Free IgG Research Articles

Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes

Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (∼18 IgG per 100 nm liposome) exhibited long circulation times (MRT=8.5 h, Cl=0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRT≤0.7 h, Cl≥7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab′ fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.

Characterization of Porcine Platelet Glycoproteins Recognized by Human Natural “Anti-Gal” Antibodies

Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain.

Cerebrospinal fluid free kappa light chains versus IgG findings in neurological disorders: qualitative and quantitative measurements

In this study free kappa light chains in cerebrospinal fluid (CSF) were determined both by an affinity mediated capillary blotting technique after isoelectric focusing (IEF) in agarose gel and by a quantitative enzyme linked immunosorbent assay (ELISA). The free kappa results were compared with the IgG findings in 4 neurological patient groups with a distinct CSF IgG pattern: (1) CSF without oligoclonal IgG bands, (2) CSF with serum derived IgG bands, (3) CSF restricted IgG bands and (4) CSF restricted and serum derived IgG bands. Oligoclonal free kappa bands are nearly absent in CSF of groups 1 + 2, and present in 88% of group 3 and 84% of group 4 patients. We could also establish free kappa indices from specimens in the 4 groups in analogy to IgG indices. Group 1 had a median free kappa index of 1.1, group 2: 1.0 and groups 3 + 4: 10.0. The correspondence between immunoblot and index findings for free kappa is better than for IgG. Free kappa index is more sensitive but somewhat less specific than IgG index for establishing intrathecal immune production.

Detection of antigens in immune complexes by a dot blot assay

A simple assay is described for detection of antigens as a part of immune complexes. The complexes together with free IgG molecules are absorbed on protein A (or G)-Sepharose. The Sepharose beads are transferred to nitrocellulose membrane where all IgG including their complexes with antigens are eluted by acidic buffer directly on the surface of the membrane. The antigens immobilized on the membrane after elution are detected by corresponding labeled antibodies. The validity of the procedure was assessed by experiments with complexes of avidin and rabbit antibodies. The assay was used also for detection of DNA in immune complexes in mice with experimental systemic lupus erythematosus (SLE).

Prolactin-immunoglobulin G complexes from human serum act as costimulatory ligands causing proliferation of malignant B lymphocytes.

Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.

Novel delivery of antioxidant enzyme catalase to alveolar macrophages by Fc receptor-mediated endocytosis.

Excessive production of reactive oxygen species by alveolar macrophages (AMs) in response to inhaled toxic substances is a major cause of oxidative lung injury. Therapeutic approaches designed to protect the lungs from oxidative injury by administering native antioxidant enzymes such as catalase and superoxide dismutase have been suggested. However, problems associated with poor penetration of these enzymes to the intracellular target sites have limited their effective use. The present study reports a drug targeting method based on receptor-mediated endocytosis of the antioxidant enzyme catalase to the AMs. This method employs molecular conjugate consisting of a cognate moiety, in this case IgG which recognizes the macrophage Fc receptor, covalently linked to the enzyme catalase via the reversible disulfide linkage. The uptake efficiency of the enzyme conjugate and its protection against oxidative injury were evaluated microfluorometrically using the intracellular oxidative probe dichlorodihydrofluorescein BSA: anti BSA antibody complex (DCHF-IC), and the cell viability indicator propidium iodide. The DCHF-IC-stimulated macrophages exhibited a dose- and time-dependent increase in intracellular fluorescence with a half maximal response dose of approximately 120 micrograms/ml. Free catalase (50-500 U/ml) failed to inhibit the DCHF-IC-induced oxidative burst and had only a marginal protective effect on AM injury. In contrast, the catalase-IgG conjugate (50-500 U/ml) strongly inhibited both the DCHF-IC-induced oxidation and injury in a dose-dependent manner. Effective inhibition was shown to require both the antioxidant catalase moiety ant the cognate moiety for the cell surface receptor. Specific internalization of the conjugate through the Fc receptor was also investigated by competitive inhibition using free IgG.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysaccharide on free attractants, specific IgG autoantibodies and immune complexes.

We recently found that normal human sera contain IgG antibodies against two chemoattractants, neutrophil attractant protein-1 (NAP-1/IL-8) and monocyte chemoattractant protein-1 (MCP-1), as well as immune complexes of these proteins. Intravenously administered LPS was reported to cause a sharp rise in serum NAP-1 concentration. Our study was designed to determine if LPS also caused an increase in MCP-1 and to measure associated changes in concentrations of antibody and immune complex. LPS caused a rise to peak within 2 to 3 h in serum concentrations of free NAP-1 and MCP-1, followed by an almost equally rapid fall toward base-line levels by about 5 h postinjection. MCP-1 concentration in sera from the 11 subjects rose to a peak of 330 +/- 52 pM. The peak value for NAP-1 was 80 +/- 11 pM. In 10 of the 11 subjects, free IgG autoantibody to MCP-1 decreased from a mean pre-LPS value of 1820 +/- 660 pM to a mean low of 53% of the respective initial values. Corresponding data for IgG anti-NAP-1 were a pre-LPS concentration of 216 +/- 7 pM, which decreased to a mean low of 44% of the respective initial values. The finding in some subjects of a rapid rise in free antibody after the nadir suggests the possibility of acute regulation of autoantibody secretion rates. Although the results suggested that LPS-induced chemoattractant combined with free antibody, serum concentrations of MCP-1-IgG or NAP-1-IgG did not increase, which points to an as yet unknown mechanism for trapping and elimination of the immune complexes.

Increased synthetic peptide specificity of tissue-CSF bound anti-MBP in multiple sclerosis

Free and bound hydrosoluble protein extracts were prepared from four anatomical areas of a multiple sclerosis (MS) cerebrum and from corresponding anatomical areas of a normal (non-MS) control. Increased levels of IgG and anti-myelin basic protein antibodies (anti-MBP) were detected in all MS samples and they were undetectable in the controls. IgG and anti-MBP from free (unbound) hydrosoluble protein extracts are defined as free IgG and free anti-MBP while IgG and anti-MBP from tissue bound protein extracts are defined as bound IgG and bound anti-MBP. IgG was purified from free protein extracts by protein G Sepharose affinity chromatography and anti-MBP was further isolated from purified IgG by antigen specific (MBP) Sepharose affinity chromatography. Free and bound anti-MBP were reacted with 20 synthetic peptides of human MBP prepared by the Fmoc method. Free anti-MBP, whether in the context of whole protein extracts, or as purified IgG or as purified antibody was completely neutralized by peptides #12, #15, #56 and #56 ∗ containing overall residues 75–106, partially neutralized by peptides #27, #16 and #21 containing overall residues 61–83 and did not react with the remaining 13 peptides. Tissue bound anti-MBP was completely neutralized only by peptides #12, #15, #56 and #56 ∗ (overall residues 75–106) and showed no reactivity towards the remaining 16 peptides including peptides #27, #16 and #21. Synthetic peptide specificity of free anti-MBP purified from MS cerebrum was identical to previously reported specificity of free anti-MBP from MS cerebrospinal fluid (CSF), while tissue bound anti-MBP, as well as bound anti-MBP from CSF had a more restricted synthetic peptide specificity than free anti-MBP. This suggests that the most likely epitope of anti-MBP is located between residues 84 and 95 of human MBP just proximal to the tri-proline sequence (99–101).

Autoimmune haemolysis in the dog: Relationship between anaemia and the levels of red blood cell bound immunoglobulins and complement measured by an enzyme-linked antiglobulin test

A direct enzyme-linked antiglobulin test (DELAT) was used to measure the levels of red blood cell (RBC) bound IgG, IgM, IgA and C 3 in dogs with autoimmune haemolytic anaemia (AIHA). At presentation, one or more DELAT parameters was raised in each AIHA case, and the RBC were typically coated with immunoglobulin of more than one class, together with C 3. There was no relationship between the levels of RBC-bound IgG, IgM or IgA and the severity of the anaemia, although a significant negative correlation ( r s = −0.66, P < 0.02) was found between bound C 3 and blood haemoglobin concentration. These results indicate that the level of sensitisation of erythrocytes with IgG alone is not a reliable predictor of the severity of haemolysis in different cases, and that the pathogenesis of AIHA can be complex, involving multiple immunoglobulin classes and complement in the destruction of RBC. A significant relationship ( r s = 0.63, P < 0.02) was found between serum IgG concentration and haemoglobin levels, and it is suggested that this may be due to free IgG inhibiting the interaction of IgG-sensitised RBC with macrophages. Serial measurements from individual AIHA cases during treatment revealed that the levels of RBC-bound immunoglobulins fell simultaneously with improvements in anaemia. In one dog, a relapse was associated with increases in bound IgG and IgM. Transient relative reticulocytopenia at presentation was common, but was not related to the severity of the anaemia. However, in other cases there was a persistent failure to increase RBC production, which was associated with slower recovery.

The recognition of a recombinant human Fc epsilon fragment by the subclasses of IgG autoanti-IgE: disproportional subclasses distribution of complexed autoantibody.

Circulating IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma where it is suggested to be potentially involved in the removal of IgE-allergen complexes. Since such a putative role will largely be determined by the subclass profile of complexed (i.e. IgE-bound) IgG anti-IgE, a study was undertaken to determine the subclass distribution of complexed IgG anti-IgE antibody in the sera of asthmatic patients. The study exploits the heat-labile property of IgE by heating (30 min at 56 degrees C) serum to liberate bound anti-IgE, pre- and post-heated sera are then assayed for IgG subclass anti-recombinant human Fc epsilon (rFc epsilon) activities by ELISA and any heat-induced increase in antibody activity is taken as a measure of complexed anti-IgE. This has revealed a disproportionately high amount of IgG4 in complexed (but not free) IgG anti-IgE. The propensity of IgG4 to form complexes with IgE has important biological consequences, particularly with regard to the activation of C1q and Fc gamma R by other subclasses.

Glycosylation of IgG, immune complexes and IgG subclasses in the MRL‐lpr/lpr mouse model of rheumatoid arthritis

The MRL-lpr/lpr (MRL/lpr) mouse spontaneously develops a disease syndrome which, in many respects, is similar to human rheumatoid arthritis. These mice developed joint inflammation, circulating rheumatoid factors and immune complexes. We now show that the parallel with human disease extends to a glycosylation defect which is observed on IgG from rheumatoid arthritis patients. Using the lectins ricin and Bandeiraea simplicifolia II we have found that terminal N-acetylglucosamine is clearly raised in MRL/lpr IgG. Increased exposure of galactose was also detected, indicating that a second glycosylation site must be present on these molecules. Polyethylene glycol-precipitated IgG complexes bound significantly more of each lectin than did free IgG, indicating that the changes in glycosylation were associated with complex formation. The sugar abnormality was most marked in the IgG2a/IgG3 fraction from protein A IgG subclass chromatography. Our results suggest that the IgG glycosylation defect seen in rheumatoid arthritis is apparent in the MRL/lpr mouse and may contribute, through complex formation, to the pathological processes in the rheumatoid syndrome.

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies of defined specificity I. Application to naturally occurring antipolyamine antibodies in human sera

A simple covalent enzyme-linked immunoassay procedure (CELIA) is described for the routine determination of free and immune complex-bound antibodies in sera. Assays for the latter could not have been performed by adsorption ELISA due to the high ionic strength of the reassociating buffer. For the measurement in human sera of free naturally occurring IgG and IgM antibody directed against the hapten spermine, polycarboxystyrene microtiter plates with covalently coupled spermine were used. For the determination of immune complex-bound antipolyamine IgG and IgM antibody titers, serum was first dissociated at pH 2.3 in tubes and then reassociated at pH 8.1 in the wells of a microtiter plate containing covalently bound spermine. The reactivity of anti-spermine antibodies was increased from 2- to 13-fold after dissociation and reassociation compared to that of non-dissociated area. The apparent reaction constant ( R app.) of free IgG antibodies to spermine in the sera of 19 bronchopulmonary patients with cancer differed significantly from the R app. values of IgG antibodies having this specificity in ten other patients with non-malignant disease.

Enhanced CNS uptake of systemically administered proteins through conjugation with tetanus C-fragment

No other exogenous protein enters the central nervous system from the circulation as readily as tetanus toxin. We examined the capability of the non-toxic binding fragment of tetanus toxin (C-Fragment) so serve as a vehicle for transport of other proteins into the mouse CNS. Using periodate oxidation of the enzyme horseradish peroxidase (HRP), we synthesized two separate macromolecular complexes, one containing C-fragment and HRP, and the other C-fragment, HRP and a third “test” protein-human IgG. The distribution of C-fragment-HRP was typical of blood borne proteins including native C-fragment, with labeling of all neurons with known projections outside the blood-brain barrier, particularly large spinal motoneurons. C-fragment-HRP conjugates showed superior neuronal labeling to over 100-fold greater quantities of free HRP. Complexes containing C-fragment, HRP and human IgG were internalized by neurons from both intramuscular and intraperitoneal injections. The efficiency of neuronal uptake of IgG in the C-fragment conjugated form was enhanced over 40-fold compared to free IgG. Linkage of a large protein to C-fragment probably leads to enhanced endocytosis of that protein by neuronal terminals projecting outside the blood-brain barrier. C-fragment can serve as a vehicle to allow selected proteins to bypass the barrier and enter the CNS.

Association of elevated anti-sarcolemma, anti-idiotype antibody levels with the clinical and pathologic expression of chronic Chagas myocarditis.

Antibody F(ab')2 fragments derived from the sera of four patients with histology-proven chronic Chagas myocarditis [cF(ab')2]-complexed antibody F(ab')2 fragments of children with acute Trypanosoma cruzi infection [aF(ab')2] in significantly higher molar ratios than those measured with F(ab')2 antibody fragments of normal subjects [nF(ab')2] from nonendemic areas (p less than 0.05). Anti-idiotype [cF(ab')2 X aF(ab')2] immune-complex formation was significantly blunted by preabsorption of cF(ab')2 with porcine heart atria sarcolemma (PAMs) immobilized on sepharose beads (inhibition, mean, 78.1 +/- 2.4%, n = 4). cF(ab')2 X nF(ab')2] immune-complex formation was also inhibited by pretreatment of cF(ab')2 with PAMs (inhibition, mean, 48.7 +/- 7.5%, n = 4). The sera of three groups of subjects from a geographic zone endemic for T. cruzi infection in the northeast of Brazil were assayed for free and immune-complexed IgG anti-acute T. cruzi infection F(ab')2. The indexed levels of free IgG anti-idiotype antibody activity and levels of IgG anti-idiotype immune complexes (IC') were markedly elevated in hospitalized patients with severe, decompensated chronic Chagas myocarditis (n = 23), and their IC' indexes were significantly higher than those measured in asymptomatic seropositive subjects from a nearby endemic village of the northeast of Brazil (Moniz Ferreira, n = 92, p less than 0.001) and in healthy seronegative villagers (n = 84, p less than 0.001). There exists a strong correlation between elevated IgG anti-sarcolemma, anti-idiotype activity levels and the clinical and pathologic expression of chronic Chagas myocarditis.

Experimental Histoplasmosis in the Mouse: Immunoglobulin Class Response, Total Immunoglobulin A Levels, Immune Complex Formation, and Occurrence of Antigen in Serum and Urine

Biotin-avidin enzyme-linked immunosorbent assays (ELISA) were developed to study the appearance of free antibody of the IgM, G, and A classes of immune complexes (IC) containing those classes and of antigen in the serum and urine of mice with experimentally induced, disseminated histoplasmosis (histo) over a period ranging from 4 to 64 days after infection. Free IgM was detected 4 days after infection, remained at low levels, and disappeared on Day 64, whereas free IgG was first detected on Day 7 and rose to very high levels before declining on Day 64. Free IgA was detected only once, on Day 21. IC containing IgM were seen first on Day 4, remained at low levels, and became undetectable on Day 64. IC containing IgM were detected on Day 7, peaked on Day 14, and declined through Day 64. IC containing IgA wee seen at low levels on Days 7, 14, and 21. Estimation of total IgA levels, done by single radial immunodiffusion, showed a statistically significant decrease on Day 14, followed by a slightly significant increase on Days 21 and 30. Antigen was detected in as much as 80% of serum specimens and 100% of urine samples during the first 2 wk postinfection but rarely afterwards. We conclude that ELISA provides a highly sensitive way to study antibody, IC, and antigen in host body fluids during histo infection and that IgM and antigen detection can be very early indices of infection. Measurements of IC and total IgA seem to be of relatively less importance in detection of infection.

Enhancing effects of immunoactive peptide FR48217 on immunological responses to vaccination by inactivated influenza virus

The adjuvant effect of an immunoactive peptide, FR48217 (FR), was investigated in mice vaccinated with inactivated influenza virus with and without FR (designated as V+FR and V-FR, respectively). Mice were infected with influenza virus A/PR/8/34 28 days after vaccination. The survival rate indicated a strong adjuvant effect of FR on vaccination with inactivated influenza virus. No difference in interferon activity in the pulmonary lavage fluid was found between the V+ FR and V - FR groups. The titres of virus-specific IgA and IgG antibodies in the serum increased after vaccination in both V+ FR and V-FR groups and were significantly higher in the V+ FR group than in the V-FR group. On days 2 and 4 after viral infection, the serum antibody titres decreased sharply and then increased again rapidly. The free IgA and IgG antibody levels in the pulmonary lavage fluid of vaccinated mice started to increase after day 4. It is proposed that one of the mechanisms by which FR has the adjuvant effect on vaccination is the increase of virus-specific IgA and IgG antibodies in the serum, which are diffused into the infection sites of the epithelial tissues in the respiratory tract to neutralize the virus at the early stages of viral infection.

Evaluation of Production and Characterization of Monoclonal Antibodies to Human IgG of Four Subclasses

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.

Studies on the mechanism of C3b inhibition of immune complex induced C1 activation

We had previously demonstrated that in normal human serum (NHS) nascent C3b inhibited C1 activation by immune complexes (IC). We have now investigated the mechanism of this feedback inhibition. For these studies, EA-IgG were added to solutions containing physiological concns of purified C1, C1-In, C2, C3 and C4. Mixtures were then incubated at 37° C for 30 min. Western blot and autoradiographic analyses revealed that almost half of the IgG molecules had become covalently linked to C3b in a 1:1 complex with the C3α' chain of C3b being bound to the heavy chain of IgG. IgG-C3b and free IgG were separated by ion exchange chromatography and immune complexes were formed with each. The consumption of complement in NHS by EA-IgG and EA-(IgG-C3b) were then compared. The results indicate that binding of C3b to IgG did not significantly inhibit the C1 activating potential of the IgG. Thus feedback inhibition is not due to the binding of C3b to IgG. An alternative mechanism was next explored. After incubation of EA-IgG with C1 through C3, EA were separated from supernantant fluid by centrifugation. It was determined that one-third to one-half of the IgG had been released from the erythrocytes. Release appears not to have been due to C3b binding to IgG, since the released IgG-C3b readily bind to fresh sheep erythrocyte (E), and since IgG that was free of C3b was also released from EA by complement, it is more likely that C3b binding to the E caused the dissociation of antibody. These results indicate that under physiological conditions, the C1 activating potential of an immune complex is greatly reduced as the result of the binding of nascent C3b to the antigen moiety of the IC, thereby causing the displacement of complement activating antibody. In addition to IgG, IgG-C3b is also released from the IC.

Hydrolysis of methotrexate-immunoglobulin conjugates by liver homogenates and characterization of catabolites.

Methotrexate (MTX) linked to antitumor antibodies inhibits tumor growth better than free MTX, free antibody, or MTX linked to normal rabbit IgG (NRG), in spite of the less effective inhibition of the target enzyme dihydrofolate reductase (DHFR) by conjugated MTX. In addition to the demonstrated higher uptake of MTX linked to antitumor antibodies (compared with the uptake of free MTX or nonspecific IgG conjugates), a contributory factor to the superior tumor inhibitory action of MTX-IgG conjugates may be the prolonged release of active drug from the internalized conjugate. Therefore, we have investigated whether an MTX-IgG conjugate could be hydrolyzed to release free MTX or fully active MTX-containing fragments after incubation with liver homogenates and have characterized the catabolites according to the presence of free MTX and their capacity to inhibit DHFR. Catabolism was optimal at pH 4.6, activated by dithiothreitol, and inhibited by antipain and N-alpha-p-tosyl-L-lysine chloromethyl ketone, thus implicating lysosomal enzymes. Liver homogenates produced an MTX-containing, low-molecular-weight fraction that was isolated by gel filtration. Further purification of this fraction by DEAE-cellulose chromatography gave two MTX-containing peaks, neither of which migrated as free MTX on thin-layer chromatography or inhibited DHFR more effectively than the parent conjugate. However, the presence of amino acid residues in these catabolites could contribute to their observed prolonged intracellular retention and superior antitumor action.

Chronic small airway disease in the horse: immunohistochemical evaluation of lungs with mild, moderate and severe lesions.

The peroxidase anti-peroxidase technique was used to demonstrate free and intracellular immunoglobulin (Ig) within the lungs of 23 horses with chronic small airway disease. Histologically, all the horses had chronic bronchiolitis; however, the lesions varied in degree from mild in eight horses, to moderate in nine horses and severe in six horses. Lungs from three horses which had no gross or histopathological lesions were used as controls. In comparison with control horses, horses with mild chronic bronchiolitis had increased numbers of Ig A-containing and non-immunoglobulin staining cells around the vasculature and bronchioles. As the severity of the lesions increased so did the number of IgA-, IgG(Fc)- and in several cases non-immunoglobulin staining cells around the vasculature, bronchioles and in the alveolar septa. In severely affected horses, large amounts of free IgG(Fc) were observed interstitially and in alveoli. In areas of mucosal epithelial hyperplasia and metaplasia large amounts of free IgA and IgG(Fc) were sometimes observed interepithelially in a pattern which differed from that in control horses.