index. 4) .. 4) .org on October 25. k. The mean of these F. In an attempt to further study the roles of allotype-linked and other Downloaded from jem.rupress. BALB/c mice had higher anti-NBrP titers than Fine Specificity Characteristics in Congenic and Recombinant Inbred Strains of Mice .4. Finally we studied three congenic strains carrying different H-2 alleles in a C57BL/10Sn background . The correlation turned out to be absolute. or d (Fig. or NCIP could have been used alone for the demonstration of the anti-NBrP genetic marker . CB20 antibody was indistinguishable from the anti-NBrP of the I9C H donor and very different from the BALB/c antibody . All Ig-111b mice had an index of < 0. When the indices of individual animals were plotted (Fig . 2) the three groups of strains could again be distinguished. Mice with C57BL/10Sn background were indistinguishable regardless of whether their H-2 allele was a. 3) . The FSIs of the anti-NBrP antibodies of 21 BALB/c and 18 C57BL/6 mice were distributed as two nonoverlapping populations (Fig.95 in C57BL/6. 2009 . 3.4 while all Ig-lbb homozygotes had an index of >0. The gap between these two distributions made a satisfactory basis for testing crosses between these two strains. These results indicate that the fine specificity of anti-NBrP antibodies is controlled by one (or more) allotype-linked gene(s) . There was no overlap between mice of categories 1 and 2 while mice of category 3 were located between them. It shows that any one of the haptens NNP.026 in the BALB/c and 0. C x BD. The Bailey recombinant strains. The specificity of the secondary response anti-NBrP antibodies in these strains was exclusively determined by a gene(s) linked to the Ig-1 locus (Fig . and C x BK (Ig-l') produced anti-NBrP that closely resembled C57BL/6 anti-NBrP while only C x BJ and C x BG strains (Ig-la) had low specificity indices characteristic of BALB/c . and the Bailey recombinant inbred strains that have different combinations of C57BL/6 and BALB/c genes (13) . Backcrossing into the recessive C57BL/6 strain produced 52 offspring. Antibody Concentrations . Table V presents the geometric mean of ISO values for each hapten in mice of the tested genotypes. C x BE. Ig-lab heterozygotes and Ig-bb homozygotes were separated in Fig. hybrid mice tested suggested complete dominance of the BALB/c trait (Fig.033 compared to the mean of 0 . mice was 0 . The frequency distribution of 24 F. NP.Published THEREZA IMANISHI AND O. The distribution of the specificity indices was bimodal with distinct peaks coinciding either with BALB/c or C57BL/6 peaks..

C57BL/6 mice at the different stages of immunization (Table VI) .Published 84 6 I INHERITANCE OF ANTIBODY SPECIFICITY.01 0.1 1 fine specificity 10 index ~. It was dominant over the C57BL/6 allele and segregated in the backcross generation in linkage with the allotype (Fig .rupress. Frequency distribution of FSIs (see text) in anti-NBrP of different mouse strains.N pl _ on October 25. 5 and Table VI) . II CBA C3H BALM MA/J C57L o 7:L ST/W IAH i a k a k a d a k a b a k a Downloaded from jem. 2009 U E 0 XiC7 C57BL/6 C57BL/Ka C578L/Ks LP/J SJL/J N b b b d b d b b b s c k~ or RF/J DBA/2 WMM OMEN I cl dI AKR AM CE/J e a f k "m 0. 2. The high secondary response of the BALB/c was associated with fine specificity in all but one type of mice tested . Antibodies were obtained 12 days after the second injection of NBrP-CG. In the congenic and recombinant strains high response was associated with Ig-1a allotype and low response with Ig-l b allotype in 10 cases. but one strain behaved . Each mouse is represented by a box whose horizontal location indicates the FSI.

3 . and CE/J had a high response and a low FSI.8 for BALB/c) . NCIP. Inheritance of the fine specificity . CBA and C3H strains (the third category) were intermediate between the first and the second category. Associated with the fine specificity polymorphism was a polymorphism in the magnitude of the anti-NBrP response . NP. mouse strains could be divided into three categories which correlated strongly but not absolutely with the Ig heavy chain allotype . and all non-a and non-b strains tested belonged to the second category . NNP. On the basis of the fine specificity patterns of their anti-NBrP. DBA/2N. m .Published THEREZA IMANISHI AND O . 1 1 1 to fine specificity index Fic. The differences were demonstrated by determining the relative affinities of the antibodies to related haptens NIP. Apart from the data in Tables III and VI. All allotype a strains. The AKR strain could not be reliably classified. For explanations see Fig.~ m as C57BL/6 ca E 847 bb Ff (BALB/cxC57BL/5) 0 mom ab Ft XC57BL/5 mmm s . except CBA and C3H. All five strains of allotype b belonged to one category. Discussion We found that mice of different strains produced qualitatively different antibodies to hapten NBrP when they were immunized by NBrP conjugates of BSA or CG. and all allotype Ig-l' strains except B10-Br a low response . RF/J . XC57BL/5 bb Downloaded from jem. An exception was AKR but here the number of mice was low (Table VII) .9 for C57BL/6 and 3. 2 . and DIP.iol a. 2.rupress. The difference in anti-NBrP titers between C57BL/6 and BALB/c mice persisted throughout the primary and the secondary responses. Mice of the non-a and non-b allotype. All allotype Ig-la strains had a high response as had the BALB/c. 2009 o. MOM ab F. A/J. we found a similar difference at day 7 of the primary response (log means. only five mice were tested .org on October 25. Mouse strains of the first fine specificity category produced lower antibody titers than mice of the second and the third category. The B10-Br mouse strain that carries the Ig-lb allotype and had the corresponding high FSI was a high responder . differently (Table VII) . This was interesting since at that time all antibody was 2-mercaptoethanol (2-ME) sensitive and poorly . The backcross generation was divided into those heterozygous or homozygous for the heavy chain allotype . 01. MAKELA BALB/o .

and the recessive C57BL/6 could be divided into bb allotype homozygotes and ab heterozygotes. hybrid animals between a category 1 strain (C57BL/6) and a category 2 strain (BALB/c) were indistinguishable from BALB/c mice . II Downloaded from jem. 2009 on October 25. Homozygotes were indistinguishable from C57BL/6 parents .rupress. 4. inhibitable by free hapten .Published 848 INHERITANCE OF ANTIBODY SPECIFICITY. This is clearly not the case with the H-2-linked Ir-genes (14). Frequency distribution of FSIs among mice of different genotypes . thus it probably was IgM. From this we conclude that allotype-linked immune responsiveness genes control the IgM response too. Hybrids between the F. For explanations see Fig . 2 . F.

and the fine specificity resembled BALB/c .org on October 25.8 2.5 3.7 2 .* values for: TABLE V 84 9 NBrPNIPNNPNPNC1Paminocap aminocap aminocap aminocap aminocap 5 .7 1 .2 9.7 0. 2 suggest that CBA and C3H mice may . progeny BALB/c x C57BL/6 Backcross progeny F.6 1.5 4.9 2.6 1.2 1.6 1. 11 such strains were immunized.5 16 2.400 84 120 1. Data of Table 11 and Fig.7 6.8 7 .1 5.9 3.6 49 51 3.5 2. x C57BL/6 Congenic strain (Ig) CB20 Bailey Recombinant inbred strains C x BG C x BJ C x BD C x BE C x BH C x BI C x BK Congenic strains (H-2) B10-D2 H-2d B10-A H-2a B10-Br H-2k IgC H allotype (Ig-1) as bb bb ab ab bb bb as as bb bb bb bb bb bb bb bb No .2 2.3 32 69 10 4 . MAKELX An Allotype-Linked Gene Controls the Fine Specificity of Anti-NBrP Antibodies Strain Parental strains BALB/c C57BL/6 C57BL/Ka F.3 6. The data indicate that the amount and fine specificity of the anti-NBrP antibodies of the BALB/c mice mark a V H gene .1 9.6 2.rupress.4 2. 2009 21 18 14 23 23 29 12 9 14 8 15 15 12 11 8 8 8 * Animals were bled 12 days after the second injection .3 1 . x C57BL/6 F. These data suggested that both observed polymorphisms were controlled by an allotype-linked gene(s).2 0 . Whenever these mice carried the Ig-1a allotype of the BALB/c they were high responders to NBrP.9 2. All strains carrying the C57BL allotype produced anti-NBrP with C57BL type fine specificity regardless of the other genes .4 1. This V gene is dominant over the C57BL/6 allele .8 1.2 5.4 0.600 60 68 910 1. and heterozygotes indistinguishable from the BALB/c mice with regard to the fine specificity and the magnitude of the anti-NBrP response .4 4.2 2. All these strains but one (B10-Br) were low responders .7 67 4. Eight of them contained C57BL and BALB/c genes in various combinations.9 9.000 170 69 96 120 82 47 110 75 15 140 200 10 18 180 107 8 10 350 180 230 310 107 100 315 140 Downloaded from jem.8 13 4. . We cannot explain this exception . NBrP-T4 phage was used for HPII .1 1.Published THEREZA IMANISHI AND 0 .2 5. Values are geometric means of nanomolar hapten concentrations causing 50% reduction of the phage inactivation .900 2. This was confirmed by immunization experiments of congenic and recombinant inbred strains of mice .9 1.2 2.3 1.5 5. of mice 1.

. its product should have had a higher affinity for the immunogen than the product of the recessive allele . Sera were obtained 12 days after the second injection of NBrP-CG. As the BALB/c allele was dominant in both fine specificity and the quantity of the response. Mean anti-NBrP titers of mice with different genotypes. It could be demonstrated in anti-NBrP antibodies induced by NBrP-BSA (Table 111) . This "low" affinity was dominant over the "high" affinity of the C57BL/6 antibody in FI. This allele would be recessive to the BALB/c allele since the F I mice were like BALB/c (unpublished experiments) but a proper study was not conducted since both strains belong to allotype a and a marker for the Ig-1 locus was not available . A corresponding affinity difference between BALB/c and C57BL/6 could be demonstrated by using radioactive antigen-binding test (unpublished data) and in the primary response to 1`?BrP-CG. II Downloaded from jem. Therefore the low affinity of the BALB/c antiNBrP antibodies seems to be exclusively controlled by the V H gene that causes a high response to NBrP and a low FSI . have a third allele of the same locus . Anti-NBrP antibodies of the BALB/c mice had a lower affinity for NBrP than those of the C57BL/6 mice. 2009 FIG. The only possible explanation we can offer at this stage is that whenever the BALB/c allele was present in a hybrid mouse it was expressed in much more numerous B lymphocytes than the C57BL/6 allele . 5 .rupress. and it was linked to the allotype in the backcross mice (Table V) . The dominance of the low affinity is difficult to explain as is its concordance with high total response .org on October 25.Published 850 INHERITANCE OF ANTIBODY SPECIFICITY .

The NBrP gene is one of the several known V genes (4-9) linked to the heavy chain allotypes . CBA and C3H were clearly different from the other five allotype a strains studied . MAKELA TABLE VI 85 1 Control by an Allotype-Linked Gene of the Concentration of Anti-NBrP Antibodies No.27 t 0 . All five allotype b strains had a high FSI while five out of seven studied allotype a strains had a low FSI .24 L 4 . gene is more important for the antibody than the V.13 d d d as as 17 29 12 sec§ 17 29 12 sec 12 sec 12 sec 12 sec b b b bb bb bb ab ab bb L 3 . 16) .. or allotype Ig-1° and low FSI the frequency of crossing-overs between the Ig-1 and the NBrP loci is likely to be less than 5% .09 H 6.20 t 0 . one of them allotype linked and the other perhaps H-2 linked) the data begin to suggest that the V. This was a priori unexpected since the variability of the light chain V regions is great and it correlates with antibody specificity as does the V  polypeptide sequence (15. x C57BL/6 8 8 21 7 7 18 23 23 29 H-2 Igc  Days allotype after (Ig-1) immunization as HPI titer* H H H 4 . (7) and of Blomberg et al (6) .org on October 25. $ Log means t standard error. One crossing-over event between the two loci seems to be on the record however : the BAB/14 strain that is a homozygotized hybrid between the C57BL/Ka and BALB/c strains seems to combine the allotype from C57BL/Ka and the NBrP gene from the BALB/c (Makela et al . Since NBrP and NP are related haptens the possibility must be considered that the BALB/c gene described in this paper and the C57BL/6 gene of a pre- .rupress. marked with the letter L. unpublished observation) .Published THEREZA IMANISHI AND O . complex gene (Sher and Cohn [9) reported that expression of S107 idiotype was regulated by two genes. 2009 db H 6..09 6 .1-3-dextran marker from the BALB/c (6.. As there are no published reports on inherited idiotypes or fine specificity characteristics unlinked to the IgC E. of mice Parental strains BALB/c C57BL/6 F. progeny BALB/c x C57BL/6 Backcross progeny F.09 L 5. This suggests that the IgC  chromosomal region of a common ancestor of CBA and C3H may have received a V-gene portion from a non-a mouse through recombination . .09$ 5 .20 t 0 .10 * Titers marked with the letter H are high and differ significantly (P < 0. This is also suggested by a linkage diseuuilibrium between the two loci.55 t 0 . As none of the 53 backcross mice tested recombined allotype Ig-la and high FSI. § sec.10 Downloaded from jem.14 t 0 . Both these closely related strains were likewise different from other allotype a strains with regard to the unrelated V-gene marker of Lieberman et al. 17) . It also has inherited the alpha.00 t 0 .29 L 5 .53 t 0 .005) from the titers of the corresponding group.18 f 0.32 t 0 . x C57BL/6 F. secondary immunization .

19 6.26 6.10 5 .40=0. This weakly suggests that the NP and the NBrP markers are not alleles of one cistron since both strains are homozygous .org on October 25.74=0.23 = 0.96 t 0.06 6.12 5.54 t 0.12 6.13 5.35 t 0.14 = 0. only a meiotic recombination between the two markers could demonstrate that they belong to different cistrons.12 5 . vious paper (8) are alleles of the same cistron .38 = 0 .09 6.13 5.05+0 . Ig-ld ll 6 5 6 a Ig-le k Ig-1 7 10 All mice were bled 12 days after the second injection of NBrP-CG.7410. however. If this were true.20 t 0.17 5 . then the BALB/c allele is dominant in NBrP immunization but the C57BL/6 allele is dominant in NP immunization (unpublished experiments) .10 6.16 5.53+0 . II Anti-NBrP Antibody Concentration in the Sera of Different Strains of Mice Strain CBA C311 BALB/c MA/J C57L ST/bJ IAH CxBG CxBJ C57BL/6 C57BL/Ka C57BL/Ks LP/J SJL/J CB20 CxBD CxBE CxBH CxBI CxBK B10-D2 B10-A B10-Br H-2 Igc  allotype Ig-11 No .55 = 0.43 t 0. C57BL/6 and SJL/J strains produce anti-NP antibodies of different but anti-NBrP antibodies of indistinguishable fine specificity types . of mice 10 12 21 4 6 HPI titer* 6.12 5.17 5 .48 = 0.21 5. .94=0.rupress.70 t 0.17 t 0.23 t 0 .22 5 .45 f 0.12 RF/J DBA/2N AKR A/J CE/J k d Ig-1° Ig-1 .Published 852 INHERITANCE OF ANTIDOBY SPECIFICITY .19 5.49 f 0.33 5 .13 5. 2009 d d b s d d b b b a k d d 18 14 5 14 10 12 8 15 15 12 11 8 8 8 5.03 = 0.20 0.08 6.11 TABLE VII k k d k k b b b b Ig-11 Ig-la 6 14 Ig-1 ° 8 9 Downloaded from jem.21=0. * Log means t standard error.11 5.74=0.94 t 0.10 5.26 6 . while true allelism is impossible to prove .40 = 0.17 6. The evidence is inconclusive.16 5.80 = 0.

recombinant inbred. Nisonoff. Exp . J. MAKELA 853 There are at least four types of Mendelian genes that control the magnitude of specific immune responses . editor . 1973 . 6 . W . Received for publication 19 December 1974 . This gene was dominant over the C57BL/6 allele . B . B . Exp . Humphrey.. Mage. It is genetically controlled but several genes are involved .) . T . and W . This category included most of the tested strains . New Biol . Sela. . Pawlak. New York. E. J. Potter. 230 :63 . 3 . 1972 . Kindt. J. In The Antigens .. (T15 idiotype) marker in the mouse regulating natural antibody to phosphorylcholine . Young-Cooper. 139:983 . Med . 23) . Idiotype expression and the inheritance of mouse antibody clones . Eichmann. One type includes the H-2-linked Ir genes controlling antigen recognition by thymus-derived lymphocytes (18. Mushinski. B. R.. Downloaded from jem. 4 . Mage. Academic Press. Genetics of the antibody response to dextran in mice Science (Wash . The fourth type is manifested by a simultaneous low or high response to several antigens (22. R. 1973 . K . 0. and M . D. J .5-din itrophenyl) acetyl (NNP) but low for (4-hydroxy-5-cloro-3nitrophenyl)acetyl (NCIP) . Blomberg. and backcross mice showed that both the fine specificity and the magnitude of the anti-NBrP response of BALB/c mice were controlled by an allotype-linked gene . When Ir genes of this type are being studied without influence of other genes the magnitude and the fine specificity (affinity) are intimately connected. Med . Lieberman. J. Exp . D . Anti-NBrP antibodies of all allotype b mice (five strains tested) had a high relative affinity for (4hydroxy-3. 2009 References 1 . 19) . mice of allotype b had lower titers than the other mice .. Immunoglobuli n allotypes . Genetic control of variable and constant regions of heavy chains . Terry . M . and S. The third type was demonstrated by antigen-specific responsiveness or unresponsiveness that was controlled by one Mendelian gene unlinked to the H-2 and to allotype (20. L. W . F. 137:603 . 177 :178. Jr . R. (6) and by the data presented above.. 137 :22 . 1972 . 7 . M . 108 :1110 . and W . Evidence for the linkage of the IgC  locus to a gene controlling the idiotypic specificity of anti-pazophenyl-arsonate antibodies in strain A mice. A. 1974 . Associated with fine specificity characteristics were anti-NBrP titers.. Med . The third category (CBA and C3H strains) had an intermediate fine specificity . L. Recombinatio n of genes coding for constant and variable regions of immunoglobulin heavy chains . B. Another category was characterized by high relative affinity for NCIP but low for NNP . The second type is exemplified by the data of Blomberg et al. Lack of recombinant mice in the backcross generation on one hand and a linkage disequilibrium between allotypes and fine specificity patterns on the other suggest close linkage between the two genes . 5 . Rudikoff. G. 21) . 1971 . E. R. Summary Mice of 17 inbred strains produced anti-(4-hydroxy-5-bromo-3-nitrophenyl)acetyl (NBrP) of three different fine specificity types. 1 :299 . and M . Weigert . 1973 . R. Studies of congenic..rupress. Alexander . Nat . Lieberman. 2 .. C . Immunol . Genetics of a new IgV. Mandy .org on October 25. Potter. M . Potter. J . Inc .Published THEREZA IMANISHI AND 0 .. Geckeler. Mushinski. and C.

V . Asof'sky. B . C . 16 . D . New York . Downloaded from jem. Riblet. Weir. 1969 . Benacerraf. and H . and M .. and B . Makela . Med . Immunological cross-reactions within a family of related haptens . Benacerraf.. 1966 . 140 :1498 . M . and M . Sela . Sher. 1972 . O .. 1974 . 1974 . Joslin. Cohn and M . Cross-idiotypi c specificity among monoclonal IgM proteins with anti --y -globulin activity . A . F . Jaton. Immunogenetics . F . R. 1967 . and H . 22 . J. Immunol . J. A . Capra . J . 1969 . R . 1971 ..) 18 . 135 :110 . K . 1054 . In press . 23 . M . 1972 . 11 :325 . McDevitt. 1974 . Segregation data and localization to the fifth linkage group of a gene affecting antibody production . R . 17 . Hatcher. Dunham. Benacerraf . Eur. and J . G . G . D . 1 . Landy. J . Transplantation (Baltimore) . W . 9 . 15 . Academic Press. Genetic control of the immune response : the effect of non-H-2 linked genes on antibody production .) . L . Imanishi. D . England . I . 1972 . R . II 8 . 10 :465 . D . 172 :273 . Biozzi. Cohn . 137 :331 . Genetic factors controlling anti-sheep erythrocyte antibody response and immunoglobulin synthesis in backcross and F Z progeny of mice genetically selected for "high" or "low" antibody synthesis . editor . 1974 . J . 103 :66 . J . Genetic control of the immune response . C .. Weigert . D . J . G . Mouton. H . H . H . 14 . 10 . J. Kunkel. Science (Wash . Inheritance of antibody specificity . Linkage analysis of the dextran response gene . Med . and O . Exp . 11 . G . In Handbook of Experimental Immunology . McDevitt. Similaritie s in the light chains of anti -y-globulins showing cross-idiotypic specificities . 136 :790 . Med . P . 139 :128 . 1972 . Chemical and serological studies with an iodine-containing synthetic immunological determinant 4-hydroxy-3iodo-5-nitrophenyl-acetic acid (NIP) and related compounds .. 2009 . Blackwell Scientific Publications Ltd . Localize d hemolysis in gel . H .. 9 :1139 . Anti-(4hydroxy-3-nitrophenyl)acetyl of the mouse primary response . A selective defect in immunologic (IgG) memory in nonresponder mice . Bailey. Inc . Grumet. J. The nature of the antigenic determinant in a genetic control of the antibody response . Lieberman. Inheritance of an idiotype associated with the immune response of inbred mice to phosphorylcholine . Winchester. on October 25. Wortis . A . Immunol .Published 854 INHERITANCE OF ANTIBODY SPECIFICITY .. O . C . 19 . Med . 2 :319 . 20 . Mitchison. Gasser.. Stiffel. Johnson. Capra . V . J. Dresser. D . Makela . Kunkel. Wincester. Exp .. and M . Recombinant-inbred strains . J. C . Exp . J. T . Pitt-Rivers . Immunochemistry . M . D . J. Joslin. E . 1972 . D .. Dorf. Mozes. Exp . Med . 21 . 112 :1329 .rupress.. Brownstone. 130 :493 . (Abstr . N . 1973 . Exp . Exp . R. Immunology . Genetic control of the immune response in mice . W . and O . H . 12 . In Genetic Control of Immune Responsiveness . O . and R . and B . Oxford. 13 ... E . Histocompatibility-linked immune response genes . editors . Med . G . Agnello. F . Immunol . E . and J .. 1972 .