RESUMEN
The synthesis and secretion of immunoglobulin induced in cultured B-lineage cells is of interest for several reasons: (i) analysing the B-cell repertoire, (ii) recall of immunological activity retained in the circulating lymphocyte population, and (iii) study of factors needed for clonal expansion, immunoglobulin class switching, IgV-region mutation and maturation of cells to Ig secretion. Methods available are outlined and alternative procedures for cell separation and purification, helper cell provision and Ab/Ig assay systems are discussed. The aim is to provide practical guidance for those who intend to begin work in what is a vitally important, but experimentally difficult, area. There are a bewildering number of methods described in innumerable publications, old and new. The review provides a personal assessment of the present state of knowledge and prospects for improvements when all the new observations relating to cell-cell interactions and cytokines are integrated into existing technologies. The survey is chiefly concerned with physiologically based procedures, but artificial auxiliary methods are also briefly mentioned.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulinas/biosíntesis , Animales , Linfocitos B/citología , Biomarcadores , Diferenciación Celular , División Celular , Células Cultivadas , Humanos , Cambio de Clase de Inmunoglobulina , Región Variable de Inmunoglobulina , Activación de LinfocitosRESUMEN
By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L-cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR-LON-HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130kD found by SDS-PAGE analysis. This may be partly accounted for by the non-globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.
Asunto(s)
Receptores de Complemento 3d/sangre , Animales , Células Dendríticas/química , Humanos , Ratones , Receptores de Complemento 3d/análisis , Células Tumorales CultivadasRESUMEN
The immunotoxin BU12-SAPORIN was constructed by covalently coupling the single-chain ribosome-inactivating protein saporin to the anti-CD19 monoclonal antibody BU12 via a disulphide linker using the heterobifunctional reagent SPDP. The immunoreactivity and specificity of BU12-SAPORIN was identical to that of unmodified native BU12 antibody. BU12-SAPORIN was selectively cytotoxic in vitro in a dose-dependent manner for the CD19+ human common acute lymphoblastic leukaemia (cALL) cell line NALM-6 but exhibited no toxicity for the CD19- T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2. The survival of severe combined immunodeficient (SCID) mice with disseminated NALM-6 leukaemia was significantly prolonged compared with sham-treated control animals by a course of therapy with BU12-SAPORIN but not with the irrelevant anti-CD7 immunotoxin HB2-SAPORIN. BU12-SAPORIN had no therapeutic effect in SCID mice with disseminated CD19- HSB-2 leukaemia. These preclinical studies have clearly demonstrated the selective cytotoxicity of BU12-SAPORIN for CD19+ target cells both in vitro and in vivo. This, taken together with the lack of expression of the CD19 molecule by any normal life-sustaining tissue and its ubiquitous and homogeneous expression by the majority of cALL and B-NHL cells, provides the rationale for undertaking a phase I trial of systemic therapy with BU12-SAPORIN.
Asunto(s)
Antineoplásicos/farmacología , Inmunotoxinas/farmacología , Leucemia de Células B/tratamiento farmacológico , N-Glicosil Hidrolasas , Proteínas de Plantas/farmacología , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/farmacología , Antígenos CD19/inmunología , Antígenos de Neoplasias/inmunología , Antineoplásicos/análisis , Antineoplásicos Fitogénicos/análisis , Antineoplásicos Fitogénicos/farmacología , Linfocitos B/metabolismo , Linfoma de Burkitt/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Inmunotoxinas/análisis , Leucemia de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones SCID , Proteínas de Neoplasias/biosíntesis , Trasplante de Neoplasias , Proteínas de Plantas/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Dodecil Sulfato de SodioRESUMEN
Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti-IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed.
Asunto(s)
Anticuerpos/farmacología , Linfocitos B/metabolismo , Inmunoglobulinas/biosíntesis , Interleucina-10/farmacología , Interleucina-2/farmacología , Activación de Linfocitos/fisiología , Linfocitos T/fisiología , Animales , Anticuerpos/fisiología , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Complejo CD3/inmunología , Antígenos CD40 , Comunicación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Humanos , Inmunoglobulina D/inmunología , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/inmunología , Interleucina-4/farmacología , Ratones , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Antigens such as CD21 and CD23, which express only one copy of an epitope require two monoclonal antibodies (mAbs) for their detection and estimation. This requirement is exploited in two ways in a technique based on the chromic chloride haemagglutination test. For simple titration of antigen two portions of red cells each coated with one of a pair of synergising mAbs are used in a 1:1 combination. For testing the antigenic specificity of a mAb and assessing its region of epitope binding, the mAb under test is serially diluted in fluid containing a standard amount of antigen and red cells are added to which have been attached a different mAb. If the red cell-bound mAb recognises a determinant topographically distinct from that of the soluble mAb, red cell agglutination to high titre occurs. In titrations of ascitic fluid containing approximately 1 mg/ml mAb, titres of log2(9) to log2(16) were recorded from a starting dilution of 1 in 200. Hence the test is very sensitive and only minute amounts of a mAb are required for testing. The same test system can be used for assessing the relative display of epitopes on antigen obtained from different sources, e.g., culture supernates and body fluids. The method is of general applicability to monomeric antigens and its use is illustrated by analysis of CD21 and CD23 antigens and antibodies.
Asunto(s)
Epítopos/inmunología , Receptores de Complemento 3d/inmunología , Receptores de IgE/inmunología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Linfocitos B/inmunología , Sitios de Unión , Línea Celular , Complemento C3d/inmunología , Eritrocitos/inmunología , Pruebas de Hemaglutinación , Herpesvirus Humano 4/inmunología , Humanos , Ratones , Proteínas/inmunología , Ovinos , SolubilidadRESUMEN
Efficient immunoglobulin (Ig) production was induced when human B cells were cultured with autologous T cells activated by immobilized CD3 mAb in cultures supplemented with IL-2. Negatively purified B cells or B cells positively selected with mAb to CD19, CD21 or CD72 surface antigens produced IgM, IgG and IgA, whereas B cells selected for surface IgD or IgM produced predominantly IgM indicating that little or no isotype switching was occurring. Results are compared with reports describing high levels of mu to gamma and mu to alpha switching in single B cell systems. The limited proliferation of B cells in our culture system may account for the difference. When untreated T and B cells were cultured together in the presence of immobilized CD3 mAb, B cell numbers peaked at 6-10 days whereas T cells continued to proliferate maximally. All 52 T cell clones tested induced the production of IgM, IgG and IgA from unselected or CD19 selected B cells, but efficiency of production of Ig overall and of the different isotypes varied with different T clones. All T clones which induced high IgM, IgG and IgA production induced IgE production too, but some less active T clones also induced IgE production under non-switching conditions indicating that direct contact with activated T clone cells efficiently induces IgE as well as IgG and IgA production from B cells already expressing these isotypes. Less Ig was produced with optimal numbers of untreated T clone cells than with X-irradiated cells, confirming that proliferating T cells can inhibit as well as activate Ig production from B cells.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Cambio de Clase de Inmunoglobulina/fisiología , Inmunoglobulinas/biosíntesis , Subgrupos de Linfocitos T/inmunología , Animales , Células Clonales , Eritrocitos/inmunología , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Inmunoglobulinas/sangre , Ovinos/inmunologíaRESUMEN
It is confirmed that large amounts of IgM, IgG, and IgA are produced when human B cells are cultured with T cells activated by immobilized CD3 antibody (CD3 system). IL-2 was essential; lower levels of Ig production with different isotype ratios were obtained if IL-4 or IL-6 replaced IL-2. Depletion of sIgG+ or sIgA+ cells from the B population to be cultured markedly reduced production of IgG or IgA. Cultures of B cells selected with the pan-B markers CD19, CD72, or CD21 contained similar levels of Ig of all three isotypes, whereas B cells selected for sIgM or sIgD expression produced IgM but very little IgG or IgA indicating that little isotype switching was occurring. Production of IgG or IgA from cells expressing these isotypes was more efficient than production of IgM from IgM+IgD+ cells. These results are considered in the light of the demonstration by others of the production of multiple isotypes from single sIgM+-selected B cells. Cloned human T cells from a single donor induced production of all three isotypes, but the proportions varied indicating that the potent T-B cell interactions inducing B cell activation may override and conceal the operation of isotype specific cell interactions. Some T clones used at an optimal dose were as effective untreated as X-irradiated, whereas with other clones maximum Ig production was not achieved without irradiation.
Asunto(s)
Linfocitos B/inmunología , Comunicación Celular , Cambio de Clase de Inmunoglobulina , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Células Cultivadas , Células Clonales , Humanos , Inmunoglobulinas/biosíntesis , Interleucina-2/farmacología , Ratones , Linfocitos T Colaboradores-Inductores/efectos de la radiaciónRESUMEN
A soluble form of complement receptor number 2 (sCR2) found in human serum closely resembles that produced in culture by B lymphoblastoid cells. Epitope analysis with a panel of CD21 monoclonal antibodies revealed only minor differences between antigen from the two sources. Purified sCR2 from both sources bound to C3dg prepared from human or mouse serum and to u.v.-inactivated Epstein-Barr virus. SDS-PAGE analysis of culture supernates of B-lymphoid cells labelled by growth in medium containing 35S-methionine revealed a major component of molecular weight approximately 130 kDa and another band at 30 kDa. Incubation with endoglycosidase F reduced the size of the high molecular weight component. Gel filtration of untreated serum or culture supernate revealed that, in its native state, sCR2 behaved as a molecule or complex of apparent molecular weight 320 kDa. Possible explanations are discussed.
Asunto(s)
Receptores de Complemento 3d/química , Linfocitos B/química , Linfocitos B/inmunología , Sitios de Unión de Anticuerpos , Línea Celular , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Epítopos/análisis , Epítopos/inmunología , Herpesvirus Humano 4 , Humanos , Receptores de Complemento 3d/inmunología , Solubilidad , Células Tumorales CultivadasRESUMEN
A soluble form of CD23 (sCD23) was found in the urine from 12 normal individuals but was not present in 20 normal sera, suggesting that sCD23 produced by cells in tissues is eliminated in the urine. The sCD23 from urine differed in physicochemical properties from the sCD23 found in supernates from B-lymphoblastoid cell lines (B-LCL) and in the sera of patients with B type chronic lymphocytic leukaemia (B-CLL). On SDS-PAGE analysis under reducing conditions urinary sCD23 showed two bands corresponding to molecular weights of 45-60 kD and 28-35 kD indicating that sCD23 may be excreted in combination with another molecule. When subjected to gel filtration in its native state, sCD23 from urine showed a major peak at approximately 150 kD and a minor peak (probably a breakdown product) at 21 kD. Urinary sCD23 was more strongly held by DEAE-cellulose and required 0.5 M buffer pH 8.0 for elution, suggesting that it is more anionic than sCD23 from culture supernates. Five MoAbs recognizing different epitopes on sCD23 from B-LCL supernates were tested on urinary sCD23. Four of the MoAbs were reactive but one (EBVCS-1) was not. Urinary sCD23 did not bind to IgE. The level of sCD23 found in normal urine (approximately 0.02-0.05 micrograms/ml) was exceeded in 17 of 24 cases of B-CLL. In one case with a high cell count and a serum concentration of 10 micrograms/ml, the urine contained 80 micrograms/ml sCD23. In another case a high serum sCD23 was not matched by a high urinary level. In this case the gel filtration pattern was closer to that found with urine sCD23 rather than the B-LCL pattern found with sera of other B-CLL patients.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Leucemia Linfocítica Crónica de Células B/orina , Receptores Fc/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos B/química , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Epítopos/análisis , Humanos , Inmunoglobulina E/metabolismo , Leucemia Linfocítica Crónica de Células B/inmunología , Peso Molecular , Receptores Fc/química , Receptores de IgERESUMEN
Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.
Asunto(s)
Membrana Celular/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Anticuerpos Monoclonales , Linfocitos B/inmunología , Sitios de Unión de Anticuerpos , Western Blotting , Linfoma de Burkitt , ADN/genética , Epítopos/análisis , Escherichia coli/genética , Citometría de Flujo , Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Técnicas de Inmunoadsorción , Leucemia de Células Pilosas , Linfoma de Células B Grandes Difuso , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales CultivadasRESUMEN
The mechanisms controlling immunoglobulin production have been studied in two types of immunoglobulin-secreting cell generated from human B cells. The first type (I) was produced by activation and transformation of B cells by co-culture with cells of an Epstein-Barr-virus-positive (EBV+) lymphoblastoid cell line (EBV-B-LCL). The second type (II) consisted of human/mouse hybrid cells produced by fusing human tonsil B cells with cells of a mouse plasmacytoma line. Both these methods, singly and in combination, have been widely used for initiation of cell lines secreting human monoclonal antibodies (MoAbs). The two cell types were of quite different phenotype with respect to human B cell antigens. In type I cells MHC Class II and the pan B antigens CD19 and CD37 were expressed at levels typical of cells at the B cell stage. The antigens CD23 and CD39 were expressed at the high levels characteristic of EBV-transformed B cells. Type II cells expressed few B cell antigens. MHC Class II, pan B and the CD23 and CD39 antigens were very weakly expressed and by 119 days post-fusion only CD38 was detectable on cells of the three lines studied; CD9 was on two and CD19 on only one of the three lines. Thus the phenotype of type I cells was influenced by EBV transformation but was otherwise typical of activated B cells. Whereas the human B antigen expression of the hybrid (type II) cells was at the low level encountered on human plasma cells. It is suggested that fusion of a human B cell to a mouse cell which is at the plasmacytoid stage of differentiation results in a switching off of the expression of human peripheral B antigens by a differentiation-linked mechanism. These results are considered in relation to the practical aspects of the production of human MoAbs and the theoretical aspects of control of the passage of B cells to a secretory stage of differentiation.
Asunto(s)
Antígenos de Superficie/análisis , Linfocitos B/inmunología , Transformación Celular Viral , Células Híbridas/inmunología , Inmunoglobulinas/biosíntesis , Animales , Células Cultivadas , Herpesvirus Humano 4 , Humanos , Ratones , Tonsila Palatina/inmunología , Plasmacitoma/inmunología , Factores de TiempoRESUMEN
Two new cell lines with the phenotype of terminally differentiated B cells have been derived from the presentation bone marrow of a patient with plasma cell leukaemia. They express the same immunoglobulin (A1-kappa) as the original bone marrow cells. JJN-1 is an hypodiploid, slow-growing line with a plasmacytic morphology, which grows in medium with 15-20% fetal calf serum. When JJN-1 was stimulated with a supernatant ('ESG') containing B cell stimulatory factor 2 (BSF-2/IL-6), a hypotetraploid sub-line, JJN-2, was selectively stimulated. JJN-2 is dependent on ESG for survival. The stimulatory effect of ESG can be completely abrogated by an anti-BSF-2 monoclonal antibody. However, purified BSF-2 alone only produces sub-maximal stimulation of the lines. Both lines show complex karyotypic abnormalities, including 14q- and del(6q). JJN-1 and JJN-2 may be useful for the study of late B cell differentiation and for use as immunogens for the generation of anti-plasma cell monoclonal antibodies.
Asunto(s)
Inmunoglobulina A/inmunología , Interleucinas/farmacología , Leucemia de Células Plasmáticas/inmunología , Antígenos de Superficie/análisis , Linfocitos B/inmunología , Línea Celular , ADN de Neoplasias/análisis , Femenino , Humanos , Cadenas kappa de Inmunoglobulina/inmunología , Interleucina-6 , Cariotipificación , Persona de Mediana Edad , Mitosis/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Cells of 7 EBV-B-LCL, 10 Burkitt lines and 13 EBV-B-LCL/Burkitt line hybrids have been phenotyped for antigens of the major B cell clusters and for some other antigens. High levels of CD23 and CD39 and low levels of CD38 (T10) were characteristic of EBV-B-LCL; the converse was true for Burkitt lines. In hybrids the EBV-LCL phenotype was dominant. The phenotype of Burkitt-line cells correlated strongly with that of germinal centre B cells in tonsil sections, but differed markedly from that of marginal zone B cells or follicular mantle cells. The results are discussed in relation to the origin of Burkitt tumours of "sporadic" and "endemic" type, in particular to histopathological evidence that Burkitt lymphomas develop in germinal centres. Recent studies on the location of the breakpoints of Burkitt chromosomal translocations are also considered to be compatible with this concept, even though different regions of the immunoglobulin heavy chain locus are involved in the two types of BL.
Asunto(s)
Antígenos CD , Linfocitos B/inmunología , Linfoma de Burkitt/inmunología , Herpesvirus Humano 4 , Tejido Linfoide/inmunología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Anticuerpos Monoclonales , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación de Linfocitos B/análisis , Línea Celular , Humanos , Glicoproteínas de Membrana , Fenotipo , Receptores Fc/análisis , Receptores de IgERESUMEN
Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking.
Asunto(s)
Antígenos de Neoplasias/análisis , Mieloma Múltiple/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos de Superficie/análisis , Citoplasma/inmunología , Técnica del Anticuerpo Fluorescente , Humanos , Leucemia de Células Plasmáticas/inmunología , Formación de RosetaRESUMEN
The origin of the raised serum IgG3 in primary biliary cirrhosis has been examined. Blood lymphocytes from patients with PBC and from age- and sex-matched controls were cultured, and the culture supernatants were assayed for IgG and IgG3. Lymphocytes from PBC patients spontaneously synthesized a higher percentage IgG3/total IgG than did control lymphocytes, as determined by ELISA. The increased synthesis of IgG3 in culture correlated with serum IgG3 in the patients. This strongly suggests that the raised serum IgG3 in these patients is due to increased synthesis of this isotype. Following PWM stimulation, the proportion of IgG3/IgG synthesized by normal (and most PBC) lymphocytes increased and the difference in IgG3 synthesized by PBC and control lymphocytes became less marked. The kappa/lambda light chain ratio of the IgG3 was assayed by ELISA but no evidence was found for clonally restricted synthesis of IgG3 by PBC blood lymphocytes.
Asunto(s)
Inmunoglobulina G/biosíntesis , Cirrosis Hepática Biliar/inmunología , Linfocitos/inmunología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina M/biosíntesis , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/biosíntesisRESUMEN
Specific and sensitive ELISA to quantitate the human IgG subclasses in cell culture supernatants are described. These assays detect a minimum of 5 ng/ml IgG1, 90 ng/ml IgG2, 8 ng/ml IgG3 and 8 ng/ml IgG4 and can generally measure IgG subclasses in lymphocyte cultures containing a minimum of 200 ng/ml of total IgG. The isotype specificity of these ELISA is demonstrated and each individual ELISA shown to react with a number of paraproteins of the relevant subclass independently of their light chain type or their (major Caucasian) allotype. These assays have been used to determine the IgG subclass response of normal human lymphocytes to pokeweed mitogen in vitro.
Asunto(s)
Anticuerpos Monoclonales , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/análisis , Isotipos de Inmunoglobulinas/análisis , Especificidad de Anticuerpos , Medios de Cultivo/análisis , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Alotipos de Inmunoglobulinas/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Activación de Linfocitos , Linfocitos/inmunología , Masculino , Paraproteínas/análisisRESUMEN
Anti-idiotope antibodies produced by mice immunized with a human IgG paraprotein complexed with various mouse monoclonal antibodies (McAbs) have been measured. All animals receiving more than one injection of the paraprotein (free or complexed with a mouse McAb) produced antibodies to the idiotypes of the paraprotein. Complexing with a McAb, especially an anti-Fc-gamma McAb, enhanced the response. Antibodies to the idiotopes of mouse McAbs were more difficult to produce and their production was very dependent on the mode and schedule of the immunization. The best antisera were produced by mice receiving a course of injections of pre-formed complexes of the IgG paraprotein and McAbs. Four of five mice produced antibodies to the idiotopes of an anti-light chain McAb (C4) after a course of immunization (one primary plus four boosts) of an IgG-C4 complex. Two of the six mice receiving a similar course of injections of the paraprotein complexed with an anti-gamma McAb (A55) produced high titres of antibodies to A55 idiotypes. Responses were enhanced when complexes were prepared with a pool of McAbs. It is probable that the formation of large multi-cross-linked complexes containing the McAb under study is important in generating the response. Once a response is initiated, very high titres may be achieved.