Isolation of specific IgM monoclonal antibodies by affinity chromatography using alkaline buffers

Comparative studies on the efficacies of various buffers in eluting absorbent-bound antibodies revealed the denaturative effect of 3 M KSCN on all three IgM antibodies but not on an IgG protein, and the generally weak eluting power of glycine-HCl buffer, pH 2.5, on these monoclonal antibodies. A recently described medium consisting of 50% (v/v) ethylene glycol in an alkaline buffer, pH 10.5, was found to be relatively efficient for elution in all cases. However, subsequent studies on one of the IgM antibodies showed that alkali alone could effect elution, with recovery of active protein improving on increasing the pH, till the maximum (38%) at pH 11.0, after which denaturation occurred. Addition of ethylene glycol to the medium facilitated the elution; however, at pH greater than 10.0, the solvent potentiated the denaturative effect of the medium. Since pH 11.0 was found to be the highest pH in which all three IgM antibodies examined were stable, 0.1 M glycine-NaOH buffer, pH 11.0, may be a useful eluent for IgM (and other) antibodies in general.     

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E

Commercial vaccines against Aujeszky's disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72h post-infection using 10(4)pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis.     

Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies

Summary Ten monoclonal antibodies (MAbs) were raised against dengue 1 (DEN 1, Hawaii) virus E glycoprotein. Specificity of the MAbs was tested by ELISA and immunofluoresence. Eight were DEN 1 type-specific, one was DEN group-reactive (DGR) and one was flavivirus cross-reactive (FCR). Two of these type specific MAbs exhibited haemagglutination-inhibition (HI) and neutralized (N) DEN 1 virus in vivo (HS). These two MAbs showed 100% protection against a challenge of 100 LD₅₀ of DEN 1 virus in adult Swiss albino mice. The remaining six MAbs were HI negative, N negative and non-protective against challenge (NHS). Of these only three were reactive in the CF test. The DGR, FCR and one of the NHS MAbs (NHS-3) did not react with DEN 1 virus grown in Vero cells, whereas they reacted with DEN 1 virus grown in LLC-MK2 and C6/36 cells in immunofluorescence, probably indicating a difference in the synthesis/processing of viral proteins in these different cell lines. An epitope map of the E gp was drawn using a computer programme based on the additivity index values. The epitope map delineated five domains, a) S-I representing type-specific, HI positive, N positive and protecting MAbs. b) S-II representing type-specific, HI negative, N negative MAbs. c) S-III representing type-specific HI/N negative MAb, but distinct from S-II. d) DGR representing HI/N negative DEN group reactive MAb. e) FCR representing HI/N negative flavivirus cross-reactive MAb. Epitope analysis of a number of different DEN 1 strains isolated in India over a period of 30 years showed that the domains S-II and S-III which react with HI negative, DEN-1 specific MAbs were variable. The DGR domain and the S-I domains were conserved.     

Purification of murine monoclonal antibodies by affinity chromatography using protein A-Sepharose

Summary A simple method is described for the isolation of murine monoclonal immunoglobulin G subclasses using protein A-Sepharose affinity chromatography.     

Immunological characteristics of Aujeszky's disease virus glycoprotein

A panel of monoclonal antibodies (MAbs) specific to glycoprotein D (gD) of Aujeszky's disease virus (ADV, Suid herpesvirus 1) was produced and characterized. MAbs were used to identify 9 topologically different epitopes and epitopic groups on gD. The majority of the identified epitopes were conformational. Most gD-specific MAbs possessed virus-neutralizing activity in the presence and absence of the complement. MAbs neutralized the virus at the stage of its penetration into the cell and inhibited the cell-to-cell spread of viruses. Two immunodominant epitopes and one immunodominant domain that induce the most prominent humoral immune response were identified when the animals were infected and immunized. A method was developed for affinity purification of ADV glycoprotein D. Immunization of mice with affinity-purified gD induced a strong humoral immune response and protected mice against lethal ADV challenge. In passive immunization, the majority of gD-specific MAbs protected mice against infection. The findings confirm the important role of ADV glycoprotein D in inducing protective anti-ADV immunity.     

Purification of tick-borne encephalitis virus by affinity chromatography using monoclonal antibody

The application of a simple technique for purification of tick-borne encephalitis (TBE) virus is described. TBE virus was grown in chick embryo cell (CE) cultures and the virus was concentrated by differential centrifugation. Final purification was made by the filtration through Sepharose column to which monoclonal antibodies to TBE virus had been bound. The method was effective in eliminating avian retroviruses.     

Characterization of neutralizing domains on varicella-zoster virus glycoprotein E defined by monoclonal antibodies

Summary. The genome of varicella-zoster virus (VZV), encodes at least six glycoproteins and they elicit the formation of complement-independent, complement-dependent, and non-neutralizing antibody responses. We have used our library of MAbs to VZV glycoprotein E (gE) to determine the neutralizing epitopes of gE, and shown that gE has 3 distinct neutralizing domains. In this report we have used the baculovirus expression system to identify the antigenic domains of gE. We have generated 3 recombinant baculoviruses, expressing the full-length gE and two overlapping truncated forms (the amino-terminal and the carboxy-terminal) of gE. By immuno-fluorescence and immunoblotting we have explored the physical interactions of Mabs to gE on these constructs. Our panel of MAbs revealed 3 district antigenic domains on gE. All MAbs reacted with the full-length gE; MAbs with high titered complement-dependent neutralizing activities reacted with the N-terminal truncated gE; MAbs with low titered or non-neutralizing activities reacted with the C-terminal truncated gE; MAbs with complement-enhanced neutralizing activities reacted with both truncated constructs. However, although the antibody binding in immunofluorescence and immunoblotting was carried out under denatured conditions, whereas the neutralization is under non-denatured conditions, still the antigenic mapping was similar in both conditions. Received April 24, 1996 Accepted July 29, 1996     

Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.     

Monoclonal blocking ELISA detecting serum antibodies to the glycoprotein gII of Aujeszky's disease virus

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Aujeszky's disease virus (ADV) in porcine serum was developed. This ELISA is based on the reaction between virus antigen immobilized in a microdilution plate and a monoclonal antibody (MAb) reactive with a highly stable epitope on a glycoprotein complex, gII, of ADV. The viral epitope was expressed by 18 European field, laboratory and vaccine strains of ADV. The MAb used in the test was selected among 15 MAbs all reactive with viral epitopes apparently recognized by the porcine immune system as well. Good agreement was found when serum samples from 375 pigs were tested in both a polyclonal and the monoclonal blocking ELISA.     

Cell-binding properties of glycoprotein B of Aujeszky's disease virus

The glycoprotein B (gB) of Aujeszky's disease virus (ADV) has a role in virus entry and cell-to-cell spread. In this report we examined the cell-binding properties of native ADV gB purified from the virus envelope by affinity chromatography. The binding of gB to the surface of susceptible cells BHK-21 and MDBK was specific, dose-dependent, and nearly saturable, which is characteristic of conventional receptor-ligand interactions. The purified gB was shown to specifically bind to immobilised heparin. The addition of soluble exogenous heparin and heparinase treatment of cells inhibited the binding of gB to the cells. Cell-associated gB could also be dissociated from the cells by soluble heparin. The results indicated that ADV gB binds specifically to cellular heparan sulphate. The binding of gB to cells inhibited the attachment of virus to cells and thus the formation of viral plaques. The results suggest that ADV gB may have a function in the initial attachment of ADV to the surface of susceptible cells.     

Purification of anti-epithelial mucin monoclonal antibodies by epitope affinity chromatography.

Monoclonal antibodies against the protein core of epithelial mucins have been found to react with the immunodominant sequence P D T R P A P (Burchell et al., 1989; Price et al., 1990a). Two immunoadsorbent matrices were prepared by linking the peptide A P D T R P A P G to CNBr-activated Sepharose and by linking the peptide C A P D T R P A P G to activated thiol-Sepharose, so that each immunoadsorbent contained the immunodominant motif. Anti-epithelial mucin antibodies (anti-breast carcinoma antibodies, anti-purified mucin antibodies and anti-human milk fat globule antibodies) were examined for reactivity with these preparations. The initial tests indicated that the substituted CNBr-activated Sepharose displayed lower non-specific antibody binding and this matrix was selected for further investigation. The anti-mucin antibodies were shown to react specifically with this affinity matrix and irrelevant antibodies failed to bind. A Sepharose-peptide immunoadsorbent column was examined for its capacity to purify several of these anti-mucin antibodies and it was determined that this procedure was highly efficient--purified IgG and IgM antibodies could be isolated from either hybridoma tissue culture supernatants or ascitic fluids. The capacity of the column was in excess of 40 mg antibody protein per ml of gel for the IgG3 antibody, C595 (anti-urinary mucin) and at least 10 mg antibody protein per ml of gel for the IgM antibody, NCRC-11 (anti-breast carcinoma). The procedure described permits the efficient purification of anti-mucin antibodies and provides a product which would be suitable for further investigations requiring highly immunoreactive antibodies (e.g., for radioimmunotherapy or immunoscintigraphy in patients with malignant disease).     

Development of immunoenzyme methods for detecting antibodies to Aujeszky's disease virus gB glycoprotein in swine serum

Four ELISA methods have been developed for detecting antibodies to Aujeszky's disease virus (ADV) glycoprotein gB. Indirect ELISA is based on affinity-purified gB (affi-gB-ELISA); three blocking ELISAs: indirect blocking ELISA (lbgB-ELISA), direct blocking ELISA (db-gB-ELISA), and two-site "sandwich" ELISA (sb-gB-ELISA) are based on monoclonal antibodies to conservative immunodominant epitopes of gB. The specificities and sensitivities of ELISAs were compared with each other and with indirect ELISA based on purified ADV virions (vir-ELISA). Affi-gB ELISA, db-gB-ELISA, and sb-gB-ELISA possess 100% sensitivity, ib-gB-ELISA 98% sensitivity, and vir-ELISA 93% sensitivity. Affi-gB ELISA, ib-gB-ELISA, db-gB-ELISA, and sb-gB-ELISA possess 100% specificity and vir-ELISA 92% specificity. The efficiency of detection of ADV-specific antibodies by affi-gB ELISA, db-gB-ELISA, and sb-gB-ELISA was comparable to that of analogous commercial test. Since db-gB-ELISA is easier to perform than affi-gB-ELISA or sb-gB-ELISA, it is concluded to be the most appropriate test for detecting pigs infected with ADV among non-vaccinated animals.     

Isolation of specific and common antibodies to staphylococcal enterotoxins A and E by affinity chromatography.

Specific and common antibodies to staphylococcal enterotoxins A and E (SEA and SEE) were isolated from anti-SEA and anti-SEE antisera by affinity chromatography. Anti-SEA and anti-SEE antisera were passed through a column with the cross-reacting enterotoxin coupled to a CNBr-activated Sepharose 4B matrix. Specifically bound common antibodies were eluted with NaSCN. The isolated specific antibodies reacted only with the homologous enterotoxin, whereas the common antibodies gave a reaction of identify with both enterotoxins in double gel diffusion plates. The common antibodies had a higher titer against SEE than against SEA. The significance of the isolation of antibodies common to two separate protein molecules is discussed.     

Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies

Immuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infectivity titration system, in an ELISA, in a haemagglutination assay and by negative contrast electron microscopy to quantify CPV or CPV antigen. The degree of purification was further estimated by testing the fractions for total protein content in a colorimetric method, for bovine serum albumin content in an ELISA and by SDS-PAGE. Over 99% of the contaminating proteins proved to be removed, and 20% or 70-90% of infectious CPV or CPV antigen, respectively, was recovered.     

Detection of antibodies to Aujeszky's disease virus in whole blood by Elisadisc

A rapid method for the detection of antibodies in whole blood by ELISA was developed. A punched filter paper disc configuration, the Elisadisc, was loaded with blood, dried, and applied directly to the Aujeszky's disease ELISA, with minimal pretreatment. Rapidity, throughput and sensitivity were equal to that of the serum ELISA. No loss of activity could be detected in the discs after storage at +4 degrees C for almost 1 yr.     

Enhanced protection of mice against Japanese encephalitis virus infection by combinations of monoclonal antibodies to glycoprotein E

In the present study, the protective effect of various combinations of four monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis virus (JEV) on the JEV-infected mice was studied. The MAbs were characterized as hemagglutination-inhibition-positive and JEV-specific (Hs). In the protective experiment, mice were first administered single MAbs or their combinations intraperitoneally (i.p.) and 24 hrs later infected with the virus intracerebrally (i.c.). The results showed that single MAbs protected the mice to the extent of 45-65%, while combinations of two or three MAbs gave 85-90% or 100% protection, respectively. The enhanced effect of combinations of several Hs MAbs might be due to the sharing of neutralization epitopes recognized by the Hs MAbs. These results suggested that a combination of at least three epitopes represented by the Hs MAbs should be included in an effective JEV vaccine.     

The antigenic structure of glycoprotein E2 in the Venezuelan equine encephalomyelitis virus studied using rat monoclonal antibodies]

A collection of 21 rat hybridomas secreting high-affinity monoclonal antibodies to Venezuelan equine encephalomyelitis (VEE) virus was generated. Using a panel of 15 monoclonal antibodies to glycoprotein E2, the antigenic structure of this protein of VEE strains TC-83 and 230 was studied. A competitive radioimmunoassay suggested a new map of the antigenic structure of glycoprotein E2 in which 5 sites including 11 epitopes of monoclonal antibody binding were distinguished. Antibody to E2-2 site neutralized virus infectivity and blocked hemagglutination test and antibody to E2-3 site could only block hemagglutination. Antibodies to other E2 protein sites lacked any biological activity.     

Border disease virus: delineation by monoclonal antibodies

Summary Many ovine pestiviruses from Britain and a number of atypical porcine isolates are largely unrecognised by monoclonal antibodies (mAbs) specific for reference strains of classical swine fever virus and bovine viral diarrhoea virus (BVDV). Additional mAbs have therefore been produced using some of these unreactive pestiviruses. Two of the viruses used were atypical porcine isolates (strains 87/6 and Vosges), whilst another had been isolated from a sheep (59386). Thirty-three mAbs were selected, none of which recognised two reference strains of BVDV, but three of which recognised the Alfort strain of classical swine fever. On the basis of radioimmunoprecipitation they were considered to be directed at one of three different pestivirus proteins (gp 53, gp 48 or p 125). Three virus subgroups were evident when the mAbs were used to type 16 ovine and two atypical porcine pestiviruses. One subgroup contained the Vosges and 59386 viruses and four ovine field isolates. The second subgroup comprised the 87/6 virus, the Moredun and Aveyron reference strains of border disease virus and four further ovine field isolates. Three of four ovine viruses making up the third subgroup had been previously categorised as BVDV-like and were largely unrecognised by the new mAbs. The findings were in agreement with previous attempts to segregate some of the same viruses using partial genomic comparisons or cross-neutralization tests.