Half-life of polyreactive antibodies

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined. In the present experiments, human monoclonal polyreactive and monoreactive antibodies of different isotypes were injected intravenously into mice and the clearance from the circulation was determined. The halflife of polyreactive IgM, IgA, and IgG antibodies was 8.0, 8.2, and 9.8 hr, respectively, compared to 35.4, 26.6, and 280 hr for monoreactive IgM, IgA, and IgG antibodies, respectively. Examination of tissue sections from animals given intravenous antibody showed substantial deposition of polyreactive, but not monoreactive, antibodies in several organs, the liver being the principal site of deposition. It is concluded that polyreactive antibodies are cleared from the circulation substantially faster than monoreactive antibodies.     

The use of in vitro immunisation, as an adjunct to monoclonal antibody production, may result in the production of hybridomas secreting polyreactive antibodies.

The possibility that immortalisation of in vitro immunised splenocytes may result in hybridomas secreting polyreactive antibodies was investigated. A panel of nine murine hybridomas, secreting IgM(kappa) anti-goat immunoglobulin G (anti-GIgG), was produced by immortalising splenocytes that had been immunised in vitro with GIgG. The ability of the corresponding monoclonal antibodies (Mabs) to bind multiple antigens was investigated using two techniques. First, the affinity constants characterising the interactions of each of the nine Mabs with each of a panel of six antigens were determined. Second, the specific anti-GIgG activities of each hybridoma supernatant and its corresponding affinity-purified IgM fraction were determined and compared. In total, these experiments indicated that eight of the nine hybridomas were polyreactive.     

A secreted receptor related to M1 protein of Streptococcus pyogenes binds to fibrinogen, IgG, and albumin

An extracellular protein (eMP) of Streptococcus pyogenes M type 1 was isolated by affinity chromatography on albumin- and IgG-Sepharose. The protein was found to bind to the human plasma proteins, fibrinogen, IgG, and albumin. Analysis of eMP by Western blotting demonstrated a major band with a molecular weight of 49 kD which was responsible for binding of the three plasma proteins. The purified protein was found to bind selectively to human and primate polyclonal IgG, human and mouse albumin, as well as human fibrinogen which has been the only fibrinogen tested. Serological investigations revealed a close relation of eMP to streptococcal M1 protein. It showed a reaction of identity with cell-extracted M1 protein in immunodiffusion. Moreover, the 49 kD peptide responsible for binding, was recognized with an antiserum directed against the 20 amino acids comprising synthetic peptide (42VAL-61GLU) of the N-terminal part of the M1 protein sequence. The affinity of M protein to plasma proteins other than fibrinogen opens new approaches to its purification by affinity chromatography.     

Binding of fibronectin, fibrinogen and type II collagen to streptococci isolated from bovine mastitis

Binding of 125I-labelled fibronectin, fibrinogen and type II collagen to group B (S. agalactiae), group C (S. dysgalactiae and S zooepidemicus), group E (S. uberis) and nontypable streptococci isolated from bovine mastitis was studied. S. agalactiae and S. uberis were found to bind low levels of all three proteins, while S. zooepidemicus bound high levels. Binding of the proteins to S. dysgalactiae varied, i.e. fibronectin was high, fibrinogen moderate and collagen low. Nontypable strains showed moderate or low binding of all proteins. Both hydrophobic and hydrophilic strains were found to bind fibronectin. For S. dysgalactiae the specific fibronectin binding ranged from 70% to 10% and for S. zooepidemicus it was more than 80% and this binding was sensitive to papain treatment. The binding of 29K-fibronectin fragment to one S. dysgalactiae strain showed an affinity of KD = 2.6 x 10(-8) M and the number of binding sites per colony forming unit (CFU) was calculated at 11,000.     

Binding of polyreactive antibodies to self versus foreign antigens

Aside from their ability to bind to multiple antigens, the classic hallmark of polyreactive antibodies is their autoreactivity. Because of their ability to bind a number of common autoantigens, it has long been speculated that polyreactive antibodies are involved in the clearance of self-antigens. However, it has been demonstrated more recently that polyreactive antibodies are also capable of binding to some foreign and synthetic antigens. Although data regarding the relative reactivity of polyreactive antibodies with self versus foreign antigens is lacking, it is generally thought that both activities may play an important biological role. In this study, the relative reactivity of polyclonal human polyreactive IgM with human proteins and tissue extracts versus foreign (xenogeneic) proteins and tissue extracts was probed. The binding of affinity purified anti-ssDNA IgM from adult human serum and the binding of polyreactive IgM in human cord serum and in human adult serum were evaluated. Using competitive and direct binding assays, human polyreactive IgM were found to be generally more reactive with foreign (xenogeneic) proteins than with self or allogeneic proteins. These data shed light on the fundamental nature of polyreactive antibodies, and may provide additional insight into their putative biological roles.     

Reovirus type 3 and [125I]-iodocyanopindolol bind to distinct domains on the beta-adrenergic like receptor

Antireceptor antibodies have been developed as a probe to study the cellular receptor for reovirus type 3. Using this probe, a glycoprotein with a molecular weight of 65-67 kilodaltons and a pI of 5.8-6.0 was isolated and identified as the reovirus receptor. This protein was also structurally similar to the affinity-purified beta-adrenergic receptor from calf lung. In this report, we employ [125I]-iodocyanopindolol, a high affinity beta-adrenergic antagonist, to further characterize this protein. We show that R1.1, a murine thymoma cell line, possesses about 2,000 receptors per cell with high affinity for ICYP (kD = 3.3 X 10(-11) M). Competitive inhibition studies suggest that the receptor is of the beta-2 subtype. Solubilized receptor proteins from R1.1 cells bound to the antireceptor antibody were further purified by SDS-PAGE and electroelution from the gel. Five percent of the proteins thus obtained could bind ICYP with high affinity (kD = 1.6 X 10(-10) M). This suggests that the purification procedure produced a collection of forms of this 65- to 67-kilodalton protein, some of which retained the conformation for binding the beta ligands. We also demonstrate that the isolated receptor protein was able to bind ICYP even when the virus binding site was occupied by the anti-idiotype, suggesting that reovirus type 3 and the beta ligands bind to distinct domains on the receptor protein.     

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.     

Serological differentiation of the potato-cyst nematodes Globodera pallida and G. rostochiensis: II. Preparation and characterization of species specific monoclonal antibodies

Hybridomas producing antibodies which react with thermostable protein antigens isolated from the potato cyst nematode species Globodera rostochiensis (TSRoP) and G. pallida (TSPaP) were isolated. Three of the isolated hybridomas (WGP 1, WGP 2 and WGP 3) produce antibodies that react with preferent affinity with protein antigens isolated from G. pallida, and two (WGR 11 and WGR 12) produce antibodies which bind preferentially to G. rostochiensis. Binding constants were determined to quantitate the differences in affinity of WGP 1, WGP 2, WGP 3, WGR 11 and WGR 12 for the protein antigens from both nematode species, and to asses the similarity in affinity for either protein antigen with respect to the other non-specific antibodies. In immunoblotting experiments a binding could be demonstrated, for most antibodies, to two thermostable proteins with apparent molecular weights of 20.6/20.8 kD for G. rostochiensis and 20.5/21.0 kD for G. pallida. the reactivity of the monoclonal antibodies with thermostable protein antigens from other common cyst nematodes was also investigated. All monoclonal antibodies, which are not specific for TSRoP or TSPaP, bind to thermostable proteins of these cyst nematode species. The use of some of the isolated monoclonal antibodies to improve the diagnosis of potato cyst nematodes in soil samples is discussed.     

Mathematical modeling as accounting: predicting the fate of serum proteins and therapeutic monoclonal antibodies

This article reviews current efforts to mathematically model the half lives of serum proteins, especially antibodies. While it is recognized that the neonatal Fc receptor, FcRn, is necessary for longer serum persistence of certain proteins, particularly the high abundance IgGs and albumin, it is not clear that it is sufficient to completely determine the half lives of these proteins. More specifically, it is unclear why the high avidity (bivalent), high affinity FcRn-IgG interaction, with half saturation in the 10 to 100 nM range (at endosomal pH according to the currently proposed mechanism), would result in a salvage mechanism that saturates at serum concentrations in the 10 to 100 mg/ml range--a discrepancy of 4 to 5 orders of magnitude. Alternative explanations include the proposal that the very low affinity binding between FcRn and IgG at blood pH is also relevant to the salvage mechanism, and that factors in addition to FcRn binding modulate the maintenance and clearance of IgG from serum.     

Monoclonal antibodies to phosphotyrosine

Phosphotyrosine coupled to KLH, BSA, and OVA was used for the production and screening of antibodies to phosphotyrosine. 800 hybridomas secreting antibodies that bound to phosphotyrosine were detected by ELISA. The most reactive 100 of these 800 were tested subsequently for their ability to bind phosphotyrosine-containing proteins on Western blots. Eight stable hybridoma cell lines were selected for further study, cloned by limiting dilution, and grown as ascites. These antibodies were purified by three different methods, and it was found that affinity chromatography on phosphotyrosine-affigel provided the most rapid and effective method. Many phosphotyrosine-containing proteins were detected by using these antibodies in Western blotting and immunoaffinity purification procedures. Binding of anti-phosphotyrosine antibody could be competed by phosphotyrosine or phenylphosphate but not by phosphoserine, phosphothreonine, or free phosphate. These antibodies should be useful for the identification and purification of proteins phosphorylated on tyrosine residues in transformed and growth factor-treated cells.     

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7−

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or β-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7⁻ cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Specific purification of monoclonal anti-DNA antibodies from culture medium using a DNA-coupled Sepharose 4B affinity column

Human monoclonal antibodies against DNA were specifically purified from the culture medium of an EBV transformant of SLE patients' lymphocytes using a DNA-coupled Sepharose 4B affinity column. The monoclonal antibodies were eluted from the column with 5% dimethylsulfoxide (pH 10.7) containing 0.5 M NaCl without loss of immunological activity and without contamination by other proteins.     

Somatic mutations can lead to a loss of superantigenic and polyreactive binding

Although antibodies have been assumed to bind a specific antigen, evidence exists showing that a single antibody can bind to multiple unrelated antigens. We previously studied a human monoclonal antibody expressing a mutated form of the V(H)3-73 gene and displaying anti-tubulin activity in a patient suffering from an immunocytic lymphoma. Despite its expression of a V(H)3 family member, this immunoglobulin failed to react with protein A (SpA), suggesting that somatic mutations could account for its change in specificity. To examine this possibility, we produced recombinant Ig expressing germ-line (IgM kappa-Germ) or the mutated form (IgM kappa-PER) of the V(H)3-73 fragment. Comparison of the respective affinities of the two Ig demonstrated that IgM kappa-Germ restores its SpA-binding capacity, and shows a moderate decrease in its affinity for tubulin. Interestingly, IgM kappa-Germ displayed polyreactive specificity for different autoantigens, which contrasted to the monospecific binding of IgM kappa-PER to tubulin. These results suggest that the monoreactive IgM kappa-PER antibody may be derived from a natural polyreactive antibody through somatic mutation. In addition, both temperature modification and mild denaturation succeeded in recovering the polyreactivity of IgM kappa-PER, which favors the view that conformational modifications of the tertiary structure of antibodies may play a key role in the genesis of polyreactivity.     

Binding of Human IgA Myeloma Proteins to Protein a. Evidence for Different Binding Mechanisms

The binding of fifteen IgA myeloma proteins to protein A was studied using affinity chromatography on protein A-Sepharose CL-4B. The percentage to which the proteins bound was variable from 1% to 11% with the exception of IgA(GED) with a binding capacity of 22%, and IgA(KLO) with a binding capacity of 84%. The binding of the proteins IgA(GED) and IgA(KLO) was studied further. IgA(GED) was a monomer, IgA(KLO) a polymer. The characteristics of the binding of these proteins were different. The protein A-reactive fraction of IgA(KLO), but not of IgA(GED) remained fully reactive on repeated protein A chromatography. Furthermore, the binding of IgA(GED) could be reduced to about 3% by either a decrease in pH to 4.5 or an increase in NaCl concentration to 2.OM, whereas the binding of IgA(KLO) was similarly reduced by a decrease in pH but its binding only reduced to half of the original value on a similar increase of NaCl concentration. In addition, F(ab')2 fragments of IgA(KLO), but not of IgA(GED), bind to protein A-Sepharose CL-4B, whereas IgA1 protease-produced Fab fragments of neither of the proteins did so, nor did pepsin-produced Fab' fragments. This suggests that the binding of F(ab')2 fragments of IgA(KLO) needs an intact hinge region.     

Antigen-binding B cells and polyreactive antibodies

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigenbinding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and β-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%–57%) by both homogeneous and heterogeneous antigens. Both CD5⁺ and CD5^? antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5⁺ antigen-binding (85%) as compared to the CD5^? antigen-binding (50%) population. Comparison of CD5⁺ B cells that bound antigens with CD5⁺ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.     

In vitro production of a hybrid monoclonal antibody that preferentially binds to cells that express both HLA-A2 and HLA-B7

A hybrid mouse monoclonal IgGl having one low affinity combining site for HLA-A2 and one low affinity combining site for HLA-B7 was made by the chemical method of Nisonoff and Palmer (Science 143:376,1964). This involved selective reduction of interchain disulphides, a splitting of the IgGl into half molecules at low pH and ionic strength, and reassociation of the half molecules by neutralization. Serologically active hybrids were separated from parental IgGl by an absorbtion procedure and recovered in about 10% yield. The hybrid discriminated between cells that express either HLA-A2 of HLA-B7 from cells that express both A2 and B7. This is because it could bind bivalently to the cell with both A2 and B7 but could only bind with a single combining site to cells expressing A2 or B7. The consequence of these different modes of attachment was to give up to sevenfold greater binding to the cell expressing A2 and B7 in comparison to the cell expressing only A2 or B7.     

Anti-anti-idiotypic (Ab3) antibodies that bind progesterone-11alpha-bovine serum albumin differ in their combining sites from antibodies raised directly against the antigen

Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antiprogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone response in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-50-times lower than that of DB3, their steroid-binding specificity showed considerable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal antibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progesterone conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound free progesterone, although with low affinity and very broad cross-reactivity. Like DB3, 1A4 is encoded by a heavy-chain variable region (VH) gene segment from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11alpha-BSA. In contrast, 3B11 binds progesterone-11alpha-BSA but not free progesterone and is encoded by an unrelated VH gene from the J558 family. The light chain variable region (VL) of 1A4 lacks the intradomain disulphide bridge owing to replacement of CysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short complementarity determining region (CDR) H3 loops, comprising three and four amino acids, respectively. Modelling of the combining site of 1A4 from the X-ray crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.     

A simple and efficient scheme for the purification of insulin-like growth factor II from human bone matrix extract

Human insulin-like growth factor II (IGF-II) has been purified to homogeneity from bone which contained 10-15 times more IGF-II than insulin-like growth factor-I (IGF-I). After extraction of IGF-II by demineralization of human bone powder with 10% EDTA containing 4M guanidine HCl at pH 7.4, IGF-II was separated from IGF binding proteins by hydroxylapatite chromatography in the presence of 4M guanidine HCl. The hydroxylapatite unbound fraction containing IGF-II was purified by affinity chromatography using Sm 1.2. monoclonal antibodies, which bind both IGF-I and IGF-II. The final purification of IGF-II was achieved by FPLC mono S ion-exchange chromatography in which IGF-II was separated from IGF-I. Human IGF-II thus purified was shown to be pure by (1) HPLC reverse-phase chromatography, (2) SDS-PAGE, and (3) N-terminal amino acid sequence. From 300 g of bone, 0.18 mg IGF-II was obtained with an overall recovery of 42%. These studies demonstrate the usefulness of (1) bone as a source of IGF-II purification and (2) antibodies that cross-react with both IGF-I and IGF-II for affinity purification of IGFs.