The isolation of the antibody moieties of immune complexes from serum by the pepsin digestion of conglutinin-anti-conglutinin complexes.

A technique is described which allows the antibodies of circulating immune complexes to be isolated as their F(ab')2 fragments. The method is based on the precipitation of the complexes by the sequential addition of conglutinin and anti-conglutinin, and the subsequent digestion of these precipitates by pepsin. Using this technique it has been possible to show antibodies to Epstein-Barr (EB) virus antigens in the immune complexes of patients with Burkitt's lymphoma and to microbial antigens in two patients with nephritis. By substituting DNAase for pepsin it has also been possible to show antibodies to DNA-containing nuclear antigens in the serum of patients with systemic lupus erythematosus.     

Bovine conglutinin binds to an oligosaccharide determinant presented by iC3b, but not by C3, C3b or C3c.

Bovine conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of conglutinin to iC3b, a fragment of the complement component C3. It was reported that conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.     

Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography

A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab')(2) antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab')(2) from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab')(2) antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab')(2) product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab')(2) antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab')(2) product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.     

A time-resolved immunofluorometric assay for quantification of the bovine collectin conglutinin

A high capacity time-resolved immunofluorometric assay (TRIFMA) for the bovine collectin conglutinin was developed. The TRIFMA was constructed as a non-competitive sandwich assay based on polyclonal antibodies as the capture reagent and a novel monoclonal antibody raised against conglutinin as the detection reagent and was set up to run on an automatic analyzer designed for the TRIFMA detection system. Polyclonal antibodies immobilized on microtiter plate wells were incubated overnight at 4 degrees C with diluted plasma samples, including quality controls (QC) and dilutions of a plasma with known conglutinin concentration. Conglutinin was sandwiched between the capture antibodies and the monoclonal antibody and the detection optimised with biotin-labelled secondary antibodies and streptavidin-Eu(3+). Plates were washed four times between each step and finally incubated with enhancement solution before measuring the fluorescence. The assay detection limit was 0.34 ng/ml and the working range 0.80 ng/ml-0.20 microg/ml. Intra-plate and inter-plate coefficients of variation (CV) were in the range of 5.0-8.3% and 6.2-7.2%, respectively, at concentrations of 3.4 and 150 ng/ml. Recovery was 90.9+/-2.4% and 98.8+/-2.5% when samples were spiked with 20 ng/ml and 100 ng/ml purified bovine conglutinin (BK). No circadian rhythm (24-h variation) in conglutinin plasma levels was observed across animals, indicating that the plasma levels were not influenced by, e.g. feeding. Samples could be stored at -20 degrees Celsius and were not sensitive to repeated freezing and thawing. In conclusion, the developed TRIFMA for bovine conglutinin is specific and reliable over a measurement range covering most situations.     

Association of Pepsin with Type II Collagen (CII) Breaks Control of CII Autoimmunity and Triggers Development of Arthritis in Rats

Lewis rats develop arthritis after immunization with heterologous but not homologous rat type II collagen (CII). We have observed that if the rat CII is prepared by pepsin digestion without subsequent extensive purification, it is arthritogenic in Lewis rats. To address whether pepsin in the CII preparations contributed to the development of arthritis and whether this was associated with the induction of an immune response to CII, Lewis rats were immunized with rat CII of various degrees of purity and with various pepsin contents. After immunization with a crude preparation of CII, containing relatively large amounts of pepsin, Lewis rats developed arthritis with an incidence of 80% together with a strong anti-CII autoantibody production. Further purification of the CII on DEAE-Sepharose, which removes pepsin, eliminated the arthritogenic properties and the capacity to activate CH-specific B cells. Likewise, lathyritic CII, prepared without pepsin, induced neither a CII-specific immune response nor arthritis. If, however, pepsin was added to non-arthritogenic batches of rat CII, arthritis appeared at an incidence of 40%. By using an ELISPOT technique to detect antigen-specific interferon-γ-producing T cells and antibody-producing B cells, the immune response to CII and pepsin can be evaluated. Eleven days after immunization with lathyritic CII and pepsin, a B-eell response towards both CII and pepsin was seen. Pepsin-specific T cells were also seen at day 11, but CII-specific T cells did not appear until day 14 after immunization. In addition, a weak CII -specific proliferative response of the T cells could be demonstrated at day 14 but not at day 11 or 12. These data show that pepsin plays an important role in the triggering of a CII-specific immune response. We suggest a carrier-hapten mechanism where pepsin acts as a carrier and CII as a ‘hapten’ which will activate CII-specific B cells. Subsequently these CII-specific B cells will break the T-cell tolerance and evoke a T-cell-mediated immune response towards CII.     

Purification of soluble immune complexes from serum using polymethylmetacrylate beads coated with conglutinin or C1q. Application to the analysis of the components of in vitro formed immune complexes and of immune complexes occurring in vivo during leishmaniasis.

A procedure for the isolation of immune complexes from human sera has been developed. Two steps are involved: (1) lipid-free serum is precipitated by polyethylene glycol; (2) the solubilized precipitate is absorbed on a column of polymethylmethacrylate beads coated with conglutinin (K) or C1q; the column is washed, the complexes are then eluted, using 0.02 M EDTA (for K column) or 0.5 M NaCl (for C1q column). This procedure permitted the purification and the characterization of soluble 125I-BSA-anti-BSA, 125 I-tetanus toxoid-anti-tetanus toxoid, and 125-I-HBsAg-anti-HBsAg complexes made in vitro in the presence of fresh human serum. The isolated complexes were shown to contain antigen, antibody, C1q, C1r, C1s and C3. When normal human serum was submitted to such a procedure, no detectable amount of protein was present in the final eluted fraction. Immune complexes formed in vivo were also purified by conglutinin column from the serum of a patient with disseminated leishmaniasis. The isolated material was found to contain IgM, IgG, C1q, C1r, C1s, C3c and C3d. The purified complexes dissociated at acid pH were found to contain anti-IgG and anti-leishmania antibodies.     

Structural similarity between bovine conglutinin and bovine lung surfactant protein D and demonstration of liver as a site of synthesis of conglutinin.

Conglutinin is a Ca(2+)-dependent, carbohydrate-binding, serum protein which contains an N-terminal collagen-like region and a C-terminal, C-type lectin domain. To date, conglutinin, which appears to play an important role in defence mechanisms, has been fully described, by protein sequence analysis, only in the bovine system. To allow comparison of lung surfactant protein D (SP-D) with conglutinin, within one species, a full-length cDNA clone for SP-D has been isolated from a bovine lung library. The derived amino acid sequence for bovine SP-D shows a higher (78%) level of identity to the sequence of conglutinin than to the sequence of human or rat SP-D (67 and 65% respectively). However, SP-D and conglutinin are known to have different carbohydrate-binding specificities, therefore some of the 16 residues conserved in the C-type lectin domains of all three species of SP-D, but which are not conserved in conglutinin, appear likely to be involved in determination of specificity. The use of a polymerase chain reaction (PCR)-derived DNA probe for bovine SP-D in Northern blotting studies yielded a signal from bovine liver mRNA as well as the expected signal from bovine lung mRNA. Since SP-D appears to be a lung-specific protein, it seems probable that the liver is the primary site of synthesis of conglutinin.     

检测循环免疫复合物的牛胶固素酶联免疫吸附试验在临床的应用

本文介绍了一种检测循环免疫复合物的牛胶固素酶联免疫吸附试验。方法简便、稳定,可供临床应用。使用该方法检测了285份免疫复合物病患者血清,阳性率为56.2％。     

Conglutinin Binds The HIV-1 Envelope Glycoprotein gpl60 and Inhibits its Interaction with Cell Membrane CD4

The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160 was calcium-dependent, which is characteristic of the binding of a C-type lectin to its ligand, and the binding was inhibited in a dose-dependent manner with N-acetyl-D-glucosamine. Deglycosylation of rgp160 abrogated the conglutinin binding. In addition, conglutinin exerted a dose-dependent inhibition of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein.     

The Conglutination Phenomenon: XIV. The Resistance Enhancing Effect of Conglutinin and Immuno-Conglutinin in Experimental Bacterial Infections

The effect of conglutinin and immuno-conglutinin preparations on the resistance of animals has been studied in seven bacterial infections. In six infections —Salmonella typhimurium, Pasteurella septica, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae and Streptococcus pyogenes—higher survival rates were demonstrated in mice previously injected with the conglutinin preparations.

Infection with avirulent strains or non-pathogenic organisms in numbers large enough to cause death led to severe toxaemia against which the injection of conglutinin preparations failed to protect. Although the conglutinin preparations failed to protect against avirulent or unadapted strains, they did protect against virulent or adapted strains of the organisms.

Experiments undertaken to define the protective factor in the serum preparations indicate that the protective factor probably is the same as that which is responsible for the conglutinating activity, conglutinin.

     

Characterization of a Lectin in Human Plasma Analogous to Bovine Conglutinin

The structural characteristics of a human plasma protein analogous to bovine conglutinin were studied. The protein was previously found to bind to complement-reacted IgG in a calcium-dependent and N-acetyl-D-glucosamine-inhibitable manner and it further shows cross-reactivity with anti-bovine conglutinin antibody. By gel permeation chromatography the conglutinin activity in human plasma was localized to fractions containing proteins of Mr at around 700,000. The conglutinin was localized by one ELISA for antigen determinants and by another for biological activity. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions these fractions were shown to contain proteins of about 300,000. When human conglutinin-like protein, partially purified by affinity chromatography, was analysed unreduced by SDS-PAGE followed by western blotting, the cross-reacting anti-bovine conglutinin antibody bound to a protein with an Mr of 330,000. When the sample was reduced and alkylated before electrophoresis a band of 66,000 was immunostained. The 330,000 and 66,000 proteins were shown to be collagenase sensitive. 125I-iC3b was seen to bind to the 330,000 band when incubated with western blots of partially purified human conglutinin.     

Calcium-dependent and calcium-independent signals in the conglutinin-binding assay (KgBa) for immune complexes. Influence of anti-collagen-antibodies.

A solid phase ELISA conglutinin-binding assay (KgBa) was evaluated for the detection of circulating immune complexes. ELISA wells were coated with purified bovine conglutinin and incubated with test sera. Bound IgG was detected with enzyme labelled anti-immunoglobulin. Heat aggregated IgG which had been "solubilized" (i.e., complement treated by incubation with serum) was employed as a reference. The binding of the complement-reacted IgG to solid phase conglutinin was found to be calcium-dependent and inhibitable with N-acetyl-D-glucosamine (GlcNAc). Prolonged incubation (4 days) of aggregated IgG with serum at 37 degrees C abolished the binding to conglutinin, a finding consistent with the complete degradation of deposited C3b to C3c and C3d. The solubilized IgG that bound to solid phase conglutinin was found by gel chromatography to be of high molecular weight (greater than 600 kDa). Binding of IgG to solid phase bovine conglutinin was also observed to a variable degree in normal and pathological sera. However, in this situation the IgG binding was largely calcium-independent, was not inhibited by GlcNAc and did not decrease after prolonged incubation of the serum at 37 degrees C. The reactive IgG eluted on gel chromatography at the position of monomeric IgG suggesting binding via the antigen binding sites. Binding of this IgG was inhibited by both collagen type II and purified conglutinin. These observations suggest that the assay detects cross-reacting autoantibodies against collagen epitopes, or, alternatively, antibodies against the dietary antigen, bovine conglutinin.     

Demonstration in Human Plasma of a Lectin Activity Analogous to that of Bovine Conglutinin

Evidence of the existence in human plasma of an activity analogous to that of bovine conglutinin is presented. The human plasma component was characterized antigenically and functionally. Human plasma was shown to agglutinate complement-coated erythrocytes in the presence of Ca2+, and this conglutination was inhibited by EDTA. The molecule also binds to complement-reacted solid phase IgG and to zymosan in the presence of Ca2+. The binding to complement is not inhibited by N-acetyl-D-mannosamine, but is inhibited by N-acetyl-D-glucosamine, as previously shown for bovine conglutinin. Antibodies raised against bovine conglutinin cross-react with the human protein. The plasma concentration of the conglutinin-like protein showed a high inter-individual variation between apparently healthy donors. Unlike bovine conglutinin, the human conglutinin activity could not be demonstrated in serum but only in plasma. The activity was more stable in plasma containing metal-ion chelators like EDTA and citrate than in heparin or hirudin plasma.     

The plasma levels of coglutinin are heritable in cattle and low levels predispose to infection.

Conglutinin, like mannan-binding lectin (MBL) and CL-43, is a serum collection involved in the innate immune defence system. In man, low serum MBL concentrations, resulting from mutations in the collagen region, are associated with a common opsonic defect. Plasma levels of conglutinin in cattle were assayed by rocket immunoelectrophoresis to examine whether they were genetically determined. Samples were collected from calves (309 bull-calves and 260 heifers with complex pedigree relationships). The number of respiratory infections from the 42nd to 336th day of life was recorded. The number of infections was found to be genetically determined (heritability: h2 = 0.31 +/- 0.07). A wide concentration range of conglutinin was found in plasma (< 1.25-35 micrograms/ml for females, geometric mean 8.1 micrograms/ml, and < 1.25-47 micrograms/ml for males, geometric mean 15.5 micrograms/ml), and the concentrations was found to be genetically determined (heritability, h2 = 0.52 +/- 0.07). The analysis revealed a negative association between disease frequency and the conglutinin levels (-0.56 +/- 0.18 for female; -0.50 +/- 0.18 for male). Levels of conglutinin below the detection limit of the assay (1.25 micrograms/ml) were found in 2% of the animals. If these animals are assumed to be homozygous for a single recessive allele causing low concentrations a gene frequency of 0.15 could be calculated. These findings suggests that selection for resistance against infectious disease is possible in cattle and that the level of plasma conglutinin may be a helpful trait in such a breeding scheme.     

猪C_(19)酶联免疫吸附试验测定人血清循环免疫复合物的探讨

C_(1q)是补体系统的第一个成份,它能与被加热凝聚或同抗原结合后的IgG、IgM分子Fc段的CH_2或CH_3区结合,利用这一特性可进行循环免疫复合物(CirculatingImmune Complexes,简称CIC)检测。以C_(1q)为基础的试验是WHO所推荐的六种较敏感的CIC抗原非特异性检测法之一。由于现常采用的人C_(1q)存在某些缺点,故在一     

Peptic Digestion of Streptococcal M Protein II. Extraction of M Antigen from Group A Streptococci With Pepsin

M protein of group A streptococci was extracted by mild peptic digestion. Optimal amounts of type-specific M protein were released after 20 min of digestion with 0.02 mg of pepsin per ml at pH 5.8. Immunological analysis revealed that, unlike conventional HCl extracts, pepsin extracts lacked the surface C carbohydrate antigen and contained less non-type-specific, heat-stable cellular antigens; they also lacked detectable heat-labile T protein. Similar to HCl extracts, however, the pepsin-extracted M protein precipitated homologous-type M antisera and inhibited type-specific opsonization of homologous group A streptococci. Furthermore, the pepsin extract was capable of inducing type-specific opsonic M antibody in rabbits. This method may provide a useful initial step in the purification of M protein by reducing contaminating antigens.     

Isolation of circulating immune complexes by conglutinin and separation of antigen from dissociated complexes by immobilized protein A.

A method for the isolation of complement-fixing immune complexes from human serum and the separation of antigen from antibody is described. In order to isolate the complexes, we used soluble bovine conglutinin in a three-step procedure: (1) serum containing immune complexes is reacted with conglutinin in the presence of 10 mM calcium; (2) the conglutinin-bound immune complexes are precipitated by anti-conglutinin rabbit serum; (3) the precipitate is washed and the complexes are eluted from the precipitate by EDTA (pH 7.5) which chelates calcium and releases C3-associated immune complexes from conglutinin. To separate the antigen from the antibody, the isolated complexes are acid-dissociated (pH 3.0), and the antibody is absorbed to staphylococcal protein A conjugated to Sepharose leaving the antigen in solution. The antibody bound to Sepharose-protein A is recovered by elution with 3.5 M magnesium chloride. This procedure permitted the isolation of immune complexes from sera of hepatitis B surface antigen (HBsAg) positive chronic active hepatitis. In addition, immune complexes were isolated from sera of patients with rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis. The isolated immune complexes contained IgG, IgM, C3 and albumin. Specific antibodies such as rheumatoid factors, anti-nuclear antibodies and antimitochondrial antibodies in varying titres have been found to be present in the isolated immune complexes. The conglutinin method has proven to be a useful technique for the isolation of immune complexes and for the identification of antibody and could be applied to the identification of the antigen in immune complexes.     

Circulating immune complexes in patients with usual interstitial pulmonary fibrosis: partial characterization and relationship with Thermoactinomyces vulgaris.

Sixty-six sera were analysed by solid-phase conglutinin binding assay, to detect the levels of circulating immune complexes (CIC), and by enzyme-linked immunosorbent assay (ELISA) to show a correlation with antibodies to Thermoactinomyces vulgaris. Sixty per cent of patients with usual interstitial pulmonary fibrosis (UIP), were positive for CIC; and T. vulgaris antibodies were detected in 60% of the same patients. In comparison, there was a low frequency of positive results in bronchitis patients (5% for CIC and 35% for T. vulgaris), and in normal blood donors (0% for CIC and 30% for T. vulgaris). Furthermore 31% of patients with lung cancer were found positive for CIC, but not for T. vulgaris. Immune complexes purified on Protein A-Sepharose and by sucrose density gradient from patients with UIP, showed a sedimentation coefficient higher than 19 S. The purified material was found to contain IgG and IgM as antibodies. Binding of immune complexes, purified by sedimentation on sucrose gradient, to conglutinin was inhibited by the presence of T. vulgaris antigen; thus suggesting that this antigen might be present in the complexes.     

Collectin-43 is a serum lectin with a distinct pattern of carbohydrate recognition.

Collectin-43 (CL-43) is a C-type serum lectin and a member of the collectin family of soluble proteins that are effector molecules in innate immunity. We have investigated the binding specificity of CL-43 using as model systems a panel of structurally defined oligosaccharides in the form of neoglycolipids, and several glycoproteins derived from the complement glycoprotein C3 during activation of the complement cascade. A specificity is revealed towards fucose as part of the Lea oligosaccharide sequence, and towards mannose as found on high mannose-type chains. These are features shared with other serum collectins, conglutinin and mannan-binding proteins; a major difference is the lack of detectable binding by CL-43 to N-glycosidic oligosaccharides terminating in N-acetylglucosamine. CL-43 has a unique pattern of reactivity towards high mannose-type oligosaccharides on the two glycosylation sites of C3 and derived glycoproteins: it binds to C3c (not bound by conglutinin and mannan-binding protein) but not to hydrolysed C3 [C3(H2O)], C3b or iC3b immobilized on microwells (all bound by conglutinin but not by mannan-binding protein). When these glycoproteins are sodium dodecyl sulphate (SDS)-treated and immobilized on nitrocellulose, CL-43 (but not conglutinin nor mannan-binding protein) binds strongly to C3(H2O), iC3b and C3c. The salient conclusions are, first, that there are remarkable positive or negative effects of carrier protein on oligosaccharide presentation and these differ for the individual collectins. Second, the different though partially overlapping binding patterns among the collectins may be important for their function as circulating effector molecules with broad surveillance capabilities.     

The Alexination and Conglutination Reactions: The Reactions Between Sensitized Erythrocytes and Horse Complement and Between Alexinated Erythrocytes and Conglutinin

A method is described for the quantitative measurement of the reactions between sensitized cells and horse complement and between alexinated cells and conglutinin. The method is laborious but its application has allowed the determination of the optimal times of the reactions at various temperatures. The results obtained in these experiments indicate that the alexinated configuration with which conglutinin and immuno-conglutinin react is not one of the recognized intermediates formed during the process of immune haemolysis.