Ba Fragment Research Articles

Africanized honeybee venom (Apis mellifera) promotes human complement activation split products storm.

Complement activation split products are signatures of many immunopathological disorders. Among the laboratory findings observed in these diseases, a reduction in the level of circulating intact complement components can be mentioned, and this change has also been detected in envenomation by multiple Africanized honeybee (Apis mellifera) stings. Although envenomation by these animals elicits diverse life-threatening reactions, the capacity of bee venom (AmV) to activate the human complement system remains elusive. By coupling immunochemical and functional approaches, it was observed that AmV strongly consumes components of the alternative pathway (AP) of the complement system in normal human serum (NHS). Additionally, AmV interfered with classical (CP) and lectin pathways (LP) activities. In parallel, a high increase in Ba fragment levels was detected, suggesting that the changes in AP activity were due to its activation. Furthermore, an increase in the level of the C1s-C1INH complex and a decrease in the physiological level of MASP1-C1INH suggested that CP and LP were also activated in the presence of AmV. Strikingly, NHS exposed to increasing AmV concentrations varying from 5 to 1000 µg/mL presented a high generation of C3a, C4a and C5a anaphylatoxins, and sC5b-9 complexes assembly, thus reinforcing that AmV triggers complement activation. These results show that AmV is a strong complement activator. This activation presents a mixed profile, with a predominance of AP activation. This suggests that complement split products can play important roles in the envenomation by Africanized honeybee, as they could induce diverse immunopathological events observed in patients and may also dictate patient clinical prognosis.

Major histocompatibility complex complement (MHC) Bf alleles show trans species evolution between man and chimpanzee

HLA and disease studies by using single allele statistics have been fruitless during the last 40 years for explaining association pathogenesis of the associated diseases.Other approaches are necessary to untangle this puzzle. We aim to revisit complement alleleism in humans and primates for both studying MHC and disease association to complotypes and extended MHC haplotypes in order to also explain the positive directional selection of maintaining immune response genes (complement, MHC adaptive and MHC non-specific genes) that keeps these three type of genes together in a short chromosome stretch (MHC) for million years. These genes may be linked to conjointly avoid microbes attack and autoimmunity. In the present paper, it is obtained a new Bf chimpanzee allele, provisionaly named Patr-Bf*A:01,that differs from other Bf alleles by having CTG at eleventh codon of exon 2 in order to start the newly suggested methodology and explain functional and evolutionary MHC obscure aspects. Exons 1 to 6 of Ba fragment of Bf gene were obtained from chimpanzee. This new chimpanzee Factor B allele (Patr-Bf*A:01) is to be identical to a infrequent human Bf allele (SNP rs641153); it stresses the strong evolutive pressure upon certain alleles that are trans specific. It also may apply to MHC extended haplotipes which may conjointly act to start an adequate immune response. It is the first time that a complement MHC class III allele is described to undergo trans species evolution,in contrast to class I and class II alleles which had already been reported . Allelism of complement factors are again proposed for studying MHC complement genes, complotypes, and extended MHC haplotypes which may be more informative that single MHC marker studies.

Antimicrobial activity of the complement system

The complement system plays a key role in homeostasis and defense against pathogens. The antimicrobial activity of serum against Gram-negative bacteria is usually attributed to the action of the membrane attack complex. However, there is increasing evidence that some other components of the complement system and the products of its activation are also capable of direct killing of both Gram-negative and Gram-positive bacteria. In the course of complement activation, anaphylatoxins C3a, C4a, C5a are produced, which, in addition to their main function, can exhibit a bactericidal effect and disrupt the bacterial membrane. Recent studies have shown that in fish, complement factors D, I, as well as a Ba fragment of factor B, are able to neutralize pathogens. The triggering and amplification of complement usually occurs on the surface of pathogen cells, so the local production of antimicrobial components can potentially make a significant contribution to their elimination. The aim of this review is to outline the role of individual complement members in the elimination of pathogens through direct antibiotic action. The problem of antimicrobial protection in the context of therapeutic complement inhibition is considered.

A teleost complement factor Ba possesses antimicrobial activity and inhibits bacterial infection in fish

Complement factor B (Bf) is a component of the complement system. Following activation of the alternative pathway of the complement system, factor B is cleaved into Ba and Bb fragments. In fish, the Bf of rainbow trout is known to act as a C3 convertase, but the function of the Ba fragment is essentially unknown. In this study, we examined the expression patterns of tongue sole Cynoglossus semilaevis Bf (named CsBf) and the biological activity of the Ba fragment of CsBf (named CsBa). CsBf possesses the conserved domains of Bf and shares 39.9%–56.4% sequence identities with other fish Bf. CsBf expression was high in liver, muscle, and heart, and low in intestine, blood, and kidney. Bacterial infection significantly induced CsBf expression in kidney, spleen, and liver in a time-dependent manner. Recombinant CsBa (rCsBa) exhibited apparent binding capacities to bacteria and tongue sole peripheral blood leukocytes, and binding of rCsBa to bacteria inhibited bacterial growth. When overexpressed in tongue sole, CsBa significantly reduced bacterial dissemination in fish tissues. Together these results indicate for the first time that a fish Ba possesses antibacterial effect as well as immune cell-binding capacity, and thus probably plays a role in host immune defense against bacterial infection.

A novel antibody against human factor B that blocks formation of the C3bB proconvertase and inhibits complement activation in disease models.

The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at approximately 27A resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the alpha'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.

Conformational changes during the assembly of factor B from its domains by 1H NMR spectroscopy and molecular modelling: their relevance to the regulation of factor B activity

Factor B is a key component of the alternative pathway of complement and is cleaved by factor D into the Ba and Bb fragments in the presence of activated C3 (C3b or C3(H 2O)). The Ba fragment contains three short consensus/complement repeat domains, while the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain, all three of which are implicated in multisite contacts with C3. The upfield-shifted signals in the 1H NMR spectra of factor B, the Ba and Bb fragments, and the vWF-A and SP domains were used as sensitive conformational probes of their structures. Temperature studies and pH titrations showed that the Ba fragment and the vWF-A and SP domains had conformationally mobile structures. The comparison of the NMR spectra of the SP domains of both factor B and factor D showed that the factor D linewidths were broader than those for factor B, which may result from a range of proteolytically inactive conformations of factor D in the absence of substrate. The NMR spectra from the separate vWF-A and SP domains in combination with that of the Ba fragment generally accounted for that of intact factor B, apart from the perturbation of an upfield-shifted signal from the Ba fragment. A new upfield-shifted signal was observed in the Bb fragment that was not detected in the spectra for the vWF-A or SP domains or intact factor B. Ring current calculations based on homology models or crystal structures predicted that buried hydrophobic methyl-aromatic interactions probably accounted for the upfield-shifted signals, with many arising from the N-terminal subdomain of the SP domain to which the C terminus of the vWF-A domain is directly linked. It was concluded that: (1) the conformation of the free SP domain is better ordered in solution than that of factor D; (2) the conformation of the Ba fragment is affected by its incorporation into factor B; and (3) the proximity of the vWF-A and SP domains within the Bb fragment leads to a conformational change in which conserved charged residues may be important. Allosteric structural rearrangements in the SP domain as the result of its interactions with the vWF-A domain or the Ba fragment provide an explanation of the regulation of the catalytic activity of factor B.

Identification of the c3b binding site in a recombinant vWF-A domain of complement factor B by surface-enhanced laser desorption-ionisation affinity mass spectrometry and homology modelling: implications for the activity of factor B

Factor B is a key component of the alternative pathway of the complement system. During complement activation, factor B complexed with activated C3 is cleaved into the Ba and Bb fragments by the protease factor D to form the C3 convertase from the complex between C3b and Bb. The Ba fragment contains three short consensus/complement repeat (SCR) domains, and the Bb fragment contains a von Willebrand factor type A (vWF-A) domain and a serine protease (SP) domain. Surface-enhanced laser desorption-ionization affinity mass spectrometry (SELDIAMS) was used to investigate the reaction of factor B with immobilised activated C3(NH 3) in the presence of Mg 2+. A recombinant vWF-A domain (residues G229-Q448), the native Ba and Bb fragments and native factor B all demonstrated specific interactions with C3(NH 3), while no interactions were detected using bovine serum albumin as a control. A mass analysis of the proteolysis of the vWF-A domain when this was bound to immobilised C3(NH 3) identified two peptides (residues G229-K265 and T355-R381) that were involved with vWF-A binding to C3(NH 3). A homology model for the vWF-A domain was constructed using the vWF-A crystal structure in complement receptor type 3. Comparisons with five different vWF-A crystal structures showed that large surface insertions were present close to the carboxyl and amino edges of the central β-sheet of the factor B vWF-A structure. The peptides G229-K265 and T355-R381 corresponded to the two sides of the active site cleft at the carboxyl edge of the vWF-A structure. The vWF-A connections with the SCR and SP domains were close to the amino edge of this vWF-A β-sheet, and shows that the vWF-A domain can be involved in both C3b binding and the regulation of factor B activity. These results show that (i) a major function of the vWF-A domain is to bind to activated C3 during the formation of the C3 convertase, which it does at its active site cleft; and that (ii) SELDIAMS provides an efficient means of identifying residues involved in protein-protein interactions.

Mutations of the Type A Domain of Complement Factor B That Promote High-Affinity C3b-Binding

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.

Characterization of physiologic breakdown products of the complement fragment Ba

To better characterize the activation products of factor B which are generated under physiologic conditions Ba was purified directly from human EDTA-plasma by immunoaffinity chromatography using anti-Ba Sepharose. SDS-PAGE analysis revealed the existence of degradation products of the Ba fragment which were truncated at the carboxyterminus. A monoclonal antibody (mAb D22/3) was produced by immunizing mice with a synthetic peptide which corresponds to the Ba carboxyterminus (Glu 215-Arg 234). This mAb was found to react with an epitope (Ba neo-epitope), which is newly formed after the generation of Ba from its precursor protein factor B. This neoantigenic determinant is absent both in factor B and the desArg/Lys Ba derivatives. The conversion of Ba by carboxypeptidases in human serum was monitored using an assay which is based on mAb D22/3, revealing a half-life of Ba in serum of 150 min. Furthermore, this assay allowed to quantitate plasma levels of intact and degraded Ba in healthy probands and in patients with chronic renal failure. The processing of the Ba carboxyterminus may be of functional relevance as the biological activity of the Ba fragment which had been shown to suppress human B lymphocyte functions in vitro resides in its carboxyterminal amino acid sequence.

Localization of a pernicious anaemia autoantibody epitope on the alpha-subunit of human H,K-adenosine triphosphatase.

Four cDNA fragments encoding different portions of the alpha-subunit of human H,K-adenosine triphosphatase (ATPase) were amplified by means of the polymerase chain reaction technique, ligated into the plasmid pGEX-2T, and expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fragments A (residues 163-313), Ba (residues 360-797), Bb (residues 526-797), and C (residues 822-1031) together encompass 77% of the alpha-subunit and cover most of its cytosolic part. The reactivities of autoantibodies in the sera from patients with pernicious anaemia with the recombinant fusion proteins were analysed by immunoblotting. One autoantigenic epitope was found in the NH2-terminal part of the Ba fragment--that is, between residues 360 and 525. No epitope was detected in the other fragments. The Ba fragment was cleaved off from the glutathione S-transferase fusion protein by the action of thrombin and was then further purified. By means of enzyme-linked immunosorbent assay, 28 of 42 sera (67%) from patients with pernicious anaemia were positive against the purified Ba fragment. The present results provide a final proof that the human H,K-ATPase alpha-subunit is a major autoantigen in the parietal cell and that the major epitope is located between residues 360 to 525 on the cytosolic side of the secretory membrane.

Structural analysis of chicken factor B-like protease and comparison with mammalian complement proteins factor B and C2.

Chicken complement factor B-like protease is a glycoprotein of 95 kDa. Activation of chicken serum complement with inulin cleaved the B-like protease into an N-terminal Ba fragment of 37 kDa and a C-terminal Bb fragment of 60 kDa. The whole protein and the two fragments were purified by affinity chromatography using mAb to chicken Ba or Bb followed by ion exchange chromatography. Amino acid sequencing showed that chicken B-like protease was cleaved at a site homologous to that cleaved in mammalian complement components B and C2 on activation. Limited tryptic digestion of the B-like protease generated fragments similar to Ba and Bb. More than 200 residues of the Ba sequence and two N-linked glycosylation sites were established by amino acid sequencing of peptides derived by digestion with four proteases. Comparison of human and mouse C2 and B sequences indicated a slower evolutionary rate for B (85% sequence identity) than for C2 (74% sequence identity). Comparison of chicken Ba to human and mouse C2b and Ba showed 42 to 45% sequence identity with respect to C2b fragments, and 46 to 49% sequence identity with respect to Ba fragments. Taking the slower evolutionary rate of factor B into account, chicken factor B-like protease seems to be equally related to mammalian complement components B and C2, and the B-like protease most likely represents the present-day descendant of a common ancestral protein for mammalian B and C2. This conclusion is in agreement with the requirement for the B-like protease in both classical and alternative activation pathways for chicken complement, and with the apparent lack of a chicken serum protein with exclusive C2 activity.

Apparently non-expressed alleles of factor B (BF) code for hypomorphic proteins.

In three families with an apparent non-expressed factor B (BF) allele (BF*Q0), advanced methods of isoelectric focusing for the determination of BF F subtypes revealed different hypomorphic BF products (BF QL) with functional hemolytic activity expressed by the assumed BF*Q0 allele. A Taq I and a Msp I restriction fragment length polymorphism as well as the Ba fragment of the expression products showed banding patterns for the BF*QL alleles corresponding to BF S types, whereas an altered Bb fragment was seen in two BF QL products. In one family an intragenic recombination site within the Bb part of the BF gene was assumed. Investigations of factor B and its conversion fragments, as demonstrated by the used methods, allow to complement molecular genetic investigations of BF*Q0 alleles in heterozygous genotypes on a protein level. We conclude that apparently non-expressed alleles of factor B code for hypomorphic but functionally active proteins.

Improvement of BF typing and of BF F subtyping after neuraminidase treatment in the unconverted and converted factor B

When neuraminidase-treated sera are analyzed by agarose gel isoelectric focusing, the factor B (BF) banding pattern is reduced to predominantly one major band without cathodically positioned bands. This not only makes unequivocal typing of BF allotypes possible but also the reliable distinction of all BF F subtype phenotypes with delimitation of "BF F subtype variants". With this new method, serum aging affects the BF determination to a lesser extent than when applying methods that separate native sera. We show that sialylation is not responsible for the BF F subtype polymorphism. All of the investigated BF allotype bands, including those characteristic of the subtypes, show functional hemolytic activity. The banding pattern after removal of neuraminic acid residues ranges from pH 6.8 to 7.3 for factor B, from pH 5.3 to 5.9 for the Ba fragment, and from pH 8.2 to 8.7 for the Bb fragment. The protein structure of factor B is also discussed. Eliminating the superimposition of bands in different BF allotypes, as demonstrated by these methods, proved to be necessary for the detection of hypomorphic BF gene products (BF QL), which are expressed by assumed BF*Q0 alleles in heterozygous genotypes. This allows investigation of BF*Q0 alleles on a protein level, which complements molecular genetic approaches.

Molecular characterization of human complement factor B subtypes

While several laboratories have agreed that there are two subtypes of the BF*F alleles, no information has been available until now at the molecular level. The region of the BF gene corresponding to the Ba fragment [1.7 kilobases (kb)] of the BF*S, BF*FA, and BF*FB alleles has been sequenced after specific amplification using the polymerase chain reaction (PCR). A point mutation at codon 7 has been revealed converting a cytosine in the BF*S allele to a thymidine in BF*FB. At the translational level an arginine residue in BF*S is substituted for a tryptophan residue in BF*FB. The amino-terminal sequencing of factor B immunoprecipitated from serum has been carried out from microquantities of protein blotted onto polyvinylidene fluoride (PVDF) membranes. We have shown that the difference between the BF*FA and the BF*FB subtypes in characterized by a glutamine at position 7 in BF*FA and a tryptophan in BF*FB.

The Ba fragment of complement factor B inhibits human B lymphocyte proliferation.

Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.

Reduction of complement activation during bypass by prime manipulation

Complement activation is believed to be of importance in the development of complications arising after cardiopulmonary bypass. The effect on complement activation of priming the extracorporeal circuit with crystalloid alone, crystalloid plus albumin, or crystalloid plus the plasma expander polygeline was assessed in 36 patients undergoing coronary artery operations with cardiopulmonary bypass using a bubble oxygenator. Activation of the alternative and common complement pathways was monitored before, during, and after the bypass period by measuring concentrations of factor B and its fragment Ba and C3 and its fragment C3d. Complement activation occurred in all three groups of patients, with no difference between the crystalloid and crystalloid-albumin groups. In contrast, Ba fragment concentrations were persistently and significantly lower during and after bypass in the polygeline group, denoting reduced complement activation. C3d levels also showed a tendency to be lower in this group. Our results indicate that addition of polygeline to the priming solution reduces complement activation. Because complement activation is associated with morbidity after cardiopulmonary bypass, addition of polygeline to the priming solution may offer an inexpensive method of reducing morbidity after cardiopulmonary bypass.

Hereditary subtypic patterns detected in the Ba fragment of complement factor B: Occurrence of four common alleles in Japanese

A procedure which can detect subtype-specific minor bands of factor B (BF) by polyacrylamide gel isoelectric focusing is presented. After zymosan-mediated fragmentation of BF in serum via alternative pathway for complement activation, serum samples are subjected to isoelectric focusing in a narrow pH range (4.2-4.9). The Ba fragments are detected by using immunoblotting. In addition to the previously reported minor bands with subtypic specificities, heterogeneities are observed in other minor band group, where a single minor band corresponds exclusively to a subtype in a regular combination with the previously announced subtypic patterns. A one-to-one correspondence of a single band to each subtype provides an unambiguous determination for three subtypic phenotypes deduced from the two divided BF*F alleles, BF*FA and BF*FB. An autosomal codominant heredity is confirmed through segregation analysis. A population survey reveals that four common alleles, BF*S, BF*FA, BF*FB, BF*Fb1, occur in a Japanese population and the former three alleles, except BF*Fb1, occur in a Cambodian population. The presence or absence of a single anodal minor band was found to be the only difference after neuraminidase treatment of FA and FB, implying that an amino acid substitution responsible for the FA-FB subtypic difference is involved in an additional acquisition in FA of an oligosaccharide unit with a charged sialic acid.

Polymerization of factor B of the alternative complement pathway via disulfide bond(s) in the presence of Cu 2+ and stimulation by C3b, the major fragment of C3

Factor B (B) of the alternative complement pathway has been found to dimerize via disulfide bond(s) in the presence of CuCl 2. Poly B has no B activity. The Bb fragment was also dimerized, indicating that one free sulfhydryl group on the Bb portion might be involved in polymerization. The Ba fragment was not dimerized. C3b, the major fragment of C3, has the capacity to stimulate polymerization of B. Incubation of C3b, B and factor D in the presence of Mg 2+ and Cu 2+ resulted in the formation of poly B and diminished cleavage of B. These results suggest that polymerization of B due to Cu 2+ might be partly responsible for the impairment of C3 convertase activity of the alternative pathway.

The BF F subtypes are detectable in the Ba fragment of factor B

The unanimous recognition of the two subtypes FA and FB of the BF ∗F allele has repeatedly been challenged. In the present investigation we are reporting about the unequivocal and simple detection of the subtypes on the Ba fragment of factor B by immunofixation isoelectric focusing after conversion with inulin. The common BF phenotypes F, S, and FS could be diagnosed in addition to the subtypes of BF ∗F which were observed in two regions acidic of the F major band. By comparison of standard phenotypes the subtypes in the Ba fragment corresponded to those of native factor B. All BF bands could be attributed to the Ba fragment by developing Western Blots with monoclonal antibodies directed against Ba. The distribution of the major BF phenotypes and alleles and the BF F subtypes in a population sample of 527 unrelated individuals from F.R.G. was in Hardy-Weinberg equilibrium. The allele frequency was determined to be 0.0731 for BF ∗FA, and 0.1053 for BF ∗FB. The advantages of determining the subtypes on the Ba fragment are: broadening of the FA FB corridor, a more reliable diagnosis of phenotypes, improved distinction between homozygous FA and heterozygous FAFB types, and recognition of common BF phenotypes as well as subtypes in aged sera. It is suggested that the problem in the designation of BF F subtypes by different groups should be resolved by an international reference typing.