Ig Fractions Research Articles

Sprouting inducing activity of human sera: low capacity in sera from patients with cranial polyneuropathy.

Cranial idiopathic acute peripheral facial nerve paralysis (Bell palsy) has been used as a model disease in the present study mainly to investigate trophic capacity of serum components. The ability to induce sprouting from chick embryo dorsal root ganglia was used as the bioassay for trophic activity. Sera from normal patients induce neuritic outgrowth from dorsal root ganglia of chick embryo in the absence of any exogenous factor. Sprouting, to a very limited extent or none, has been induced by 80% of the sera derived from the Bell palsy patients. Further analysis of the biochemical nature of the reduced activity has revealed that in sera of Bell palsy patients the substances that are fractionated within the Ig fraction are responsible for the reduced capacity to induce sprouting without loss of supportive molecules.

Differentiation of the humoral immune response in inflammatory diseases of the central nervous system.

The three main immunoglobulin classes obey the basic principles of passive protein transfer at the blood-CSF barrier and the serum-derived portions could therefore be quantified with the help of the permeability marker albumin. The Ig fractions secreted into the CSF by sessile plasma cell clones have been determined in various inflammatory diseases of the central nervous system. The humoral immune response in multiple sclerosis and chronic encephalitis of unknown cause was dominated by IgG antibodies. In most other inflammatory diseases IgA and IgM were concomitantly synthesized, e.g. in neurosyphilis and meningoencephalitis caused by viruses of the herpes group. In tick-borne meningopolyneuritis Bannwarth, only IgM and in bacterial meningitis only IgA may be produced locally. The detection of a secretory immunoglobulin fraction in the CSF may be the sole laboratory parameter in chronic inflammatory processes of the nervous system.

The involvement of the pentose shunt in thyroid metabolism after stimulation with TSH or with immunoglobulins from patients with thyroid disease. II. The reoxidation of NADPH and stimulation of hormone synthesis.

Reducing equivalents derived from the tissue re-oxidation of NADPH (NADPH-diaphorase) have been implicated in the peroxidation that is involved in the organification of iodine in the production of thyroid hormones. Immunoglobulin (Ig) fractions from patients with thyroid diseases and from normal controls, in a standard dose of 125 micrograms/ml and 0.3 microunits/ml thyrotrophin (TSH) were incubated with segments of guinea-pig thyroid gland maintained in vitro. A quantitative cytochemical study was made on how these fractions influenced the enzyme activity. A good correlation was found between the ability of such Ig fractions to stimulate the NADPH-diaphorase activity and (1) the degree of hyperthyroidism in the patients and (2) the amount of T3 secreted by the thyroid segments in vitro.

Osteosarcoma Patients: Isolation of Serum Antibodies by Affinity Chromatography<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Antibodies specific for membrane-associated antigens of human osteosarcoma cells were isolated from sera of 12 patients with osteosarcoma (OS). Affinity columns were prepared by coupling purified membrane antigens from cultured human OS cell lines (TE-85 or LM) to CBrN-activated Sepharose 4B. The antigens were prepared by discontinuous sucrose gradient ultracentrifugation, papain digestion, and DEAE column chromatography. Diluted serum was passed over the affinity columns, and the adsorbed proteins were eluted with 2.5 M MgCl2 (pH 6.5). Immunodiffusion, indirect immunofluorescence, and complement fixation were used to assay antibody activity in the eluate. Specific anti-OS activity was found in the immunoglobulin (Ig) fraction isolated from the sera of the 12 OS patients, as confirmed by blocking experiments. No anti-OS antibody activity was found in sera from healthy individuals or patients with breast carcinoma, clear cell liposarcoma, or leukemia in this study. The anti-OS activity of the isolated Ig from OS patients was abolished after absorption with cultured human OS cells from lines LM, TE-85, or G292 but not after absorption with cells from lines WI-38 (embryonic lung), TE-32 (rhabdomyosarcoma), CAMA-1 or SW527 (breast carcinoma), or M-14 (melanoma). Absorption with rabbit antihuman IgG but not with rabbit antihuman IgM immunobeads completely eliminated the antibody activity.

Natural antibodies against tubulin, actin, myoglobin, thyroglobulin, fetuin, albumin and transferrin are present in normal human sera and monoclonal immunoglobulins from multiple myeloma and Waldenström's macroglobulinemia may express similar antibody specificities

Sera from a pool of 800 healthy donors and from 3 individual healthy donors were passed through tubulin, actin, thyroglobulin, myoglobin, fetuin, transferrin and albumin immunoadsorbents. Proteins were eluted in all the immunosorbents and were found to be essentially composed of the three major Ig classes and albumin. The isolated Ig fractions were shown to react specifically, via their Fab fragment with the antigens and were specifically inhibited by them. These results strongly suggest that natural antibodies against the seven antigens are present in normal human serum, and probably against a high variety of self antigens. These results prompted us to search in the sera of patients with monoclonal gammapathies, paraproteins having natural antibody-like function. Among the 62 sera studied 3 were shown to react with actin and 1 with tubulin. Most important, these 4 monoclonal immunoglobulins exhibited similar specificities to that found with natural antibodies. This seems to indicate, that at least for some patients, the monoclonal immunoglobulins produced may represent the expansion of a clone producing a natural antibody.

Mechanism of increased antitumor immunity by a synthetic polymer

The antitumor effect of a synthetic polymer NED 137 was assessed and the mechanism of its action was explored. The polymer produces enhanced immunity to tumor and heterologous erythrocytes for at least 60 days after administration. If it was administered repeatedly at six weekly intervals to animals—postexcision of primary or recurrent tumor for four doses—the animals remained free of tumor for an indefinite period of time. The antitumor effect is related to a serum factor which is specific to the tumor with which the animal was in contact at the time of administration of the polymer. It was present in the Ig fraction of the serum. No acute or chronic toxicity was observed in animals. A Phase I clinical trial with metastatic gastrointestinal cancer was initiated in February 1979. The results with the first 94 patients are reported.

Sex-specific glycoproteins in Chlamydomonas flagella an immunological study

To identify mating type-specific glycoproteins associated with the flagellar membrane of Chlamydomonas eugametos, which could be involved in sexual agglutination, antibodies were raised in rabbits against purified gamete flagella of either mating type. The immunoglobulin (Ig) fractions exhibited partial mating-type specificity in agglutinating gametes, in the indirect immunofluorescence test and in the crossed immunoelectrophoresis test. This specificity was strongly enhanced by absorbing the fractions with flagella of the opposite mating type. Absorbed Ig fractions produced a single precipitation line with Triton extracts of gamete flagella in the crossed immunoelectrophoresis technique. On polyacrylamide gel electrophoresis this line appeared to contain two flagellar glycoprotein fractions, PAS 1 and PAS 4. Polyacrylamide gels of flagellar extracts incubated with these Ig fractions, followed by staining with peroxidase-anti-rabbit Ig resulted in the staining of only the PAS 1 and PAS 4 bands, which confirms that these components of the flagellar membrane are mating type-specific antigens.

Antigen receptors on major histocompatibility complex-restricted T lymphocytes. I. Preparation and characterization of syngeneic antisera against trinitrophenyl-activated T cell blasts and demonstration of their specificity for idiotypes on cytotoxic T lymphocytes.

This paper describes the specificity of AKR anti-(a) [AKR anti-trinitrophenylated AKR (AKR-TNP)] [AKRa (AKRaAKR TNP)] antisera raised in syngeneic AKR mice against AKRaAKR-TNP cell populations enriched for H-2-restricted aTNP cytotoxic lymphocytes (CTL) by blast-cell isolation. The activity of the antisera resided in the Ig fraction. All antisera were shown to reproducibly react with AKRaAKR-TNP-CTL-containing cell populations in indirect immunofluorescence and all removed the major fraction of CTL in complement-dependent lysis causing a considerable depression of cell-mediated lympholysis. The antisera were nonreactive with alloreactive AKRaC57BL/6 CTL and other H-2-restricted AKR CTL against fluorescein-isothiocyanate-conjugated AKR-target cells. It could be excluded that the antisera contained contaminating antibodies against TNP, TNP-neoantigenic determinants (NAD), or processed CTL-receptor-bound TNP-NAD, thus demonstrating specificity for determinants on T cell receptors of AKRaAKR-TNP CTL. These receptors were produced by the CTL themselves. These observations are interpreted to suggest that AKRa (AKRaAKR-TNP) antisera contain anti-idiotypic antibodies directed against specificity-associated determinants (idiotypes) on T cell receptors of H-2-restricted AKRaAKR-TNP CTL. The antisera provide a new tool to study the genetic control of idiotype expression on H-2-restricted CTL, the biochemistry of T cell receptors, and the regulation of the generation of H-2 restricted CTL on the idiotype level.

Binding of anti‐tumor immunoglobulins and their daunomycin conjugates to the tumor and its metastases. In vitro and in vivo studies with lewis lung carcinoma

The present study was undertaken in order to evaluate the possibility of using anti-tumor antibodies as specific carriers of chemotherapeutic drugs to tumor metastases. Xenogeneic and syngeneic antisera were prepared against a metastasizing C57BL tumor, the Lewis lung carcinoma (3LL). The binding of absorbed xenogeneic and syngeneic anti-3LL sera or their Ig fractions to 3LL tumor cells was assayed by direct and indirect methods. Antisera prepared against tumor cells from a local growth reacted to a similar extent with those obtained from the lung metastases. Radioiodinated immunoglobulins from syngeneic antisera injected into mice bearing metastases localized preferentially in metastasis-bearing lungs as compared to several other organs tested. No such preferential uptake was observed either in mice bearing metastases injected with iodinated normal Ig or in normal mice injected with iodinated anti-3LL Ig. This relative accumulation in the metastasis-bearing lungs was observed 3-4 days after the inoculation, when the whole Ig fraction was used, whereas a specific antibody-enriched preparation localized as early as 24 h after injection. Daunomycin--anti-3LL-Ig conjugates were effective inhibitors of tumor cells from both local and the metastatic growths. They were more active than either the free drug or drug conjugates of normal Ig, in in vitro assays.

Expression of Ia Antigen on Adult and Neonatal B Lymphocytes Responsive to Thymus-Independent Antigens

Abstract Spleen cells from normal adult or neonatal mice were treated with anti-Ia and C and in vitro responses to two thymus-independent (TI) antigens, trinitrophenyl-Brucella abortus (TNP-BA) and TNP-Ficoll were studied. TNP-BA, a TI antigen that is known to stimulate neonatal B cells, was used in these experiments with the intent of stimulating an early appearing population of B lymphocytes, which might be anticipated to express surface immunoglobulin (sIg+) but not Ia. Cells treated with C and antisera with activity to I region determinants coded for by genes in I-A, I-E, or I-E/C were unable to mount significant responses to TNP-BA or TNP-Ficoll. These diminished responses could not be reconstituted with x-irradiated cells as a source of macrophages. When neonatal spleen cells, which allegedly possess a high fraction of Ig+ Ia- cells, were treated with A.TH anti-A.TL, there was a marked impairment in their ability to respond in vitro to TNP-BA. Thus, even primitive TI responses depend largely on cells that have already expressed Ia. To assess whether spleen cells treated with anti-Ia and C contained small numbers of immunocompetent cells that might not have been detected in in vitro systems employed, we transferred antisera and C-treated (CBA/N × DBA/2)F1 female spleen cells to B cell defective (CBA/N × DBA/2)F1 male mice and challenged the recipients with TNP-Ficoll 7 or 14 days later. Anti-TNP PFC were measured 5 days after antigen challenge. There was no detectable anti-TNP response seen in F1 male mice reconstituted with anti-Ia + C-treated cells whereas cells treated only with C were able to transfer significant responses. Responses to TNP-Ficoll were shown to be more sensitive to anti-Ia and C treatment than were responses to TNP-BA suggesting that the subset of B cells responding to TI-2 antigens may bear a higher density of surface Ia than does the B cell subset responding to TI-1 antigens.

Simple purification of goat IgG1 and IgG2 subclasses by chromatography on protein A-sepharose at various pH

Two ml of goat serum are applied on a 2 × 20 cm column of protein A-Sepharose at pH 9.1. Elution at the same pH yields two protein peaks, the second being highly purified IgG1. A minor Ig fraction, eluted at pH 6.5, is discarded. Pure IgG2 is then eluted at pH 5.9. Yields of both subclasses average 70 %.

Radioimmune analysis of an unexpected mouse allotype.

We have used a radioimmune assay to confirm our earlier findings of an unexpected immunoglobulin allotype in Igb-congenic BALB/c mice. Although these mice were bred to exclude the IgG2a allotype of BALB/c (Ig-la), an Ig-la-like antigen was detected in the 7S Ig fraction of two (of five) pooled serum samples, it represented 0.1--0.3% of the total 7S protein and was indistinguishable from a reference Ig-la. The detection of putative Ig-la in Igb-congenic mice is inconsistent with the notion that allotypes are products of allelic structural genes. It appears rather that expression of Ig-la is controlled by allelic regulator genes and that its low and transient production in Igb-congenic mice results from incomplete negative regulation.

Tumour antigens and alloantigens. I. Relation of a rat tumour-specific antigen with normal alloantigens of the host strain.

AbstractIt has been shown that tumour‐specific antigen from a chemically‐induced rat hepatoma is capable of binding to immunoadsorbent columns made from the Ig fractions of antisera raised in allogeneic animals against a variety of normal tissues. This reactivity is observed with antisera directed against normal syngeneic liver, spleen and lymph‐node cells but it is not detected in normal sera, F, hybrid sera or an antiserum directed against syngeneic erythrocytes. This phenomenon is specific since soluble extracts of normal syngeneic liver, but not of normal altogeneic liver, are capable of inhibiting the binding of hepatoma‐specific antigen to an immunoadsorbent column of Ig from an allogeneic anti‐normal‐liver antiserum. Syngeneic and allogeneic rats were immunized with immune complexes made from xenogeneic sera, alloantisera or syngeneic immune serum and soluble extracts of normal tissue and affinity‐chromatography‐purified hepatoma‐specific antigen. The resulting sera were examined by means of the membrane immunofluorescence test for reactivity against a panel of syngeneic and allogeneic cells. Xeno‐antisera directed against normal syngeneic tissue precipitated with purified hepatoma antigen and this material generated tumour‐specific antibody following immunization into syngeneic rats. The phenomenon of cross‐reactivity of one chemically‐induced rat hepatoma‐specific antigen with normal syngeneic alloantigens is not a unique phenomenon as preliminary results indicate that two other immunologically distinct hepatomas show similar characteristics.

Isoelectric focusing of CSF proteins in known or probable infectious neurological diseases and the Guillain-Barré syndrome

The CSF and serum proteins of 120 patients with known or probable infectious neurological diseases or the Guillain-Barré syndrome were examined with thin-layer IEF. All but two of these patients exhibited one or combinations of different CSF-protein aberrations in the acidic and alkaline range. Aberrant non-Ig fractions (including transferrin, the tau-fraction and gammatrace protein) were found in frequencies varying between 4 and 48%. CSF Ig components of restricted heterogeneity, i.e. oligoclonal bands and/or regional increases of gammaglobulins, were more frequent in patients with (meningo-)encephalitic or (meningo-)myelitic/radiculitic disorders (respectively 69 and 48%) than in subjects with meningitis or Guillain-Barré syndrome (17%). The occurrence of such Ig abnormalities was higher in subacute or chronic than in acute disease and in subjects examined ⩾ 4 weeks after the onset rather than earlier. Ig-band spectra with marked anodal extensions were found predominantly in (meningo-)encephalitic disorders with infratentorial symptoms. Age and sex were not found to influence the occurrence of abnormal Ig fractions. Such components could be detected in spite of pronounced blood-CSF barrier defects.

CSF protein examinations with thin-layer isoelectric focusing in multiple sclerosis

Thin-layer IEF, due to its extremely high resolving capacity, has been found to be quite valuable for CSF protein examinations, one important advantage of the technique being its excellent capacity for separation of immunoglobulins. The CSF and serum proteins of 230 patients with clinically verified or probable MS and 20 subjects with optic neuritis were examined with thin-layer IEF and the findings were compared with clinical data and results of other CSF examinations. All but 3 of the MS patients and about two thirds of the subjects with optic neuritis exhibited one or combinations of different CSF protein aberrations in the acidic and alkaline range. Oligoclonal bands and/or regional increases of Ig fractions, changes compatible with intrathecal Ig synthesis, were detected in respectively 95 and 80% of patients with clinically verified and probable MS and 30% of subjects with optic neuritis. Other aberrant CSF protein fractions (including transferrin, the taufraction and gamma-trace protein) were found in about half of the cases; some of these fractions had the highest occurrence in patients with the most extensive Ig abnormalities. The diverse CSF protein aberrations seemed to be influenced by the duration and course of the disorder as well as the probable sites of lesions; further factors might be the release of decomposition products from destroyed tissues, the genetically determined reactivity of the individual and the presence of possible agents.

Tumor-Associated Antigens of Rat Moloney Sarcoma Cells

Abstract Rat Moloney sarcoma cells (MST) were pulsed with 35S-l-methionine for 10 and 60 min and lysed by vortexing in 0.5% deoxycholate, 0.5% NP40, 0.02 M Tris, 0.05 M NaCl, pH 7.5, for 30 sec. The lysate was centrifuged at 16,300 × G for 10 min and the supernatant was co-precipitated with Ig fractions of normal BN serum, normal Lewis serum, BN antiserum to Moloney sarcoma cells (BNaMST), BN antiserum to tumor-associated antigens (BNaTAA), BN antiserum to murine leukemia virus (BNaMuLV), BN antiserum to p30 (BNap30), BN antiserum to gp70 (BNagp70), Lewis antiserum to BN (LeaBN), and BN antiserum to BC5 tumor (BNaBC5). With BNaTAA and BNaMST, a cytoplasmic TAA with m.w. 85,000 was detected. In addition, BNaTAA detected three other species of cytoplasmic TAA with m.w. 220,000, 170,000, and 39,000.

ANTIBODIES TO SYNTHETIC POLYRIBONUCLEOTIDES IN SPOUSES OF PATIENTS WITH INFLAMMATORY BOWEL DISEASE

Serum from patients with inflammatory bowel disease (I.B.D.) and their unaffected spouses bound significantly more synthetic single-stranded (poly·rA) and double-stranded (poly·rA·poly·rU) R.N.A. but not KB cell D.N.A. than did serum from age-matched controls and their spouses. Binding activity resided in Ig fractions and was predominantly of IgM class. A significant correlation was observed between the amount of double-stranded but not single-stranded R.N.A. bound by serum from patients and from their respective spouses. These findings provide indirect support for the presence of R.N.A. viruses in patients with I.B.D. and the transmission of such agents to their close personal contacts.

Characterization of Murine Colony-Forming B Cells

Abstract With appropriate culture conditions, numbers of proliferating foci are a linear function of the number of B cells placed in mitogen-stimulated semisolid cultures, and colony formation is not obviously suppressed or enhanced by T cells. This made it possible to determine the fraction of Ig+ B cells in different tissues and stages of development which was capable of colony formation. No evidence was obtained that these functional cells were enriched among either mature or immature B cell populations. Cytolysis with A.TH anti-A.TL serum and C before culture was used to study expression of Ia antigens on colony-forming cells. By this criterion, multipotential stem cells, granulocyte-macrophage progenitors, and fetal colony-forming B cells were Ia negative, whereas all clonable B cells in adult tissues were Ia positive. Colony formation by adult lymph node cells was inhibited by an average of 79% by the addition of rabbit anti-mouse IgD to the cultures, and extremely small quantities of antiserum were required. Adult spleen and bone marrow contained a higher percentage of antiserum-resistant and presumably δ- B cells, and clonable B cells in fetal tissues were not significantly inhibited. An additional subpopulation of late appearing B cells whose proliferation was resistant to anti-Ig antibodies was identified with a particular set of culture conditions. Colony-forming cells were also heterogeneous in their resistance to preincubation with anti-immunoglobulin antibodies. Under conditions that did not significantly affect adult splenic B cells, clonable cells in fetal and neonatal tissues were substantially inactivated. It appears that colony-forming B cells comprise an equivalent proportion of the B cells in different sites and stages of development and include cells that differ in maturity by the criteria employed here. However, they do not seem to be representative of B cells as a whole, and when considered in relationship to the defect in CBA/N mice, the possibility emerges that the cloning technique preferentially detects a lineage of functionally specialized B cells.

Use of antibody-coated red cells for the sensitive detection of antigen and in rosette tests for cells bearing surface immunoglobulins

Conditions for an improved chromic chloride method for the attachment of antibody to red cells are described. The method, which is applicable to whole Ig fractions as well as affinity-purified antibodies, is reproducible and has a sensitivity for detection of antigen in the nanogram range. The coated cells may be used in a rosette assay for the detection of cell surface-bound antigens.

Cellular and Humoral Immunity in Non-Hodgkin’s Lymphoma: Correlation of Immunodeficiencies with Clinicopathologic Factors

Immune function was evaluated in 28 non-Hodgkin's lymphoma patients in an attempt to correlate the occurrence of immunodeficiency with the prognostic clinicopathologic factors, lymph-node histology, and clinical stage of disease. Anergy to a battery of recall antigens occurred infrequently (4/28) and only in patients who had Stage IV disease (4/8) (p = less than .004), but did not correlate with lymph-node histology. In contrast to anergy, cellular immunodeficiencies were often detected by lack of response to keyhole limpet hemocyanin immunization in patients regardless of stage. Reductions in at least two of three Ig fractions were found in a third of the patients, with, again, a significantly greater incidence in Stage IV patients (p = less than .005). No significant correlation with histologic type was possible. The response to phytohemagglutinin in vitro was reduced in the patients, but this was of no correlative value.