Evaluation of nine different fixatives. 2. Preservation of IgG, IgA and secretory component in an artificial immunohistochemical test substrate

An artificial substrate was developed for quantitative testing of the ability of various fixatives to preserve the reactivity of IgG and IgA isotypes (gamma and alpha chains) and the secretory component (SC) of secretory IgA as model antigens. Polymerized normal rabbit serum was used as matrix and defined amounts (10-0.1 g/l) of antigen were incorporated into it by diffusion before fixation and paraffin embedding. The various fixatives comprised alcohol, routine formalin, glutaraldehyde(1%)-formalin, Baker's formol calcium, formol sublimate, acetic acid(2%)-formol saline, Bouin's fluid, Susa fixative, and carbodiimide. The detection sensitivity afforded by these fixatives was defined as the immunofluorescence staining end point. Compared to the reference value obtained with alcohol (gamma and alpha chains, 0.06 g/l of IgG and IgA; SC, 0.12 g/l of colostral IgA), an antigen concentration at least 8 times higher was necessary for detection with most of the cross-linking fixatives. Bouin's and Susa fixatives were peculiar in that they required more than 150 times higher antigen concentration for detection of IgG but only 3-8 times higher for IgA. The determined sensitivities were compared with the immunofluorescence performance results obtained on human tissues prepared with the same fixatives; excepting carbodiimide (which produced unacceptable autofluorescence of the substrate matrix) a remarkably good correlation was found with regard to IgG- and IgA-producing cells (especially of the former isotype) and secretory epithelium (IgA and SC). However, the latter result depended on pronase treatment of the tissue sections to unmask epithelial antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of nine different fixatives

Summary An artificial substrate was developed for quantitative testing of the ability of various fixatives to preserve the reactivity of IgG and IgA isotypes ( and chains) and the secretory component (SC) of secretory IgA as model antigens. Polymerized normal rabbit serum was used as matrix and defined amounts (10–0.1 g/l) of antigen were incorporated into it by diffusion before fixation and paraffin embedding. The various fixatives comprised alcohol, routine formalin, glutaraldehyde(1%)-formalin, Baker's formol calcium, formol sublimate, acetic acid(2%)-formol saline, Bouin's fluid, Susa fixative, and carbodiimide. The detection sensitivity afforded by these fixatives was defined as the immunofluorescence staining end point. Compared to the reference value obtained with alcohol ( and chains, 0.06 g/l of IgG and IgA; SC, 0.12 g/l of colostral IgA), an antigen concentration at least 8 times higher was necessary for detection with most of the cross-linking fixatives. Bouin's and Susa fixatives were peculiar in that they required more than 150 times higher antigen concentration for detection of IgG but only 3–8 times higher for IgA. The determined sensitivities were compared with the immunofluorescence performance results obtained on human tissues prepared with the same fixatives; excepting carbodiimide (which produced unacceptable autofluorescence of the substrate matrix) a remarkably good correlation was found with regard to IgG- and IgA-producing cells (especially of the former isotype) and secretory epithelium (IgA and SC). However, the latter result depended on pronase treatment of the tissue sections to unmask epithelial antigens. It was concluded that detection sensitivity as determined with this artifical substrate may give a good idea about the immuno-histochemical performance obtained on tissue prepared with the actual fixative; but the degree of antigenic masking in the various tissue compartments has to be taken into account if the comparisons are to be meaningful.This study was supported by the Norwegian Cancer Society, The Norwegian Research Council for Science and the Humanities, and Anders Jahre's Fund. The results were in part presented at the Symposium on Immunocytochemistry of Lymphomas, Micro 82 Conference, The Royal Microscopical Society, London, July 1982 (Histochem. J. 15:655–689, 1983)     

Double labeling of SIgG+ cells harboring a lymphotropic herpesvirus by avidin-biotin complex immunoperoxidase and immunogold-silver staining techniques

By reversing the usual order of double-staining procedures, we obtained optimal conditions for simultaneous detection of guinea pig lymphotropic herpesvirus (GPHLV) and surface immunoglobulin G-positive (SIgG+) cells in paraffin-embedded tissue sections. Of the different fixatives tested, Bouin's solution gave the best preservation of morphology and cell surface IgG. Double immunolabeling was best achieved when avidin-biotin complex immunoperoxidase (ABC-IP) staining was performed first for detection of intracellular viral antigen, followed by immunogold-silver staining (IGSS) for localization of cell surface IgG.     

Immunohistochemical demonstration of estrogen receptors (ER) in formalin-fixed, paraffin-embedded human breast cancer tissue by use of a monoclonal antibody to ER

We describe an immunohistochemical method using a monoclonal antibody to localize estrogen receptors (ER) in formalin-fixed, paraffin-embedded tissue. The avidin-biotin-peroxidase complex method was used, preceded by trypsin treatment to expose antigenic sites. In 111 breast cancer specimens studied simultaneously by a dextran-coated charcoal (DCC) assay and the paraffin section method, agreement on receptor status was found in 101 (91%) specimens. Quantitative staining features showed a high degree of correlation with the results of the steroid binding assay (r = 0.81). Studies on the influence of fixation on ER localization done in rabbit uteri showed that fixatives mainly composed of coagulating reagents (Carnoy's, Zenker's, Bouin's, Lilly's AAF, Helly's, ethanol) precluded ER staining, whereas cross-linking fixatives (formaldehyde, glutaraldehyde) preserved antigenic sites, although the immunoreactivity of the receptor was somewhat decreased. Studies on the effect of enzyme preincubation showed this to increase antigenic expression of ER in formaldehyde-fixed breast tumors and in formaldehyde-, glutaraldehyde-, and Zamboni-fixed rabbit uteri.     

IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.     

A human myeloma cell line that does not express immunoglobulin but yields a high frequency of antibody-secreting hybridomas

We selected an 8-azaguanine-resistant variant of a human myeloma cell line (RPMI 8226) by cloning the parental cells on a feeder layer of mouse spleen cells in the presence of increasing concentrations of 8-azaguanine. Culture media and cellfree extracts of both the parental and variant (8226 AR/NIP4-1) cell lines were assayed for production of immunoglobulin heavy and light chains by double immunodiffusion and for lambda-chain by radioimmunoassay. Secretion of free lambda-chain by the parental cell line was confirmed. In contrast, no immunoglobulin heavy or light chains were detected in culture medium of the variant cell line by either immunodiffusion or radioimmunoassay. No intracellular lambda-chain could be detected in the variant cells by radioimmunoassay of cellfree extracts or by immunofluorescence of fixed cells. Hybridomas were produced by fusion of 8226AR/NIP4-1 cells with lymphocytes from a mesenteric lymph node recovered at surgery from a hypertransfused renal transplant recipient. Twenty hybrid culture supernatants were assayed for immunoglobulin by double immunodiffusion, and 15 contained either IgG (lambda) or IgG (kappa). None produced IgM or IgA. An IgG (kappa)-producing hybridoma was shown by immunofluorescence not to express lambda-chain. A second fusion between the variant cell line and spleen cells from a renal transplant patient produced a stable hybridoma secreting IgM (lambda) antibody specific for the I antigen.     

Unlabeled antibody peroxidase-antiperoxidase method combined with direct immunofluorescence

Paired staining with the unlabeled antibody peroxidase-antiperoxidase (PAP) method and direct immunofluorescence (DIF) differentiated distinctly between gastrin- and somatostatin-producing cells in the human gastric antrum. Similar paired staining of complexed lambda and alpha chains in immunoglobulin (Ig)A myeloma cells, of kappa and free J chains in IgG myeloma cells, and of secretory IgA and its epithelial transport protein, the free secretory component (SC), in colonic crypt cells, demonstrated that PAP staining inhibits subsequent DIF staining of an antigenic determinant present on the same molecule as the antigen revealed by the brown color of diaminobenzidine (DAB) or present or an unassociated molecule in the same cell. A quenching effect of the DAB reaction product was noted for both fluorescein (green) and rhodamine (red) emissions. In addition, a blocking effect of the DAB deposits has been demonstrated and is assumed to be the principal methodological basis for the paired PAP-DIF staining approach omitting intermediate antibody elution, as well as for the more time-consuming sequential PAP staining with DAB substrate for the first and 4-chloro-1-naphthol (CN) for the second antigen. The quenching and blocking effects limit in practice the paired PAP-DIF method to the localization of antigens present in separate cells.     

Optimal fixation conditions for the immunoperoxidase identification of human J chain from tissue sections

Summary J chain can be used as a marker of plasmablasts and plasma cells at an earlier stage than intracellular immunoglobulin. Immunoperoxidase techniques were used to study optimal fixation conditions for the preservation of human J chain antigenicity in paraffin-embedded tissue sections. The most constantly positive staining for J chain combined with good morphological integrity was obtained with Bouin's fluid for 1.5 h at 20°C. All other fixatives studied showed less consistent staining results.Studies supported by the Sigrid Jesélius Foundation     

Immunoglobulin light and heavy chain isotypes in skin diseases: restricted distribution in bullous pemphigoid and linear IgA bullous dermatosis

To assess the heterogeneity of immunoglobulins involved in various skin diseases, direct and indirect immunofluorescence studies of skin biopsies and sera, respectively, for kappa and lambda light chains, were performed. The anti-basement membrane zone (anti-BMZ) antibodies of patients with bullous pemphigoid showed a predominance of kappa light chains, and patients with linear IgA bullous dermatosis showed a predominance of one light chain that was sometimes kappa and sometimes lambda. The bullous pemphigoid autoantibodies were then studied for IgG subclass distribution; a predominance of IgG4 was found. Although other explanations are possible, the light chain restriction in bullous pemphigoid most likely reflects heavy chain restriction and preferential association of heavy and light chain isotypes. The basis of the heavy chain restriction is not apparent. The light chain restriction in linear IgA bullous dermatosis may represent a restricted idiotypic repertoire.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessing the efficiency of free light chain assay in monitoring patients with multiple myeloma before and after autologous stem cell transplantation along with serum protein electrophoresis and serum protein immunofixation

Monoclonal gammopathies are a group of disorders, referred to as paraproteinaemias, dysproteinaemias or immunoglobulinopathies, associated with monoclonal proliferation of plasma cells. Monoclonal immunoglobulin secreted by these cells is an indicator of clonal proliferation. The aim of this study is to analyze the efficiency of three methods: serum protein electrophoresis (SPE), serum protein immunofixation (IFE) and FLC (free light chain) assay for the diagnosis and monitoring of the tumor burden in multiple myeloma. In this study we have presented the dynamic evolution of 7 patients with intact immunoglobulin multiple myeloma (IIMM) (2 IgG, kapa; 3 IgG, lambda; 1 IgA, kappa; 1 IgA, lambda) and 2 patients with light chain multiple myeloma before and after autologous peripheral blood stem cell transplantation (PBSCT). All 7 patients fulfilled the four criteria for the diagnosis of IIMM: bone marrow plasma cells exceeding 20%, lytic bone lesions, identification and quantification of M protein by scanning densitometry of electrophoresis gels, IFE (immunofixation protein electrophoresis) confirmed and typed the M protein. All patients had been given cytotoxic chemotherapy (VAD or VELCADE) before autologous (PBSCT). In two of the patients with IIMM both SPE and kappa/lambda ratio fell towards normal range after autologous PBSC and both reported a relapse of the disease after 23 months and 19 months respectively. SPE could not normalize after chemotherapy and transplantation in three patients with IIMM, the kappa/lambda ratio being the only marker used to monitor the tumor kill. In one patient the kappa/lambda ratio could not normalize even after PBSCT still indicating the presence of plasma cell disorder at the time when IFE was still negative. 16 months after PBSCT both SPE and FLC indicated a relapse of the disease. Classical SPE failed to demonstrate the presence of M-protein in light chain multiple myeloma, the diagnosis being established by using IFE and the FLC assay. Because IFE is a qualitative method and its interpretation may be sometimes subjective, FLC was the only method used to follow the disease course. The measurement of kappa/lambda ratio proved to be more sensitive than SPE, IFE and the levels of free light chains kappa or lambda individually indicating whether the treatment is effective or not.     

Production of immunoadsorbents based on cellulose carbonates and their application to the isolation of specific immunoglobulin light chains

The purification of rabbit immunoglobulin molecules expressing kappa (κ) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (κ) IgG. Cellulose trans-2.3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preservng immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit a1a3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline celulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (κ) IgG, thus separating immunoglobulin, expressing kappa light chain, from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations.     

Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.     

Dissociation of κ- and λ-chains from reduced human immunoglobulins

1. The light chains of human immunoglobulin (Ig) exist in two forms, kappa (type K) and lambda (type L). The two types of chains can be partially separated by taking advantage of the fact that lambda-chains, for the most part, dissociate from reduced Ig at higher pH than do the kappa-chains. The same difference in dissociation of type K and L chains was observed with myeloma IgG and IgA proteins, but not with pathological IgM proteins. 2. When analysed in urea-glycine starch gels, pH7, both kappa- and lambda-chains show ten electrophoretic bands having the same mobilities as those of the whole light-chain subfractions. Normal kappa- and lambda-chains show similar differences in overall amino acid composition to those previously found with myeloma kappa- and lambda-chains and type K and L Bence-Jones proteins.     

Chicken secretory immunoglobulin: chemical and immunological characterization of chicken IgA.

1. Chicken IgA purified from biliary fluids was chemically and immunologically characterized. 2. Chicken IgA was determined to be the only immunoglobulin class present in bile. Gel filtration studies reveal polymeric IgA e.g. 17-19S. 3. Antigenically, chicken IgA is distinct from chicken IgG, and IgM. 4. Chicken IgA does not show antigenic homology to human IgA. 5. SDS poly-acrylamide gel electrophoresis revealed IgA to possess heavy chains of 60,000 and light chains of 24,000 mol. wt, respectively. 6. Peptide mapping of tryptic digests of chicken alpha chains reveals approximately 35 peptides. The peptide map pattern is distinct from chicken gamma chains.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Sensitivity and efficiency of four immunohistochemical methods as defined by staining of artificial sections

Direct (DIF) and indirect (IIF) immunofluorescence, indirect immunoperoxidase conjugate (IPC) and unlabelled antibody peroxidase antiperoxidase (PAP) staining was performed on sections of artificial substrate containing different concentrations of human immunoglobulin (Ig)A or IgG. Detection sensitivity, in terms of the lowest amount of discernible antigen, was evaluated by direct microscopy and by microphotometry. Staining efficiency (signal-to-noise ratio) was evaluated by microphotometry. Only minor differences in antigen detection sensitivity were found when IPC and PAP were compared with DIF and IIF under appropriate conditions. The sensitivity of DIF was only marginally improved by raised conjugate concentration and prolonged incubation time. Microphotometry of DIF on ethanol-fixed IgA substrate revealed that the staining intensity increased proportionally with the antigen concentration whereas on formaldehyde-fixed substrate a progressive masking of the antigen was indicated which, however, could be overcome by applying raised conjugate concentration and prolonged incubation time. Such antigenic self masking was of relatively little importance to IPC and PAP staining, probably because of the inherent amplification in these methods. An additional masking effect due to extraneous protein was revealed by DIF when ethanol-fixed sections had been soaked in bovine serum albumin and postfixed with formaldehyde; unmasking was achieved by proteolytic treatment of the sections.     

Development of monoclonal IgA and an apparent IgG in a patient with macroglobulinemia: sharing of individually specific antigenic determinants among IgM, IgA, and IgG.

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.