Biomedical subjects

B Parhami-Seren

Publications and source records attributed to B Parhami-Seren.

At least 19 recordsLinked to original sources

PubMed2006

Immunologic quantitation of tissue factors.

The large number of conflicting reports on the presence and concentration of circulating tissue factor (TF) in blood generates uncertainties regarding its relevance to hemostasis and association with specific diseases. We believe that the source of these controversies lies in part in the assays used for TF quantitation. We have developed a highly sensitive and specific double monoclonal antibody fluorescence-based immunoassay and integrated it into the Luminex Multi-Analyte Platform. This assay, which uses physiologically relevant standard and appropriate specificity controls, measures TF antigen in recombinant products and natural sources including placenta, plasma, cell lysates and cell membranes. Comparisons of reactivity patterns of various full-length and truncated TFs on an equimolar basis revealed quantitative differences in the immune recognition of TFs by our antibodies in the order of TF 1-263 > 1-242 > 1-218 > placental TF. Despite this differential recognition, all TF species are quantifiable at concentrations < or = 2 pM. Using a calibration curve constructed with recombinant TF 1-263 and plasma from healthy individuals (n = 91), we observed the concentration of TF antigen in plasma to be substantially lower than that generally reported in the literature: TF antigen in plasma of 72 individuals (79%) was below 2 pM (quantitative limit of our assay); TF antigen levels between 2.0 and 5.0 pM could be detected in six individuals (7%); and in 14% (13 plasmas), the non-specific signal was higher than the specific signal, and thus TF levels could not be determined. These differential recognition patterns affect TF quantitation in plasma and should be considered when evaluating plasma TF-like antigen concentrations.

Antibodies, Monoclonal↗

PubMed2004

Factor VIIa replacement therapy in factor VII deficiency.

Factor (F)VII deficiency is an autosomal recessive disorder for which a replacement therapy is not universally available; recombinant FVIIa has been utilized as a therapeutic substitute. As FVII competes with FVIIa for binding to tissue factor in initiating the extrinsic pathway of blood coagulation, a lower dose of FVIIa replacement in cross-reacting material-negative (CRM-) individuals can achieve hemostasis. Three coagulation models (computational, synthetic and in vitro whole blood) were used to predict the FVIIa levels needed to provide apparent hemostasis in a non-bleeding state. Our whole blood results show that a 'normalized' coagulation profile for FVII-deficient individuals has an initiation phase that ends at 5.8 +/- 0.5 min (clot time) and the propagation phase of thrombin generation (thrombin-antithrombin III) yields a maximum concentration of 380 +/- 29 nmol L(-1). When CRM- FVII-deficient subjects were infused with a prophylactic dose of 23 micro g kg(-1) of recombinant FVIIa, 6-8 h postinfusion resulted in a comparable normalized whole blood profile. This FVIIa concentration (0.3-0.7 nmol L(-1)/equivalent dose: 0.8-1.8 micro g kg(-1)) is approximately 1/10 that currently used in treating FVII-deficient individuals and suggests that therapies should be altered relative to the concentration of the FVII zymogen.

Adolescent↗

PubMed2001

Structural analysis of mutants of high-affinity and low-affinity p-azophenylarsonate-specific antibodies generated by alanine scanning of heavy chain complementarity-determining region 2.

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain. However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.

Alanine↗

PubMed1999

Monoclonal antibodies that distinguish between two related digitalis glycosides, ouabain and digoxin.

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.

Animals↗

PubMed1999

A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator.

Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH(2)-terminal "catalytic switch" that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSKDelta59) fusion protein lacking the 59 NH(2)-terminal residues was no longer capable of fibrin-independent Pg activation (k(cat)/K(m) decreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH(2)-terminal peptide rSK1-59. Deletion of the NH(2) terminus made rSKDelta59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSKDelta59 activator complex apparently resulted from selective catalytic processing of fibrin-bound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSKDelta59) of the SK NH(2)-terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSKDelta59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH(2) terminus are responsible for SK's unique mechanism of fibrin-independent Pg activation. Because deletion of the NH(2) terminus alters SK's mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK's therapeutic efficacy.

Catalytic Domain↗

PubMed1998

Structural requirements for a specificity switch and for maintenance of affinity using mutational analysis of a phage-displayed anti-arsonate antibody of Fab heavy chain first complementarity-determining region.

We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30-36 in Ab 36-65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36-65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36-71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V(H) sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.

Animals↗

PubMed1997

Sequences of antigenic epitopes of streptokinase identified via random peptide libraries displayed on phage.

Though streptokinase (SK) is widely used to treat humans with thrombotic disease, it is antigenic and anti-SK antibody causes allergic reactions and neutralizes SK's therapeutic effects. To pinpoint the fine structure of two immunodominant, continuous epitopes in SK, we used unconstrained 15 and 6-mer random peptide libraries displayed on phage (theoretical complexity of 3.2 x 10(19) and 0.64 x 10(8) unique sequences). The first epitope, recognized by both human Ab and murine monoclonal (m)Abs, was previously localized to the amino terminus of SK. Repeated panning and selection experiments against a 15-mer peptide phage library, using a representative mAb (A2.5) to this epitope, identified a dominant structural motif (GP[R/L]WL) corresponding to amino acids 3 to 7 of native SK, which was consistent with previous epitope mapping. These findings were further confirmed by: (1) the fact that a synthetic peptide spanning the epitope of A2.5 (AGPEWLL) specifically inhibited the binding of A2.5 to SK and (2) the finding that mAb 9D10, which competes with mAb A2.5 for binding to SK, independently selected, from a different random hexamer library, an epitope sequence spanning residues 4 to 9 that overlaps the A2.5 epitope. Similar studies of the second epitope in SK, which is immunodominant for murine but not human antibodies, identified a consensus sequence KS(K/L)P(F/Y) corresponding to amino acids 59 to 63 of SK; this was confirmed by epitope peptide binding experiments. This epitope is cleaved and destroyed when SK reacts with human but not murine plasminogen. Thus, pinpointing the sequences of antigenic epitopes of SK: (1) provides a potential explanation for species differences in SK's antigenicity, (2) demonstrates the overlapping fine structure of epitopes recognized by competitive mAbs, (3) confirms previous epitope mapping studies and (4) has the potential to identify antigenic sequences that lead to allergic reactions in patients treated with SK.

Amino Acid Sequence↗

PubMed1996

Contribution of heavy chain junctional amino acid diversity to antibody affinity among p-azophenylarsonate-specific antibodies.

We showed previously that heavy chain gene junctional amino acid differences among unmutated p-azophenylarsonate (Ars) Abs that share a unique gene segment combination encoding these V regions, termed "canonical," alter affinity. To determine the contribution of junctional amino acid differences to binding, we introduced, by site-directed mutagenesis, various amino acids at position 100 and/or 107 (sequential numbering) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved or showed increased Ars binding (1-to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (< or = 0.5-fold). As much as a 150-fold difference in Ars binding was observed between 2 Abs with different sets of junctions (Asn100/Tyr107 and Val100/Lys107). Thus, amino acid replacements at D gene junctions can produce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially, can result in preferential selection of Abs containing certain junctions during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are known to be recurrent in vivo and are associated with increased Ars affinity. The mutant Abs all showed increased affinity, indicating that despite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. These additive effects reflect the "adaptability" of the canonical gene segment combination in sustaining somatic mutations leading to affinity maturation.

Amino Acid Sequence↗

PubMed1996

Structural characterization of immunodominant regions of streptokinase recognized by murine monoclonal antibodies.

In order to determine the nature of the antigenic and functional determinants of streptokinase (SK), we produced monoclonal antibodies (MAbs) by immunizing A/J mice with native SK protein. By virtue of their differential binding to a large panel of recombinant SK truncated proteins, and their effect on the formation of functional SK-plasminogen activator complex (SKPAC), these MAbs were found to recognize 6 unique and minimally overlapping epitopes on the SK protein. The fine epitope specificity of the anti-SK MAbs derived from A/J mice was compared with that of MAbs derived from BALB/c mice. A number of MAbs from both inbred strains of mice were directed against the same sequences of SK (1-13, 1-253, 120-352) suggesting that these regions of the molecule contain peptide sequences that are immunodominant. Two of the "immunodominant" sequences of SK protein appeared to be important for SK function, since the formation of SKPAC could be inhibited by MAbs against these sequences.

Animals↗

PubMed1995

Identification of a plasminogen binding region in streptokinase that is necessary for the creation of a functional streptokinase-plasminogen activator complex.

Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.

Base Sequence↗

PubMed1995

Modulation of antibody affinity by an engineered amino acid substitution.

The crystal model of the complex of the somatically mutated anti-p-azophenylarsonate (Ars) Ab 36-71 F(ab) with phenylarsonate reveals that six residues (Asn35, Trp47, Tyr50, Ser99, and Tyr106 in the H chain and Arg96 in the L chain) contact hapten. Further study of this model suggested that H chain Phe108, which forms the base of the combining cavity, also affects Ars binding. We predicted that Trp with a bulkier aromatic side chain might be accommodated in this position and increase Ars affinity. The substitution of Phe by Trp using in vitro mutagenesis at position 108 enhanced affinity 10-fold in the germline-encoded Ab 36-65. However, the same mutation in Ab 36-71 abolished the binding. Phe108 was then mutated to different amino acids in both Abs. The results indicated that except for the Trp substitution in 36-65, all other substitutions at position 108 decrease or abolish Ars binding in both Abs. It was shown previously that the 200-fold difference in affinity between 36-65 and 36-71 could be reproduced by changing only three VH amino acids. Because the mutation of Phe108 to Trp has never been observed during in vivo affinity maturation, we constructed mutants of 36-65 in which Trp108 was combined with one or more of the "favorable" mutations of 36-71, to determine whether the mutations were additive. The results indicate that it is possible to maintain an affinity significantly higher than wild-type by such combined mutations. Thus, the failure to observe Trp108 in vivo is not due to structural idiosyncrasy, but may simply be due to codon usage at Phe108 in the germline sequence. Such limited "adaptability" of a germline sequence indicates that it is possible to achieve higher affinity Abs through protein engineering via routes that are constrained during in vivo selection.

Amino Acid Sequence↗

PubMed1995

Mapping the antigenic regions of streptokinase in humans before and after streptokinase therapy.

Streptokinase saves lives in patients suffering a myocardial infarction. However, because nearly all humans tested show antibodies against streptokinase, allergic reactions to streptokinase are common and may be severe. In this report we have analysed antibodies purified from normal blood donors and patients, before and after streptokinase therapy, to identify antigenic regions of the streptokinase molecule. Antibody to streptokinase was seen in all subjects, but there were 20-30-fold differences between individuals in the antibody titer. These individual differences in titer persisted after SK treatment, though the titer for all patients rose an average of 7-fold 1 week after streptokinase therapy. To identify the regions of streptokinase to which the antibody bound, we employed a panel of well-characterized murine monoclonal antibodies and recombinant streptokinase truncated fragments. Antibodies to three discrete regions of streptokinase could be detected in all patients. Antibodies to two other regions, at the amino terminal and carboxyl terminus of the molecule, were found in many but not in all patients. However, antibodies to a sixth region of streptokinase were uncommon and of very low titer. Interestingly, individuals receiving streptokinase tended to show the same pattern of immunoreactivity after treatment as they had prior to streptokinase. We conclude that although individual differences exist in the titers of streptokinase antibody, certain regions of streptokinase appear to be more antigenic or immunodominant.

Animals↗

PubMed1994

A single engineered amino acid substitution changes antibody fine specificity.

During the acquisition of humoral immunity, the process of somatic hypermutation introduces nucleotide substitutions into expressed antibody (Ab) V region genes. Studies employing in vitro mutagenesis have shown that recurrent mutations observed in vivo often enhance the affinity of the target Ab for Ag. Here we show that a single amino acid replacement at position 35 in the H chain of an unmutated Ab with specificity for p-azophenylarsonate (Ars) confers specificity for the structurally related hapten p-azophenylsulfonate (Sulf) while abolishing specificity for Ars. The mutant Ab binds Sulf with an affinity characteristic of Ab produced by memory B cells. The same mutation in the somatically mutated anti-Ars Ab 36-71, for which the Fab crystal structure is known, resulted in a significant shift in fine specificity from Ars to Sulf. Examination of the crystal structure suggests that the specificity change is caused by a decrease in binding site size and/or new hydrogen bond geometry. Because the mutation at position 35 had been observed in somatically mutated Ab elicited by immunization with Ars followed by Sulf, the results confirm that somatic mutation in vivo can alter Ab specificity. The results also support the potential of Ab engineering to alter antigenic specificity.

Amino Acid Sequence↗

PubMed1993

A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments.

Streptokinase (SK), a bacterial product of pathogenic Streptococcus species, is now widely used as an effective therapy for the treatment of heart attacks. Because naturally occurring antibody to SK is ubiquitous, serious allergic reactions to SK therapy are common. To begin to identify regions of the molecule that are important for the antigenicity of SK we performed studies using a panel of 51 hybridomas producing anti-SK antibodies, recombinant SK fragments, and assays of SK activity. Antibodies generated from mice hyperimmunized with wild-type SK were shown to fall into six distinct complementation groups by competitive binding studies. Recombinant SK fragments were used to determine the peptide regions recognized by these complementation groups. Correlation of the effects of the mAb on SK function, with knowledge of their SK fragment-binding pattern, suggested regions of the SK molecule that are important for the construction and the catalytic function of the SK-plasminogen activator complex.

Antibodies, Monoclonal↗

PubMed1993

Conservation of binding site geometry among p-azophenylarsonate-specific antibodies.

Murine A/J anti-p-azophenylarsonate mAb that express a dominant cross-reactive Id are encoded by a single set of germ-line VH and VL region genes. The crystal structure of the Fab of antibody 36-71, which uses this canonical set of genes but is somatically mutated, was previously determined. An Fab 36-71:phenylarsonate complex was modeled, identifying amino acid side chains that were proposed as contact residues to hapten. The remarkable conservation of these residues among canonical anti-p-azophenylarsonate antibodies suggested that the overall binding site geometry was maintained among somatically mutated antiarsonate monoclonal antibodies. To test this hypothesis, we used the germ-line-encoded antibody 36-65 to construct mutant antibodies, using oligonucleotide-directed mutagenesis, which differed only at the putative H chain hapten-contacting residues, and measured their hapten binding. A framework residue at H chain position 47 involved in a hydrogen bond network with CDR residues was also mutated. Substitution of several amino acids at each position permitted evaluation of the stereochemical requirements for binding. The results indicate the importance of aromatic stacking of two H chain tyrosine residues against the phenyl ring of the hapten in maintaining affinity, as well as strict complementarity at H chain position 35. The results are consistent with the crystal model of the combining site, and provide further evidence for conservation of the three-dimensional binding site motif among antiarsonate antibodies that bear a dominant heritable ld.

Amino Acid Sequence↗

PubMed1993

Defining the structural correlates responsible for loss of arsonate affinity in an IDCR antibody isolated from an autoimmune mouse.

Immunization of the autoimmune mouse strain (M x A) Id/lpr with Ars-KLH, has been shown to elicit a prolonged anti-Ars IdCR response similar to that found in A/J mice. Cell fusion of splenocytes from a diseased mouse previously immunized with Ars-KLH resulted in a monoclonal antibody, 1-52.30, that was found to express the strain A major cross-reactive idiotype, but failed to bind Ars. Nucleotide sequence analysis demonstrated that 1-52.30: (a) used the "canonical" combination of gene segments associated with this idiotype, and (b) exhibited a pattern of somatic mutation consistent with selection for high affinity Ars binding. Two amino acids, VL 91 and 93, were mutated in 36-65, the germline equivalent of the IdCR antibodies, to 1-52.30-like residues (91G-->D, 93T-->M). The results of the mutagenesis showed that changing a single light chain residue, VL 91, from glycine to aspartic acid, resulted in a dramatic loss of Ars binding activity.

Amino Acid Sequence↗

PubMed1990

Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype.

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.

Amino Acid Sequence↗

PubMed1990

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies.

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.

Amino Acid Sequence↗