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Immunoblotting in the serodiagnosis of infectious diseases
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cysystis carinii pneumonia in the United States: epidemiologic, clinical. and diagnostic features. Ann Intern Med 1974; 80: 83-93. Ruskin J. Parasitic diseases in the compromised host. In Rubin RH, Young LS. eds. Clinical approach to infection in the compromised host. New York: Plenum Publishing Co., 1981: 269334. Springmeyer SC, Silvestri RC, Sale GE er al. The role of transbronchial biopsy for the diagnosis of diffuse pneumonias in immunocompromise marrow transplant recipients. Am Rev Respir Dis 1982; 126: 763-5. Pifer LL, Hughes WT, Murphy MJ. Propagation of Pneumocystis curinii in vitro. Pediatr Res 1977; II: 305-11. Pifer LL. Hughes WT. Stagno S, Woods DR. Pneumocystb carinii infection: evidence for high prevalence in normal and immuno-suppressed children. Pediatrics 1978. Meyers JD, Pifer LL, Sale GE, Thomas ED. The value of Pneumocysfis carinii antibody and antigen detection for diagnosis of Pneumocystis carinii pneumonia after marrow transplantation. Am Rev Respir Dis 1979; 120: 1283-8. Jarowenko M, Pifer L, Kerman R, Kahan BD. Serologic methods for the early diagnosis of Pfleumocysfb carinii infection in renal allograft recipients. Transplantation 1986; 41: 43641. Walzer PD, Young LS. Clinical relevance of animal models of Pneumocysris carinii pneumonia. Diagn Microbial Infect Dis 1984; 2: I 6. Pifer LL, Lattuada CP. Edwards CC et al. Pneumocysris cnrinii in germ-free rats: implications for human patients. Diagn Microbial Infect Dis 1984; 2: 23-l. Milder JE, Walzer PD, Coonrod JD ef al. Comparison of histological and immunological techniques for detection of Pneumocysh carinii in rat bronchial lavage fluid. J Clin Microbial 1980; I I: 409913. Maddison SE, Hayes GV, Slemenda SB et al. Detection of specific antibody by enzyme-linked immunosorbent assay and antigenemia by counterimmunoelectrophoresis in humans infected with Pneumocystis carinii. J Clin Microbial 1982; 15: 1036. Maddison SE, Walls KW, Haverkos HW. Juranek DD. Evaluation of serologic tests for Pneumocystis carinii antibody and antigencmia in patients with Acquired Immunodeficiency Syndrome. Diagn Microbial Infect Dis 1984; 2: 6975. Pitchenik AE, Ganjei P, Torres A, Evans DA, Rubin E, Baier H. Sputum examination for the diagnosis of Pneumocysris carinii pneumonia in the acquired immunodeficiency syndrome. Chest 1985; 88: 659-62. Bigby TD, Margolskcc D, Curtis JL er al. The usefulness of induced sputum in the diagnosis of Pneumocystis cnrinii pneumonia in patients with the acquired immunodeficiency syndrome. Am Rev Respir Dis 1986; 133: 515-18.
articles
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17. Kovacs JA. Swan JC, Shelhamer J rt al. Prospective evaluation of a monoclonsl antibody in diagnosis of Pneumoc~stis carinii pneumonia. Lancet 1986; 2: I- 3.
Immunoblotting in the serodiagnosis infectious diseases
of
lmmunoblotting (“Western blotting”) has provided a major new approach to the cxamination of the serological response to infectious diseases. This technique gives a permanent rccord of the antibodies produced against individual components of a microbe, after separating these according to their molecular size by electrophoresis. Excellent reviews of the mcthodology involved are now available’ ‘. Briefly, the protein mixture extracted from the microbe is first separated by gel electrophoresis and then transferred (blotted) onto a membrane such as nitrocellulose by capillary action or, preferably. under the influence of an electric field. The latter can be provided by a number of commercially available blotting chambers and has the advantage that transfer is faster and more efficient so that the resolution of the original gel is maintained. Before proceeding with the immune reaction, free protein binding sites on the membrane must be blocked by incubation with, for example, bovine serum albumin or a mild non-ionic detergent such as Tween 20. Prolongation of the duration of the blocking procedure may promote renaturation of antigenic sites’. Subsequent incubation with the patient’s serum is carried out in the presence of the blocking protein and detergent to further prevent non specific binding. After extensive washing, antibodies present in the patient’s serum which have bound to the blot are visualised using a second labelled antibody raised against human IgG, IgM or IgA. The label may be an isotope or. more conveniently, an enzyme such as horseradish peroxidase or alkaline phosphatase. The combination of immunoblotting and reflectance densitometry has been used as a sensitive, quantitative immunoassay’,.“. The potential to quantify antibody responses with the immunoblot technique has been neglected in many studies, as also has the advantage of examining multiple sera from each patient. Serial sera from patients’ with systemic candidosis demonstrated that the amount of antibody detectable could vary greatly within a few days. For example, a patient who died from
166
Leading Articles
systemic candidosis had considerable amounts of antibody present 6 days before death, which rapidly declined to barely detectable levels. In contrast, one of the surviving patients developed very high titres of antibody to an immunodominant 47 kD antigen over a 6-day periodi. If single sera had been examined from these patients the correlation between recovery and antibody production would have been missed. Immunoblotting provides a means of determining which components of a microbe react specifically with sera from infected individuals and not uninfected controls, without the need to purify individual antigens. Such antigens may have value in the development of serodiagnostic tests. Hanff et al. used immunoblotting to show that sera from the uninfected individuals reacted weakly with three polypeptides of Trqwnema pallidurn. but that other polypeptides were recognised only by sera from patients with syphilish. Antibody to these specific treponemal antigens was largely acquired as the disease progressed from primary to secondary and early latent syphilis. Electrophoresis is usually performed on protein mixtures which have been denatured by boiling in the presence of excess sodium dodecyl sulphate, SDS (anionic detergent), and a thiol reagent such as 2-mercaptoethanol. This ensures that most components will be small enough to enter the gel as single polypeptides and they will migrate according to their size so that their molecular weight can be estimated. A single band on an immunblot may contain several polypeptides of similar molecular size and these can be determined by isoelectric focussing of the electroeluted band or by two dimensional electrophoresis. The disadvantage of denaturing the preparation prior to electrophoresis and immunoblottjng is that the analysis will be limited to those antigens which remain serologically reactive after tliis procedure. Fehniger et ul. described a large 19(),kD antigen of T. pullidum which reacted specifically with syphilitic scra but had previously been missed by immunoblot analyses’. This was probably because this antigen was converted by boiling in 0.1% SDS to a predominant 19 kD form which was serologicdlly non-reactive. The size range of the polypeptides examined using the immunoglot technique will depend on the percentage of acrylamide used during the electrophoresis step, a higher percentage being used to examine low molecular weight moieties. Nitrocellulose membrane with a pore size of
0.45 urn is the most commonly used for the transfer of proteins but low molecular weight proteins can pass straight through this. A smaller pore size of 0.1-0.2 urn may overcome this problem. Not all proteins transfer with equal ease. High molecular weight proteins may need longer transfer times or higher field strengths, in which case special cooling facilities are required. The efficiency of transfer should be checked by staining the gel after transblotting with Coomassie blue. The nitrocellulose membrane itself can be stained for the presence of proteins by amido black or colloidal gold. Transfer buffers of low ionic strength are used in the transblot chamber so that high voltages can be applied. The addition of methanol to the buffer avoids the swelling of gels which occurs with low ionic strength buffers and also increases the binding capacity of nitrocellulose for protein’.?. However, it reduces the elution efficiency of high molecular weight proteins ( > 100 kD) from SDS polyacrylamide gels. As well as being a useful tool in the development of serodiagnostic tests, immunoblotting is now being used in some centres as a diagnostic procedure. For this purpose it has been particularly widely used as a confirmatory test for the presence of antibodies to human immunodeficiency virus (HIV) in individuals with AIDS, AIDS-related complex (ARC) or asymptomatic persons at risk for AIDS. Immunoblotting will detect antibody to HIV antigens p 15---l% ~24, ~31, gp41, ~53355, p64 and gp120’. Reactivity with p24 and/or gp 41 has been suggested as the minimum requirement for HIV seropositivity’. However, there have been several reports of blood donor sera with false-positive anti-HIV immunoblot reactions”‘,” The three blood donor sera described by Biberfield et rrl.” showed strong bands at p24 and p 55 and additional specimens from the donors showed the same pattern of reactivity. The sera were positive by enzyme-linked immunosorbent assay (ELISA) kits from Organon. Pasteur and Du Pont but negative by competitive ELISA (Wellcome and Abbott) and by immunofluorescence using as antigen smeared and fixed HIV infected 119 cells. One possible cause of false-positive immunoblots for HIV antibody is infection with other human retroviruses such as HTLV-IV”. Probably a commoner cause is the presence of auto-antibodies in patients’ sera which cross react with normal cell constituents contaminating the preparation of the HIV antigen. This is a potential problem with any microbe which
Leading has to be grown in tissue culture, such as viruses. or grown up in viva in experimental animal infections, such as Pneumocystis carinii. In these circumstances it is wise to include a lane of uninfected tissue culture cell lysate or, in the case of P. carinii, uninfected lung homogenate” in the immunoblot analysis. The immunoblot technique has also been advocated as an additional diagnostic tool for differentiating between true and false-positive serological reactions for syphilis14. Whole extracts of T. pallidurn were used for these analyses but since a 17 kD component of T. pallidurn was found to be the most reliable specific antigenic marker, the authors suggest that an immunodot enzyme-linked immunosorbent assay using purified 17 kD antigen might ultimately prove more convenient. Immunoblotting may therefore be of value directly in the diagnosis of an infectious disease and indirectly in the identification of immunodominant antigens for use in the development of serodiagnostic tests. Since the technique enables us to examine the antibody responses occurring to many individual components of complex micro-organisms, it may also bring to light previously unrecognised antigens which play a central role in the immune response to the infection. Subsequent purification and characterisation of such antigens may have a role to play in an immunotherapeutic approach to the treatment and prevention of infectious diseases. RUTH Department
C. MATTHEWS
01 Medical Microbiolog? St Bartholomew’s Hospital West Smithjield London ECIA 7BE
References I.
Towbin H, Gordon J. lmmunoblotting and dot immunobinding ~-current status and &tlook. J tmmunol Methods 1984; 72: 31340.
Articles 2. Gersshoni JM. Pdlade GE. principles and application. 1983: 131: l-15.
167 Protein
blotting:
Analyt
Biochem
3. Jones A. Picking up proteins. Laboratory Practice 1986; June: 47 51. 4. Au-Young JK, Troy FA. Goldstein E. Serologic analysis of antigen-specific reactivity in patients with systemic candidiasis. Diagn Microbial Infect Dis 1985: 3: 419 32. 5. Matthews RC, Burnie JP. Tabaqchali S. Immunoblot analysis of the serological response in systemic candidosis. Lancet 1984; ii: I41 5 IX. 6. Hanff PA, Fehniger TE. Miller JN. Lovett MA.
Humoral immune response in human syphilis to polypeptides of Trrponema pallidurn. J lmmunol 1982; 129: 1287 91. 7. Fehniger TE, Walfield AM, Cunningham TM 4, ul. Purification and characterization of a cloned protease-resistant Treponenw pallidunl-specific antigen. Infect Immunity 1984; 46: 598 607. 8. Schupbach J, Popovic M, Gilden RV ct ~1. Serological analysis of a subgroup of human Tlymphotropic retroviruses (HTLV-III) associated with AIDS. Science 1984: 224: 503 5. 9. Centers for Disease Control. lipdate: Provisional public health service inter-agency recommendations for screening donated blood and plasma for antibody to the virus causing acquired immunodeficiency syndrome. MMWR 19x5; 34: I 5. IO. Saag MS, Britz J. Asymptomatic blood donor with a false-positive HTLV-III Western blot N Engl J Med. 1986; 312: I IX. I I. Biberheld G. Bredberg-Raden U. Bottiger B e( ul. Blood donor sera with false-positive Western blot reactions to human immunodehciency virus. Lancet (letter) 1986: ii: 2X9-90. 12. Kanki PJ, Barin F. M‘Boup S et ul. New human T-lymphotropic rctrovirus related to simian Tlymphotropic virus type 111 (STLV III,,,,). Science 1986; 232: 238 43. 13. Gtgliotti F. Stokes DC. Cheatham AB 01 ul. Development of murine monoclonal antibodies to Pnc~rnio~:r.c/r\ urinii. J. Infect Dis 19X6: 154: ?I 2’ 14. Hensel U, Wellensiek H. Bhakdi S. Sodium dodecyl sulphate~ polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of syphilitic infections. J Clin Microblol 19X5: 31: X2 7
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cysystis carinii pneumonia in the United States: epidemiologic, clinical. and diagnostic features. Ann Intern Med 1974; 80: 83-93. Ruskin J. Parasitic diseases in the compromised host. In Rubin RH, Young LS. eds. Clinical approach to infection in the compromised host. New York: Plenum Publishing Co., 1981: 269334. Springmeyer SC, Silvestri RC, Sale GE er al. The role of transbronchial biopsy for the diagnosis of diffuse pneumonias in immunocompromise marrow transplant recipients. Am Rev Respir Dis 1982; 126: 763-5. Pifer LL, Hughes WT, Murphy MJ. Propagation of Pneumocystis curinii in vitro. Pediatr Res 1977; II: 305-11. Pifer LL. Hughes WT. Stagno S, Woods DR. Pneumocystb carinii infection: evidence for high prevalence in normal and immuno-suppressed children. Pediatrics 1978. Meyers JD, Pifer LL, Sale GE, Thomas ED. The value of Pneumocysfis carinii antibody and antigen detection for diagnosis of Pneumocystis carinii pneumonia after marrow transplantation. Am Rev Respir Dis 1979; 120: 1283-8. Jarowenko M, Pifer L, Kerman R, Kahan BD. Serologic methods for the early diagnosis of Pfleumocysfb carinii infection in renal allograft recipients. Transplantation 1986; 41: 43641. Walzer PD, Young LS. Clinical relevance of animal models of Pneumocysris carinii pneumonia. Diagn Microbial Infect Dis 1984; 2: I 6. Pifer LL, Lattuada CP. Edwards CC et al. Pneumocysris cnrinii in germ-free rats: implications for human patients. Diagn Microbial Infect Dis 1984; 2: 23-l. Milder JE, Walzer PD, Coonrod JD ef al. Comparison of histological and immunological techniques for detection of Pneumocysh carinii in rat bronchial lavage fluid. J Clin Microbial 1980; I I: 409913. Maddison SE, Hayes GV, Slemenda SB et al. Detection of specific antibody by enzyme-linked immunosorbent assay and antigenemia by counterimmunoelectrophoresis in humans infected with Pneumocystis carinii. J Clin Microbial 1982; 15: 1036. Maddison SE, Walls KW, Haverkos HW. Juranek DD. Evaluation of serologic tests for Pneumocystis carinii antibody and antigencmia in patients with Acquired Immunodeficiency Syndrome. Diagn Microbial Infect Dis 1984; 2: 6975. Pitchenik AE, Ganjei P, Torres A, Evans DA, Rubin E, Baier H. Sputum examination for the diagnosis of Pneumocysris carinii pneumonia in the acquired immunodeficiency syndrome. Chest 1985; 88: 659-62. Bigby TD, Margolskcc D, Curtis JL er al. The usefulness of induced sputum in the diagnosis of Pneumocystis cnrinii pneumonia in patients with the acquired immunodeficiency syndrome. Am Rev Respir Dis 1986; 133: 515-18.
articles
165
17. Kovacs JA. Swan JC, Shelhamer J rt al. Prospective evaluation of a monoclonsl antibody in diagnosis of Pneumoc~stis carinii pneumonia. Lancet 1986; 2: I- 3.
Immunoblotting in the serodiagnosis infectious diseases
of
lmmunoblotting (“Western blotting”) has provided a major new approach to the cxamination of the serological response to infectious diseases. This technique gives a permanent rccord of the antibodies produced against individual components of a microbe, after separating these according to their molecular size by electrophoresis. Excellent reviews of the mcthodology involved are now available’ ‘. Briefly, the protein mixture extracted from the microbe is first separated by gel electrophoresis and then transferred (blotted) onto a membrane such as nitrocellulose by capillary action or, preferably. under the influence of an electric field. The latter can be provided by a number of commercially available blotting chambers and has the advantage that transfer is faster and more efficient so that the resolution of the original gel is maintained. Before proceeding with the immune reaction, free protein binding sites on the membrane must be blocked by incubation with, for example, bovine serum albumin or a mild non-ionic detergent such as Tween 20. Prolongation of the duration of the blocking procedure may promote renaturation of antigenic sites’. Subsequent incubation with the patient’s serum is carried out in the presence of the blocking protein and detergent to further prevent non specific binding. After extensive washing, antibodies present in the patient’s serum which have bound to the blot are visualised using a second labelled antibody raised against human IgG, IgM or IgA. The label may be an isotope or. more conveniently, an enzyme such as horseradish peroxidase or alkaline phosphatase. The combination of immunoblotting and reflectance densitometry has been used as a sensitive, quantitative immunoassay’,.“. The potential to quantify antibody responses with the immunoblot technique has been neglected in many studies, as also has the advantage of examining multiple sera from each patient. Serial sera from patients’ with systemic candidosis demonstrated that the amount of antibody detectable could vary greatly within a few days. For example, a patient who died from
166
Leading Articles
systemic candidosis had considerable amounts of antibody present 6 days before death, which rapidly declined to barely detectable levels. In contrast, one of the surviving patients developed very high titres of antibody to an immunodominant 47 kD antigen over a 6-day periodi. If single sera had been examined from these patients the correlation between recovery and antibody production would have been missed. Immunoblotting provides a means of determining which components of a microbe react specifically with sera from infected individuals and not uninfected controls, without the need to purify individual antigens. Such antigens may have value in the development of serodiagnostic tests. Hanff et al. used immunoblotting to show that sera from the uninfected individuals reacted weakly with three polypeptides of Trqwnema pallidurn. but that other polypeptides were recognised only by sera from patients with syphilish. Antibody to these specific treponemal antigens was largely acquired as the disease progressed from primary to secondary and early latent syphilis. Electrophoresis is usually performed on protein mixtures which have been denatured by boiling in the presence of excess sodium dodecyl sulphate, SDS (anionic detergent), and a thiol reagent such as 2-mercaptoethanol. This ensures that most components will be small enough to enter the gel as single polypeptides and they will migrate according to their size so that their molecular weight can be estimated. A single band on an immunblot may contain several polypeptides of similar molecular size and these can be determined by isoelectric focussing of the electroeluted band or by two dimensional electrophoresis. The disadvantage of denaturing the preparation prior to electrophoresis and immunoblottjng is that the analysis will be limited to those antigens which remain serologically reactive after tliis procedure. Fehniger et ul. described a large 19(),kD antigen of T. pullidum which reacted specifically with syphilitic scra but had previously been missed by immunoblot analyses’. This was probably because this antigen was converted by boiling in 0.1% SDS to a predominant 19 kD form which was serologicdlly non-reactive. The size range of the polypeptides examined using the immunoglot technique will depend on the percentage of acrylamide used during the electrophoresis step, a higher percentage being used to examine low molecular weight moieties. Nitrocellulose membrane with a pore size of
0.45 urn is the most commonly used for the transfer of proteins but low molecular weight proteins can pass straight through this. A smaller pore size of 0.1-0.2 urn may overcome this problem. Not all proteins transfer with equal ease. High molecular weight proteins may need longer transfer times or higher field strengths, in which case special cooling facilities are required. The efficiency of transfer should be checked by staining the gel after transblotting with Coomassie blue. The nitrocellulose membrane itself can be stained for the presence of proteins by amido black or colloidal gold. Transfer buffers of low ionic strength are used in the transblot chamber so that high voltages can be applied. The addition of methanol to the buffer avoids the swelling of gels which occurs with low ionic strength buffers and also increases the binding capacity of nitrocellulose for protein’.?. However, it reduces the elution efficiency of high molecular weight proteins ( > 100 kD) from SDS polyacrylamide gels. As well as being a useful tool in the development of serodiagnostic tests, immunoblotting is now being used in some centres as a diagnostic procedure. For this purpose it has been particularly widely used as a confirmatory test for the presence of antibodies to human immunodeficiency virus (HIV) in individuals with AIDS, AIDS-related complex (ARC) or asymptomatic persons at risk for AIDS. Immunoblotting will detect antibody to HIV antigens p 15---l% ~24, ~31, gp41, ~53355, p64 and gp120’. Reactivity with p24 and/or gp 41 has been suggested as the minimum requirement for HIV seropositivity’. However, there have been several reports of blood donor sera with false-positive anti-HIV immunoblot reactions”‘,” The three blood donor sera described by Biberfield et rrl.” showed strong bands at p24 and p 55 and additional specimens from the donors showed the same pattern of reactivity. The sera were positive by enzyme-linked immunosorbent assay (ELISA) kits from Organon. Pasteur and Du Pont but negative by competitive ELISA (Wellcome and Abbott) and by immunofluorescence using as antigen smeared and fixed HIV infected 119 cells. One possible cause of false-positive immunoblots for HIV antibody is infection with other human retroviruses such as HTLV-IV”. Probably a commoner cause is the presence of auto-antibodies in patients’ sera which cross react with normal cell constituents contaminating the preparation of the HIV antigen. This is a potential problem with any microbe which
Leading has to be grown in tissue culture, such as viruses. or grown up in viva in experimental animal infections, such as Pneumocystis carinii. In these circumstances it is wise to include a lane of uninfected tissue culture cell lysate or, in the case of P. carinii, uninfected lung homogenate” in the immunoblot analysis. The immunoblot technique has also been advocated as an additional diagnostic tool for differentiating between true and false-positive serological reactions for syphilis14. Whole extracts of T. pallidurn were used for these analyses but since a 17 kD component of T. pallidurn was found to be the most reliable specific antigenic marker, the authors suggest that an immunodot enzyme-linked immunosorbent assay using purified 17 kD antigen might ultimately prove more convenient. Immunoblotting may therefore be of value directly in the diagnosis of an infectious disease and indirectly in the identification of immunodominant antigens for use in the development of serodiagnostic tests. Since the technique enables us to examine the antibody responses occurring to many individual components of complex micro-organisms, it may also bring to light previously unrecognised antigens which play a central role in the immune response to the infection. Subsequent purification and characterisation of such antigens may have a role to play in an immunotherapeutic approach to the treatment and prevention of infectious diseases. RUTH Department
C. MATTHEWS
01 Medical Microbiolog? St Bartholomew’s Hospital West Smithjield London ECIA 7BE
References I.
Towbin H, Gordon J. lmmunoblotting and dot immunobinding ~-current status and &tlook. J tmmunol Methods 1984; 72: 31340.
Articles 2. Gersshoni JM. Pdlade GE. principles and application. 1983: 131: l-15.
167 Protein
blotting:
Analyt
Biochem
3. Jones A. Picking up proteins. Laboratory Practice 1986; June: 47 51. 4. Au-Young JK, Troy FA. Goldstein E. Serologic analysis of antigen-specific reactivity in patients with systemic candidiasis. Diagn Microbial Infect Dis 1985: 3: 419 32. 5. Matthews RC, Burnie JP. Tabaqchali S. Immunoblot analysis of the serological response in systemic candidosis. Lancet 1984; ii: I41 5 IX. 6. Hanff PA, Fehniger TE. Miller JN. Lovett MA.
Humoral immune response in human syphilis to polypeptides of Trrponema pallidurn. J lmmunol 1982; 129: 1287 91. 7. Fehniger TE, Walfield AM, Cunningham TM 4, ul. Purification and characterization of a cloned protease-resistant Treponenw pallidunl-specific antigen. Infect Immunity 1984; 46: 598 607. 8. Schupbach J, Popovic M, Gilden RV ct ~1. Serological analysis of a subgroup of human Tlymphotropic retroviruses (HTLV-III) associated with AIDS. Science 1984: 224: 503 5. 9. Centers for Disease Control. lipdate: Provisional public health service inter-agency recommendations for screening donated blood and plasma for antibody to the virus causing acquired immunodeficiency syndrome. MMWR 19x5; 34: I 5. IO. Saag MS, Britz J. Asymptomatic blood donor with a false-positive HTLV-III Western blot N Engl J Med. 1986; 312: I IX. I I. Biberheld G. Bredberg-Raden U. Bottiger B e( ul. Blood donor sera with false-positive Western blot reactions to human immunodehciency virus. Lancet (letter) 1986: ii: 2X9-90. 12. Kanki PJ, Barin F. M‘Boup S et ul. New human T-lymphotropic rctrovirus related to simian Tlymphotropic virus type 111 (STLV III,,,,). Science 1986; 232: 238 43. 13. Gtgliotti F. Stokes DC. Cheatham AB 01 ul. Development of murine monoclonal antibodies to Pnc~rnio~:r.c/r\ urinii. J. Infect Dis 19X6: 154: ?I 2’ 14. Hensel U, Wellensiek H. Bhakdi S. Sodium dodecyl sulphate~ polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of syphilitic infections. J Clin Microblol 19X5: 31: X2 7