"Single-shot" intrasplenic immunization for the production of monoclonal antibodies.
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Animals↗
Biomedical subjects
M Spitz
Publications and source records attributed to M Spitz.
Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody.
A monoclonal antibody (NIBy 142-36/8) raised against the soluble galactose-binding lectin of bovine heart muscle has been tested by solid-phase vinyl-plate radiobinding and nitrocellulose immunoblotting with homogenates of various bovine tissues, and the muscle tissues of pig, rabbit, chicken and rat. Muscle lectins of chicken, rabbit and rat differed from those of man and pig in their lack of reactivity with the 36/8 antibody. There was a good correlation of haemagglutinating activities and immunoreactivities of the bovine tissue homogenates, suggesting that the soluble galactose-binding protein is a major haemagglutinin in various tissues. Immunoblotting experiments revealed an array of antigenically active components in the homogenates in addition to the 13 and 26kDa proteins that were previously detected in preparations of purified lectin. These were in the range 36kDa to more than 200kDa, and a different spectrum of immunoreactive components was found in various cell types. Galactose-binding activity was demonstrable in 13, 26 and 36kDa components in certain bovine tissues, suggesting that the immunoreactive components of higher Mr may be inactive precursor forms of the lectin.
Animals↗
Use of 'single shot' intrasplenic immunization for production of monoclonal antibodies specific for human IgM.
We report here the use of 'single shot' intrasplenic injection of human IgM for immunization of mice to obtain splenocytes for use in the production of hybridomas secreting antibodies against human IgM. Fusion was performed 3 days after intrasplenic injection of 20 micrograms of myeloma IgM. IgM-specific antibodies were found in 12% of the fusion wells; only 1 well contained antibodies which cross-reacted with other immunoglobulin classes. Two monoclonal antibodies (McAbs) have been fully characterized as specific for different epitopes on Fc mu. These antibodies can be used to detect IgM on the surface of human B cells by immunofluorescence and in solution by solid-phase radiobinding assay or single radial immunodiffusion. Both McAbs can also detect IgM fragments by immunoblotting from non-reducing SDS-polyacrylamide gels.
Animals↗
Human B cell proliferation is stimulated by interleukin 2.
The proliferation of human B cells was studied for response to interleukin 2 (IL-2) produced in Escherichia coli using recombinant DNA technology. The IL-2 was found to be an homogenous preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the anti-IL-2 monoclonal antibody DMS-1. IL-2 was found to stimulate B cell proliferation. Activation of the B cells using anti-IgM antibodies increased this response. Resting T cells from the same donors were found to be less reactive to IL-2. The results suggest that human B cell proliferation can be stimulated by IL-2 alone.
Antibodies, Anti-Idiotypic↗
Interleukin-2 in vivo: production of and response to interleukin-2 in lymphoid organs undergoing a primary immune response to heterologous erythrocytes.
In this report, we describe experiments which demonstrate that antigenic stimulation in vivo causes the appearance of cells in both spleen and lymph node which secrete interleukin-2 (IL-2). Cells also appear in these organs which proliferate in response to IL-2. For these experiments, sheep red cells (SRBC) were injected into the spleens or footpads of mice, and cell suspensions from spleens or popliteal lymph nodes prepared at various times after antigenic stimulation. These cells were assayed for their ability to respond to IL-2, and their cell culture supernatants for secreted IL-2. The proliferative response to IL-2 steadily increased following SRBC injection to reach a peak at Day 2 for spleen cells and at Day 3 for lymph node cells. Maximal production of IL-2 was displaced from the maximal response to the lymphokine by peaking one day later for both organs. Our results strongly implicate the participation of IL-2 in the in vivo immune response and suggest the existence of in vivo regulatory mechanisms, which can control the time of IL-2 production and also the appearance of cells with receptors for IL-2.
Animals↗
Immunoblotting with monoclonal antibodies: loss of immunoreactivity with human immunoglobulins arises from polypeptide chain separation.
Immunoblotting has been used to study the antigen binding characteristics of 5 monoclonal antibodies (Mc/Abs) against human Ig (1 anti-kappa, 2 anti-gamma and 2 anti-delta chain. Of the 4 Mc/Abs only 1 (the anti-kappa chain Mc/Ab) reacted with its antigen when blotted from reducing SDS polyacrylamide gels. However, the 4 Mc/Abs which recognise immunoglobulin heavy chains were able to bind their antigens when blotted from native or non-reducing SDS gels. The lack of reactivity of the latter Mc/Abs in blots from reduced SDS gels may be attributed to the separation of Ig which occurs during electrophoresis after the -S-S- bonds are broken. It may be concluded that the conformation of Ig heavy chains is considerably altered when Ig molecules are disrupted and Ig chains separated, and several heavy chain determinants are lost during this process. Therefore determinants recognised by the anti-heavy chain Mc/Abs are most likely to be of the 'conformational' type whereas the anti-light chain Mc/Ab may well recognise a purely sequential determinant.
Antibodies, Anti-Idiotypic↗
Production and characterization of monoclonal antibodies to beta-galactoside-binding lectin of bovine heart muscle. Direct evidence that haemagglutinating activity is associated with a 13kDa protein.
With the aim of obtaining monospecific antibodies against the beta-galactoside-binding lectin of bovine heart muscle, spleen cells from Lou rats immunized with lectin were fused with the rat myeloma line Y3.Ag1.2.3. Two immunoglobulin M (IgM)-producing clones, designated NIBy 142-36/8 and NIBy 143-9/5, derived from separate fusions, were used to generate ascites containing high-titre binding activity against the 13kDa component in preparations of lectin. Direct evidence that haemagglutinating activity is associated with the 13kDa protein was obtained by the specific elution of 13kDa polypeptides with haemagglutinating activity from an immobilized antibody adsorbent. Solid-phase radiobinding assays and immunoblotting of isolated lectins and/or muscle homogenates confirmed the earlier indications with conventional antisera that the beta-galactoside-binding lectins of bovine, human and monkey muscle tissue are antigenically related.
Animals↗
Intrasplenic primary immunization for the production of monoclonal antibodies.
A novel immunization procedure for eliciting monoclonal antibodies ( McAbs ) is described. With intrasplenic inoculation only small amounts of immunogen are required. As little as 20 micrograms of protein antigen or 2.5 X 10(5) cells have been found sufficient to immunize mice or rat spleen cells for the production of specific McAbs . A high proportion of hybridomas secreting McAbs against cell surface antigens and soluble proteins has been obtained with this immunization procedure. The system could facilitate McAb production in many instances in which only small quantities of immunogen are available.
Animals↗
New poliovirus vaccines: a molecular approach.
This article summarizes recent work on the determinants of antigenicity in poliovirus type 3 and reports on experiments in progress aimed at understanding the molecular basis of attenuation in Sabin's type 3 vaccines. Ways in which this new information might be used to produce alternative, safe, inexpensive, multivalent vaccines against polio and other enteroviruses are discussed.
Amino Acid Sequence↗
Single shot intrasplenic immunization: an advantageous procedure for production of monoclonal antibodies specific for human fibrin fragments.
We describe the use of the single shot intrasplenic immunization technique as a particularly effective procedure for the production of specific monoclonal antibodies against a high molecular weight antigen. We found that with this technique several different, completely specific monoclonal antibodies could be produced against high molecular weight crosslinked fibrin degradation products. These results contrasted with those obtained using conventional multidose immunization, which only produced monoclonal antibodies that were cross-reactive with fibrinogen and/or noncrosslinked fibrin degradation products.
Animals↗
Neutralization epitopes on poliovirus type 3 particles: an analysis using monoclonal antibodies.
Monoclonal antibodies to poliovirus type 3 secreted by 51 hybridoma cell clones have been characterized in terms of (i) virus-neutralizing properties, (ii) reactivity in antigen-blocking tests with infectious, 155S ('D' antigen) and empty 80S ('C' antigen) poliovirus particles and (iii) reactivity in immunoblot tests with the isolated protein components of the poliovirus capsid. The antibodies could be separated into three groups on the basis of their reactivities with 'D' and 'C' antigens. All antibodies that reacted with both 'D' and 'C' antigen had potent neutralizing activity. Only a proportion of antibodies that reacted uniquely with 'D' antigen possessed neutralizing activity. Unexpectedly, one of 24 'C' antigen-specific antibodies inhibited virus growth. None of the antibodies that possessed virus-neutralizing activity reacted with isolated poliovirus capsid proteins, although the majority of these have been shown in previous studies to be specific for VP1 on intact virus particles. These findings suggest that antigenic determinants involved in virus neutralization do not survive the denaturing conditions required for the isolation of poliovirus capsid proteins and consequently are likely to be specified by the structural conformation of VP1 rather than by amino acid sequence alone. However, several of the antibodies which bound uniquely to 'C' antigen reacted in immunoblot tests, five with VP1 and one with VP3. Some of these antibodies also possessed heterotypic reactivity with the corresponding capsid proteins separated from other poliovirus types.
Animals↗
Elucidation of the antigenic determinant in lipotropin for two monoclonal antibodies.
Two monoclonal antibodies were found to give enhanced affinity for beta-lipotropin when mixed, as evidenced by competitive radioimmunoassay. Both monoclonals were found to react with a pentapeptide Ala-Glu-Leu-Glu-Tyr, which is a sequence of high local hydrophilicity within the N-terminal section of beta-lipotropin.
Amino Acid Sequence↗
Location and primary structure of a major antigenic site for poliovirus neutralization.
We have determined a major antigenic site for virus neutralization on the capsid protein VP1 of poliovirus type 3. Antigenic mutant viruses selected for resistance to individual monoclonal antibodies had point mutations concentrated in a region 277-294 bases downstream from the start of the region of viral RNA coding for VP1. These findings provide the basis for an improved understanding of the molecular basis of virus neutralization.
Amino Acid Sequence↗
Detection and affinity purification of beta-endorphin precursors using a monoclonal antibody.
A monoclonal antibody to porcine beta-lipotropin has been produced which binds to the N-terminal (gamma-lipotropin) portion of the molecule. The antibody can be used to detect beta-lipotropin as well as other beta-endorphin precursors (predominantly a Mr 38 000 polypeptide) using radiobinding assay or the immunoblotting technique. Purification of the peptides can be readily achieved by affinity chromatography using the monoclonal antibody covalently bound to Sepharose 4B. As the antibody recognises the N-terminal part of beta-lipotropin, it can be used to detect and purify beta-lipotropin and other beta-endorphin precursors in the presence of beta-endorphin.
Animals↗
Monoclonal antibodies specific for the Sabin vaccine strain of poliovirus 3.
Monoclonal antibodies to poliovirus type 3 secreted by nine mouse hybridoma cell lines were tested for virus-strain specificity with a collection of wild and vaccine-like type 3 viruses, the genetic characteristics of which had been determined by T1-oligonucleotide mapping. Four of the monoclonal antibodies had both virus-neutralising and antigen-blocking activity, and five had antigen-blocking activity only. Of the four virus-neutralising antibodies, two (NIBp138, NIBp165) reacted only with Sabin vaccine virus and other poliovirus type 3 strains with Sabin-like T1-oligonucleotide maps. Two antibodies (NIBp132, NIBp134) neutralised a broad range of type 3 viruses. Of five monoclonal antibodies with antigen-blocking activity only, four (NIBp135, NIBp136, NIBp140, and NIBp141) reacted exclusively with Sabin virus and strains with Sabin-like maps, and one (NIBp139) reacted with all type 3 strains tested. The vaccine-specific monoclonal antibodies, particularly NIBp138 and NIBp165, have considerable potential value in distinguishing wild type 3 viruses from those of Sabin vaccine origin. This conclusion was strongly supported by the finding that certain viruses which showed genetic evidence of being related to Sabin virus reacted well with vaccine-specific monoclonal antibodies despite having undergone genetic drift away from vaccine virus as indicated by changes in up to 30% of their oligonucleotides.
Antibodies, Monoclonal↗
Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody.
An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the Poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG anti-mouse F(ab). We have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.
Antibodies, Monoclonal↗
Genetic and antigenic variation in type 3 polioviruses: characterization of strains by monoclonal antibodies and T1 oligonucleotide mapping.
Considerable genetic and antigenic heterogeneity was detected among a collection of 17 poliovirus type 3 strains isolated between 1939 and 1958 in studies using monoclonal antibodies and by T1 oligonucleotide mapping. Heterogeneity was detected even amongst a collection of nine viruses designated Saukett and assumed to originate from the same prototype virus. The monoclonal antibodies were found to differ in their strain specificities for poliovirus type 3 strains in virus neutralization or single-radial-immunodiffusion tests. Relationships between strains detected in this way were in general consistent with those detected by oligonucleotide mapping. One of the monoclonal antibodies (NIBp 56) was able to distinguish between certain Saukett virus strains which differed by as little as a single specific oligonucleotide. The heterogeneity detected amongst Saukett viruses is of potential practical importance since these strains are used widely in the manufacture of inactivated poliovirus vaccine.
Antibodies, Monoclonal↗
Immunochemical studies of polioviruses: identification of immunoreactive virus capsid polypeptides.
Investigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and non-neutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The non-correlation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.
Antibodies, Monoclonal↗