Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH₂-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

Expression of the bovine leukemia virus envelope glycoprotein (gp51) by recombinant baculovirus and its use in an enzyme-linked immunosorbent assay

The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH2-terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.     

Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins

Summary The coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T. Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase. The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads. The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples. Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17+p28). A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID). Sera with conflicting results in the different ELISA tests were examined by Western blotting. There was a high correlation between the ELISA tests based on p17+p28 recombinant proteins and whole virus ELISA, with an estimated value of 0.92. Only 72–75% of the sera that tested positive in these two ELISA tests were positive in AGID. Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples. Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA.     

Cell Surface Display of Human Immunodeficiency Virus Type 1 gp120 on Escherichia coli by Using Ice Nucleation Protein

A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface of Escherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.     

Comparison of the biological activities of human immunodeficiency virus 1 P24 and GP41 expressed in Spodoptera frugiperda cells by use of bac-to-bac system

Recombinant transposing plasmids pFH24 and pFH41 were constructed by cloning the human immunodeficiency virus 1 (HIV-1) p24 and gp41 genes, respectively, into the transposing vector pFastBacHTa. Recombinant bacmids rBH24 and rBH41 were obtained by transposing pPolh/p24 and pPolh/gp41 expression cassettes from recombinant plasmids pFH24 and pFH41, respectively. Recombinant viruses rAcH24 and rAcH41 were generated by transfection of the Spodoptera frugiperda (Sf9) cells with the DNAs of plasmids rBH24 and rBH41, respectively. Analysis of the expressed p24 or gp41 proteins with an antiserum to HIV-1 (HIV-1 antiserum) by an enzyme-linked immunosorbent assay (ELISA) and dot blot assay showed high biological activity of these proteins; p24 was more active than gp41. Also a Western blot analysis showed stronger bands for p24 than for gp41. The high reactivities of p24 and gp41 with the HIV-1 antiserum suggest that these proteins could also be used as specific standard antigens in HIV-1 diagnostics.     

Expression of the nucleoprotein gene of rabies virus for use as a diagnostic reagent

The nucleoprotein (N) gene of rabies virus CTN strain, was cloned, sequenced and expressed in Escherichia coli as a fusion with maltose binding protein (MBP). The antigenicity of this recombinant MBP-N fusion protein was examined by Western blotting and enzyme linked immunosorbent assay (ELISA). Subsequently, an indirect ELISA was developed to detect rabies specific antibody levels. Using sera from naive and vaccinated animals the ELISA results were compared with virus neutralizing antibodies detected by a rapid fluorescent focus inhibition test (RFFIT). Neutralizing titres by RFFIT were found to correlate well with the OD values in the ELISA (r=0.9436) and the sensitivity and specificity of the ELISA were shown to be 93.4 and 100%, respectively. The data indicate that the recombinant MBP-N fusion protein can be expressed and isolated straightforwardly and may be useful as a safe and abundant source of antigen to monitor seropositivity in vaccinated canines.     

The envelope glycoprotein of HIV-1 gp120 and human complement protein C1q bind to the same peptides derived from three different regions of gp41, the transmembrane glycoprotein of HIV-1, and share antigenic homology

Gp41, the transmembrane glycoprotein of HIV-1, has been shown to be non-covalently associated with gp120. We have shown that it also binds human C1q. To analyze the interaction site(s) of gp41 with these two molecules, we established an enzyme-linked immunosorbent assay (ELISA) system using recombinant soluble gp41 [amino acids (aa) 539–684] and peptides thereof. In the cell-external part of gp41 three sites (aa 526–538, aa 590–613 and aa 625–655) were found to bind both gp120 and C1q. That gp120 and C1q use the same sites was evidenced by the fact that these proteins competed with each other for the same sites in recombinant soluble gp41 and gp41 peptides. It could be demonstrated by ELISA, that rabbit antibodies against human C1q recognized gp120, and rabbit antibodies against gp120 cross-reacted with C1q. Rabbit anti-gp120, HIV-1-positive human sera and anti-gp120 obtained from such sera agglutinated sensitized sheep erythrocytes with human C1q (EAC1q). These data suggest that in addition to functional homology between C1q and gp120 structural homology between these two molecules exists. This molecular mimicry might become the basis for immunologically relevant autoimmune phenomena.     

An Accurate Confirmation of Human Immunodeficiency Virus Type 1 (HIV-1) and 2 (HIV-2) Infections with a Dot blot assay Using Recombinant p24, gp41, gp120 and gp36 Antigens

An immunoblot assay using four recombinant proteins corresponding to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene products was developed to confirm the presence of antibodies to HIV-1 and 2 in sera reactive in screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from different categories of HIV-infected individuals (asymptomatic HIV-infected, and AIDS). A positive reaction was defined as reactivity against gag (p24) and at least one other env (either gp41 or gp120) HIV gene products; negative result was defined as no reaction with any antigen; and indeterminate result was defined as reactivity with gag (p24) or with env (gp41 or gp120) alone. None of the 180 serum samples from healthy seronegative blood donors gave a positive result, and only 4 of these samples (2.2%) gave indeterminate results. The recombinant HIV Dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the 125 HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. All seronegative and seropositive samples were tested both with the commercially available ELISA and by Western blot. The recombinant in-house HIV Dot blot assay accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did commercial Western blot (as interpreted by CDC criteria).     

Computer analysis of the amino acid sequences in gp41 of apathogenic African green monkey (AGM) virus,less pathogenic HIV-2 and highly pathogenic SIV and HIV-1 lentiviruses

The bestfit computer program was used to compare the amino acid sequence of the gp160 envelope glycoprotein of an apathogenic AGM and the pathogenic SIV_AGM monkey lentiviruses. It was found that the gp120 envelope glycoproteins of these viruses resembled each other in their functional domains. However, an insert of 40 amino acids was found in the gp41 envelope glycoproteins of the pathogenic SIV_AGM virus in the amino acid sequence between the membrane anchoring sequence and the carboxyterminus. The insert introduced a new RRIR proteolytic cleavage signal into gp41. Comparing HIV-1 gp41 to that of the pathogenic SIV_AGM virus revealed that the HIV-1 sequence contains an RR sequence that also serves as a signal for proteolytic cleavage. Comparing HIV-2 gp41 to the apathogenic and pathogenic simian immunodeficiency viruses revealed that HIV-2 gp41 lacks the above proteolytic cleavage signal. It is hypothesized that the pathogenic human and simian immunodeficiency lentiviruses can be proteolytically cleaved at the carboxyterminus of gp41, releasing two peptides: a) an immunodeficiency 58 amino acid peptide and b) an IL-2-like peptide. The apathogenic AGM virus and the less pathogenic HIV-2 lack one proteolytic cleavage signal in the gp41 amino acid sequence and therefore can release only the IL-2-like peptide but not the immunodeficiency peptide. If indeed the pathogenic SIV_AGM and HIV-1 do release an immunodeficiency peptide, then such a peptide can be regarded as a toxin. Immunization of healthy individuals or HIV-1 patients against the toxic effect of the viral gp41 toxic peptide might prevent damage to the immune system when the virus reactivation leads to ARC and AIDS in infected individuals. Synthetic peptides modeled according to the immunodeficiency peptide (the toxin) can be used to produce anti-toxin antibodies in healthy HIV-1 infected individuals. Such anti-toxin antibodies can be used for passive immunization of AIDS patients or for active immunization of HIV-1 positive individuals prior to ARC or AIDS.     

Cell surface display of human immunodeficiency virus type 1 gp120 on Escherichia coli by using ice nucleation protein.

A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface of Escherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.     

Comparison of immunoperoxidase staining with indirect immunofluorescence, ELISA, and Western blotting assays for detecting anti-HTLV-I antibodies in systemic lupus erythematosus.

Serum antibodies against human T cell leukaemia virus type I (HTLV-I) were investigated in 12 patients by four methods: indirect immunoperoxidase staining, indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA), and strip radioimmunoassay based on the Western blotting assay. Seven patients had systemic lupus erythematosus (SLE) and five various autoimmune diseases with one or more circulating autoantibodies. Serum samples from three patients were found to be HTLV-I-positive by the ELISA assay and sera from five patients showed a non-specific reaction by indirect immunofluorescence. These sera were negative when tested by indirect immunoperoxidase staining and Western blotting assay. All four methods gave positive results when tested with samples from 19 HTLV-I carriers and 16 patients with adult T cell leukaemia. Indirect immunoperoxidase staining and Western blotting assay are probably useful and more specific assays for the detection of anti-HTLV-I antibodies in samples from patients with autoimmune diseases.     

Production and Characterization of Anti‐recombinant Human Erythropoietin (rhEPO) Monoclonal Antibody

Abstract

Anti‐rhEPO McAb is valuable for the determination of recombinant human erythropoietin (rhEPO) levels for diagnosis of renal anemia and for doping control analysis. In this paper, anti‐rhEPO hybridoma was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse, using an enzyme‐linked immunosorbent assay (ELISA) method to screen the positive hybridoma. The purified McAb was characterized by ELISA, SDS‐PAGE, and Western‐blotting. Experimental results showed that the subclass and the light chain of anti‐rhEPO McAb was IgG₁ and kappa light chain. The molecular weight of anti‐rhEPO McAb was 166,000 Daltons. The affinity constant (K _aff) of anti‐rhEPO McAb with coated antigen was 5.0 × 10⁵ L/mol.     

An enzyme-linked immunosorbent assay for detection of reticuloendotheliosis virus infection in chickens

An enzyme‐linked immunosorbent assay (ELISA) for the detection of reticuloendotheliosis virus (REV) is described. The assay is based on antiserum produced in rabbits against the group‐specific (gs) antigen of REV which has a molecular weight of 30 kDa (p30). The p30 for immunisation was obtained by electro‐elution from polyacrylamide gels after separation of purified REV by electrophoresis. Western blotting indicated that the antiserum produced was specific for p30 of REV.

The double antibody sandwich ELISA developed using the rabbit anti‐p30 was found to be specific for REV and highly efficient for the detection of REV in a variety of samples when compared with the indirect immunofluorescence assay. The smallest amount of REV detectable was 2,000 fluorescence forming units. Serum, vaginal/ cloacal swab samples and egg albumens obtained from experimentally infected REV‐viraemic chickens were all consistently positive for the ¿?i‐antigen of REV. Some serum samples from non‐viraemic chickens, however, gave ‘false positive’ reactions in the ELISA. Albumens gave higher readings in ELISA than vaginal/cloacal swab samples and should therefore be the sample of choice for screening flocks for REV.     

Expression and evaluation of Chikungunya virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection

Purpose

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe-transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens.

Materials and Methods

CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA.

Results

The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%.

Conclusion

The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.     

Enzyme-linked immunosorbent assay for detecting antibodies to Borna disease virus-specific proteins.

Borna disease virus is a unique neurotropic RNA virus that causes neurologic disease in a wide variety of animal hosts. We established an enzyme-linked immunosorbent assay for the detection of antibodies to Borna disease virus on the basis of the use of three recombinant viral proteins (recp40, recp23, and recp18). This assay system is more sensitive and rapid than the methods currently used for the serologic diagnosis of infection such as Western blotting (immunoblotting), indirect immunofluorescence test, or immunoprecipitation.     

HIV antibodies in whole saliva detected by ELISA and western blot assays

Paired serum and saliva samples were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) for the presence of human immunodeficiency virus (HIV) antibodies. The study group included 36 individuals known to be HIV seropositive and 14 healthy, seronegative controls. HIV antibodies were detected in all but one of the saliva samples of the seropositive subjects. In this particular patient, seroconversion was documented 1 week earlier by sequential testings. A further saliva sample obtained 2 months later was ELISA positive for salivary HIV antibodies. Antibodies against HIV proteins gp120 and gp160 were detected by Western blot assay in all saliva specimens taken from HIV seropositive subjects (including the ELISA-negative patient who seroconverted. Antibodies against other viral proteins (p65, p55, p51, gp41, p35, p24 p18) were found in saliva haphazardly without any clear-cut correlation with the clinical stage of the disease. Pretreatment of the saliva with protease inhibitor was essential for the diagnostic use of saliva for the detection of HIV antibodies by Western blot assay. Calculation of the ratio of titres in serum to those in saliva showed the highest ratios in symptomless subjects (mean +/- SD; 1844 +/- 1412) and the lowest in patients with acquired immune deficiency syndrome (AIDS) (mean +/- SD; 811 +/- 445). The ratio of serum to saliva by ELISA showed a positive correlation with salivary flow rate, indicating a dilution of salivary HIV antibodies with increasing salivary flow rate. The gingival bleeding index was negatively correlated with the ratio, supporting the concept that salivary HIV antibodies transudate from blood to saliva via gingival fluid.     

Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2_332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2_332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2_332-452 ELISA may be a preferable method for detecting antibodies against AMDV.     

Segmentation expression of capsid protein as an antigen for the detection of avian nephritis virus infection in chicken flocks

Subclinical pathological changes in the kidneys of broiler chickens and suppression of growth caused by the avian nephritis virus (ANV) affect poultry flocks worldwide. A test for detection of virus-specific antibodies in serum would be useful for epidemiological investigations, however the poor propagation in cell cultures has restricted the development of serological tests based on the use of ANV particles as antigens. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of ANV-specific antibodies in chicken serum, using a recombinant protein antigen prepared by segmentation expression of the capsid protein antigen epitope of ANV (HM029238) transfected into Escherichia coli. The expressed fusion protein was detected by Western blotting with ANV-positive serum, and the optimal immunoreactive fusion P1 protein was determined. Using the optimized P1-ELISA, ANV-specific antibodies were detected in commercial chicken flocks aged 10-25 days obtained from the Liaoning Province, China. Out of 960 serum samples, 459 (47.8%) were positive for infection with ANV. These results indicate that the P1-ELISA is helpful for preliminary serological diagnosis of ANV infection, and could be used to for screening in ANV infection and for determining antibodies against ANV.     

Recombinant M protein-based ELISA test for detection of antibodies to canine coronavirus

The membrane (M) protein of canine coronavirus (CCoV) was cloned and expressed in E. coli. The purified recombinant protein was then evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for detection of CCoV antibodies in dog sera. Fifty serum samples, screened previously by whole virus ELISA and Western blotting, were tested. When the performance of the new test was compared with those of whole virus ELISA and Western blotting, an excellent correlation was found with the latter two assays. The ELISA based on recombinant M protein represents an alternative and valid test for detection of antibodies to CCoV in dog sera.     

A screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure using a conformation-specific monoclonal antibody.

The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in membrane fusion between viruses and target cells. The gp41 ectodomain contains two heptad repeat regions adjacent to the N and C-termini. Peptides derived from these two regions, designated N and C-peptides, are potent inhibitors of HIV-1 infection and can interact with each other to form a six-stranded coiled-coil, representing the fusogenic core structure of gp41. A monoclonal antibody was generated, designated NC-1, which specifically binds to the complex formed by the N and C-peptides, but not to the individual peptides. An enzyme linked immunosorbent assay (ELISA) was developed using NC-1 for detecting complex formed by N and C-peptides and for screening of organic compounds for antiviral agents that may interfere with complex formation and inhibit HIV-1 infection. Single point mutations in the C-peptides abolish the complex formation also eliminate their anti-HIV-1 activity. A phenylazo-naphthalene sulfonic acid derivative, designated ADS-J1, was found to inhibit both formation of NC-1 detectable complex and HIV-1-mediated membrane fusion, suggesting that the described ELISA is applicable to rapid screening of libraries of organic compounds for HIV-1 inhibitors targeted to the HIV-1 gp41 core structure.