The Experts below are selected from a list of 1257 Experts worldwide ranked by ideXlab platform

Bruce E. Kemp - One of the best experts on this subject based on the ideXlab platform.

  • simplified conjugation chemistry for coupling peptides to f ab fragments autologous Red Cell Agglutination assay for hiv 1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Simplified conjugation chemistry for coupling peptides to F(ab′) fragments: autologous Red Cell Agglutination assay for HIV-1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Autologous Red Cell Agglutination test for antibodies to feline immunodeficiency virus.
    Veterinary Immunology and Immunopathology, 1994
    Co-Authors: Kim Wilson, K.i. Mitchelhill, Wayne F. Robinson, Peter Gregory Bundesen, Bruce E. Kemp
    Abstract:

    Abstract The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous Red Cell Agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680–715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 μl of whole blood, circulating antibody to the peptide epitope caused Agglutination of the Red blood Cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680–715) peptide corresponding to the FIV, PPR strain gp40 (678–716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.

  • Kinetics of the autologous Red Cell Agglutination test.
    Journal of Immunological Methods, 1993
    Co-Authors: Bruno Catimel, Kim Wilson, Bruce E. Kemp
    Abstract:

    The kinetics of the autologous Red Cell Agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-Red blood Cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin alpha on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab' fragments were determined (n = 4.80 X 10(5) sites/erythrocyte, Ka = 0.43 X 10(7) M-1, Kd = 23 X 10(-8) M for 1C3/86, n = 4.66 X 10(5) sites/erythrocyte, Ka = 1.05 X 10(7) M-1, Kd = 9.5 X 10(-8) M for 10F7MN. The binding studies were performed under the same conditions as the autologous Red blood Cell Agglutination test. When 0.9 microgram of anti-glycophorin Fab was added to 10 microliters of blood 0.25 microgram of 1C3/86 Fab was bound whereas 0.29 microgram for 10F7MN Fab' was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 microgram of anti-peptide antibody gave half a maximal Agglutination score. This is equivalent to 10 micrograms/ml circulating antibody. Under the Agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal Agglutination occurRed when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum Agglutination score of four occurRed in the presence of 1 microgram of anti-peptide antibody equivalent to 100 micrograms/ml circulating antibody whereas an Agglutination score of 1+ was elicited by only 0.32 microgram anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin molecules per Red Cell.

  • Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.
    Journal of Immunological Methods, 1991
    Co-Authors: Kim Wilson, Peter Gregory Bundesen, D. B. Rylatt, Dale A. Mcphee, Michael Gerometta, Carmel Judith Hillyard, Bruce E. Kemp
    Abstract:

    Abstract A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on Agglutination of autologous Red blood Cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human Red blood Cells). This anti-Red blood Cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579–613). Addition of this reagent to 10 μl of whole blood results in the Fab-peptide conjugate coating the Red blood Cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, Red Cell Agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

Kim Wilson - One of the best experts on this subject based on the ideXlab platform.

  • simplified conjugation chemistry for coupling peptides to f ab fragments autologous Red Cell Agglutination assay for hiv 1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Simplified conjugation chemistry for coupling peptides to F(ab′) fragments: autologous Red Cell Agglutination assay for HIV-1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Autologous Red Cell Agglutination test for antibodies to feline immunodeficiency virus.
    Veterinary Immunology and Immunopathology, 1994
    Co-Authors: Kim Wilson, K.i. Mitchelhill, Wayne F. Robinson, Peter Gregory Bundesen, Bruce E. Kemp
    Abstract:

    Abstract The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous Red Cell Agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680–715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 μl of whole blood, circulating antibody to the peptide epitope caused Agglutination of the Red blood Cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680–715) peptide corresponding to the FIV, PPR strain gp40 (678–716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.

  • Kinetics of the autologous Red Cell Agglutination test.
    Journal of Immunological Methods, 1993
    Co-Authors: Bruno Catimel, Kim Wilson, Bruce E. Kemp
    Abstract:

    The kinetics of the autologous Red Cell Agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-Red blood Cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin alpha on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab' fragments were determined (n = 4.80 X 10(5) sites/erythrocyte, Ka = 0.43 X 10(7) M-1, Kd = 23 X 10(-8) M for 1C3/86, n = 4.66 X 10(5) sites/erythrocyte, Ka = 1.05 X 10(7) M-1, Kd = 9.5 X 10(-8) M for 10F7MN. The binding studies were performed under the same conditions as the autologous Red blood Cell Agglutination test. When 0.9 microgram of anti-glycophorin Fab was added to 10 microliters of blood 0.25 microgram of 1C3/86 Fab was bound whereas 0.29 microgram for 10F7MN Fab' was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 microgram of anti-peptide antibody gave half a maximal Agglutination score. This is equivalent to 10 micrograms/ml circulating antibody. Under the Agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal Agglutination occurRed when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum Agglutination score of four occurRed in the presence of 1 microgram of anti-peptide antibody equivalent to 100 micrograms/ml circulating antibody whereas an Agglutination score of 1+ was elicited by only 0.32 microgram anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin molecules per Red Cell.

  • Rapid whole blood assay for HIV-1 seropositivity using an Fab-peptide conjugate.
    Journal of Immunological Methods, 1991
    Co-Authors: Kim Wilson, Peter Gregory Bundesen, D. B. Rylatt, Dale A. Mcphee, Michael Gerometta, Carmel Judith Hillyard, Bruce E. Kemp
    Abstract:

    Abstract A rapid whole blood test has been developed for circulating antibodies to human immunodeficiency virus type 1 (HIV-1), based on Agglutination of autologous Red blood Cells. Evaluation of the test revealed that 100% of seropositive HIV-1 patients (both asymptomatic and AIDS cases) were detected (n = 94) with a specificity of 99.5% in healthy blood donors (n = 596). The assay uses an Fab fragment of a monoclonal antibody specifically directed against glycophorin (a transmembrane glycoprotein present on the surface of human Red blood Cells). This anti-Red blood Cell Fab is conjugated via the inter-heavy chain cysteines to a synthetic peptide corresponding to the immunodominant epitope of the HIV-1 viral coat protein gp41 (579–613). Addition of this reagent to 10 μl of whole blood results in the Fab-peptide conjugate coating the Red blood Cells with peptide. In the presence of circulating antibodies to the HIV-1 peptide, Red Cell Agglutination occurs within 2 min. The sensitivity and specificity of this reagent indicate that it is appropriate for use as a rapid diagnostic test for HIV-1 seropositivity.

H. Berger - One of the best experts on this subject based on the ideXlab platform.

  • Cold haemagglutinin disease complicatingMycoplasma pneumoniae infection in a child under cytotoxic cancer treatment
    European Journal of Pediatrics, 1992
    Co-Authors: F. M. Fink, K. Dengg, S. Kilga-nogler, D. Schönitzer, H. Berger
    Abstract:

    Acute cold haemagglutinin disease, most commonly associated with underlying mycoplasma infection, is rare in children. A 3-year-old girl who developed this auto-immune disease under intensive cytotoxic treatment for rhabdomyosarcoma is presented. Clinically, a livedo reticularis skin pattern upon exposure to cold which was reversible at room temperature and a spontaneous Red Cell Agglutination of blood samples in vitro led to the diagnosis. Together with bronchopneumonia the girl developed hyper-IgM, high antibody titres against Mycoplasma pneumoniae , as well as high titres of cold agglutinins. Laboratory signs of mild intravascular haemolysis were found. Positive direct antiglobulin test resulted from coating of Red Cells with C3d and C4. Three different antibodies were identified in serum: nonspecific cold agglutinins without complement activation, anti-I specific cold agglutinins with complement activation, as well as a weak biphasic Donath-Landsteiner haemolysin. Under antibiotic treatment and a short course of pRedisolone the clinical course was mild.

  • Cold haemagglutinin disease complicating Mycoplasma pneumoniae infection in a child under cytotoxic cancer treatment.
    European Journal of Pediatrics, 1992
    Co-Authors: F. M. Fink, K. Dengg, S. Kilga-nogler, D. Schönitzer, H. Berger
    Abstract:

    Acute cold haemagglutinin disease, most commonly associated with underlying mycoplasma infection, is rare in children. A 3-year-old girl who developed this auto-immune disease under intensive cytotoxic treatment for rhabdomyosarcoma is presented. Clinically, a livedo reticularis skin pattern upon exposure to cold which was reversible at room temperature and a spontaneous Red Cell Agglutination of blood samples in vitro led to the diagnosis. Together with bronchopneumonia the girl developed hyper-IgM, high antibody titres againstMycoplasma pneumoniae, as well as high titres of cold agglutinins. Laboratory signs of mild intravascular haemolysis were found. Positive direct antiglobulin test resulted from coating of Red Cells with C3d and C4. Three different antibodies were identified in serum: nonspecific cold agglutinins without complement activation, anti-I specific cold agglutinins with complement activation, as well as a weak biphasic Donath-Landsteiner haemolysin. Under antibiotic treatment and a short course of pRedisolone the clinical course was mild.

Bruno Catimel - One of the best experts on this subject based on the ideXlab platform.

  • simplified conjugation chemistry for coupling peptides to f ab fragments autologous Red Cell Agglutination assay for hiv 1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Simplified conjugation chemistry for coupling peptides to F(ab′) fragments: autologous Red Cell Agglutination assay for HIV-1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Kinetics of the autologous Red Cell Agglutination test.
    Journal of Immunological Methods, 1993
    Co-Authors: Bruno Catimel, Kim Wilson, Bruce E. Kemp
    Abstract:

    The kinetics of the autologous Red Cell Agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-Red blood Cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin alpha on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab' fragments were determined (n = 4.80 X 10(5) sites/erythrocyte, Ka = 0.43 X 10(7) M-1, Kd = 23 X 10(-8) M for 1C3/86, n = 4.66 X 10(5) sites/erythrocyte, Ka = 1.05 X 10(7) M-1, Kd = 9.5 X 10(-8) M for 10F7MN. The binding studies were performed under the same conditions as the autologous Red blood Cell Agglutination test. When 0.9 microgram of anti-glycophorin Fab was added to 10 microliters of blood 0.25 microgram of 1C3/86 Fab was bound whereas 0.29 microgram for 10F7MN Fab' was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 microgram of anti-peptide antibody gave half a maximal Agglutination score. This is equivalent to 10 micrograms/ml circulating antibody. Under the Agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal Agglutination occurRed when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum Agglutination score of four occurRed in the presence of 1 microgram of anti-peptide antibody equivalent to 100 micrograms/ml circulating antibody whereas an Agglutination score of 1+ was elicited by only 0.32 microgram anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin molecules per Red Cell.

K.i. Mitchelhill - One of the best experts on this subject based on the ideXlab platform.

  • simplified conjugation chemistry for coupling peptides to f ab fragments autologous Red Cell Agglutination assay for hiv 1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Simplified conjugation chemistry for coupling peptides to F(ab′) fragments: autologous Red Cell Agglutination assay for HIV-1 antibodies
    Journal of Immunological Methods, 1994
    Co-Authors: Kim Wilson, Bruno Catimel, K.i. Mitchelhill, Bruce E. Kemp
    Abstract:

    Abstract The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on Agglutination of autologous Red blood Cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous Red Cell Agglutination test is described. F(ab′)2 fragments of the anti-glycophorin antibody were prepaRed by pepsin digestion and Reduced to F(ab′) fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous Reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous Red Cell Agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

  • Autologous Red Cell Agglutination test for antibodies to feline immunodeficiency virus.
    Veterinary Immunology and Immunopathology, 1994
    Co-Authors: Kim Wilson, K.i. Mitchelhill, Wayne F. Robinson, Peter Gregory Bundesen, Bruce E. Kemp
    Abstract:

    Abstract The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous Red Cell Agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680–715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 μl of whole blood, circulating antibody to the peptide epitope caused Agglutination of the Red blood Cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680–715) peptide corresponding to the FIV, PPR strain gp40 (678–716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.