615
limiting step in the degradation of haem to bilirubin. Bilirubin is a potent antioxidant. Haem oxygenase is active only in the presence of oxygen and so would be activated during the ensuing reperfusion events; this accords with in-vitro data showing that different stress proteins are released in hypoxia and following reperfusion.7 Thus in the hypoxic joint the 32 kD protein could have an important scavenging role. The anti-rheumatic drug auranofin induces synthesis of the 32 kD protein in vitro 8 This is a direct effect, dependent on auranofin crossing the cell membrane; nevertheless, both D-penicillamine and gold(I) thiomalate can induce the formation of ROS and might indirectly induce synthesis of the 32 kD protein in vivo. A target of hypoxic/reperfusion injury is the endothelial cell. Angiogenesis, which follows myocardial infarction, is also a feature of rheumatoid synovia.1 In the presence of tumour necrosis factor alpha, which is found in rheumatoid synovial fluid, the 28 kD stress protein is phosphorylated and can activate endothelial cells,9 possibly initiating proliferation or the release of angiogenic factors. Cytoskeletal derangements occur in hypoxic conditions and the 28 kD protein associates with actin microfilaments (B. Geiger, personal communication), causing changes in morphology and perhaps in vascular permeability. The 28 kD protein may be another focus for drug development. Other cytoskeletal components may have a role in vascular permeability. The intermediate filament vimentin is much increased in synovial cells and is very sensitive to oxidative damage. ROS, like heat shock, cause collapse of the vimentin cytoskeleton around the nucleus,1O where it becomes associated with a 70 kD stress protein. Disodium aurothiomalate at 01 mmol/1 induces a similar rearrangement of intermediate filaments in fibrolasts (K. R. Rogers, unpublished). The highly conserved stress proteins control the basic biology of cellular interactions, and the complex nature of the biochemical processes involved deserves careful exploration. Moreover, apparently diverse therapeutic approaches to suppress rheumatoid disease may have in common the ability to induce stress protein synthesis. Bone and Joint Research Unit, ARC Building, London Hospital Medical College, London E1 2AD, UK
V. R. WINROW D. R. BLAKE
CR, Williams RB, Farrell AJ, Blake DR. Hypoxia and inflammatory synovitis: observations and speculation. Ann Rheum Dis 1991; 50: 124-32. 2. Allen RE, Blake DR, Nazhat NB, Jones P. Superoxide radical generation by inflamed human synovium after hypoxia. Lancet 1989; ii: 282-83. 3. Ananthan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986; 232: 1. Stevens
522-24. 4. Winrow
VR, Mojdehi G, Mapp PI, Rampton DS, Blake DR. Immunohistological localisation of stress proteins in inflammatory tissue. In: Burdon R, Rice-Evans C, Blake D, Winrow V, eds. Stress proteins in inflammation. London: Richelieu Press,
1990: 237-51.
5.de Graeff-Meeder ER, Voorhorst M, van Eden W, et al. Antibodies to the mycobacterial 65-kd heat-shock protein are reactive with synovial tissue of adjuvant arthritic rats and patients with rheumatoid arthritis and osteoarthritis. Am J Pathol 1990; 137: 1013-17. 6. Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-KDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA 1989; 86: 99-103. 7. Sciandra JJ, Subjeck JR, Hughes CS. Induction of glucose-regulated proteins after anaerobic exposure and of heat-shock proteins after reoxygenation. Proc Natl Acad Sci USA 1984; 81: 4843-47. 8. Caltabiano MM, Poste G, Greig RG. Induction of the 32-kD human stress protein by auranofin and related triethylphosphine gold analogs. Biochem Pharmacol 1988; 37: 4089-93. 9. Darbon JM, Issandou M, Tournier JF, Bayard F. The respective 27kDa and 28kDa protein kinase C substrates in vascular endothelial and MCF-7 cells are most probably heat shock proteins. Biochem Biophys Res Commun 1990; 168: 527-36. 10. Rogers KR, Morris CJ, Blake DR. Cytoskeletal rearrangment by oxidative stress. Int J Tiss React 1989; 9: 309-14.
Can disposables make matters worse? SiR,—Dr Rudin and colleagues’ letter on HIV, hepatitis, and measles in Romanian children (Dec 22/29, p 1592-93) leaves us wavering between sadness and despair. The situation is terrible: 12 of 20 orphans in hospital in Pascani, north-eastern Romania were HIV positive and most had evidence of exposure to hepatitis B virus. Rudin et al blame the "lack of disposable needles and medical
supplies and the policy of transfusion of untested blood" for this spread of bloodborne diseases among children, and they suggest an international effort to supply disposable needles and syringes. But the situation is more complex than this: a closer look reveals that this "solution" could have an effect opposite to that which might be expected. In Romania the distribution of disposable injecting equipment was a top priority of donor countries and agencies and some hospitals were quickly well supplied with such items, while in others people fought hard to obtain them. The disposable materials were enthusiastically adopted, but the lack of safe disposal points and inadequate education of the public resulted in the presence of large numbers of used, uncleaned needles and syringes in the environment. Moreover, the abundance of disposables favoured the spread of parenteral treatment outside hospitals. The discontinuity in availability of disposable needles and syringes meant frequent switching to the former practice of making do with reusable materials, but now there was antipathy to the extra work of cleaning and sterilisation. Former routines of needle and syringe sterilisation were sometimes forgotten; the media cast doubt on the efficacy of these procedures and as a result the public have tended to reject old-fashioned glass syringes. Thus pressure to re-use non-re-usable materials is at work. When people’s behaviour and the social infrastructure are the culprits, as here, a measure such as the distribution of needles and syringes should be supplemented by a programme of re-education to ensure that every type of needle and syringe is used properly and that instructions are adhered to. Whenever the focus of health intervention is an object, rather than people with human feelings, an apparently simple measure such as distributing needles and syringes will have the capacity to create explosive epidemics. As has been argued in another context, development aid may add only yet more disturbance when the ecological foundation of health is
neglected.’1 This letter could not have been written in the Ceausescu era, when it was an offence punishable by imprisonment to receive a western medical journal? Today we are fortunate to have complimentary subscriptions offered by journals, such as The Lancet and its readers3 or received by exchange agreements between our local medical journal Revista Medico-Chirurgicala and similar western publications. To cite a British opinion, "there are resources that may rescue Romania from total disaster: the intelligence, inventiveness, and wit of its people".2 This feedback is part of that
package. Pulmonary Diseases Clinic, Institute of Medicine and Pharmacy, lasi 6600, Romania
T. MIHAESCU L. VERES
1. Rijpma S, Barnes J, Meegan MK, Taylor CE. The "demographic trap". Lancet 1991; 337: 50-51. 2. Anon. Medicine in Romania. Br Med J 1990; 300: 699. 3. Heley MM. Journals for Romania. Lancet 1990; 336: 1013.
Optic neuritis associated with dideoxyinosine SIR,-2’,3’-dideoxyinosine (ddl) is being studied in patients with AIDS or AIDS-related complex who do not tolerate or who do not respond to zidovudine. The major adverse reactions to ddl are peripheral neuropathy and pancreatitis.2 We report a case of optic neuritis 6 weeks after the introduction of ddI. Although the relation between ddl and optic neuritis cannot be asserted, the stabilisation of visual loss after interruption of ddl and its deterioration when this therapy was resumed are consistent with a toxic role of the drug. A 40-year-old homosexual man with HIV infection (CDC class IVC2 [hairy leukoplakia of the tongue]) had been treated with zidovudine 1200 mg daily since 1988 because of a CD4 count of 190/ul.In June, 1990, oesophageal candidiasis developed and the CD4 count was only 70/tit. 1 month later a herpes zoster eruption was treated with acyclovir. The CD4 count was 20/ul. Zidovudine was discontinued, and in October, 1990, ddl was introduced at a dose of 8 mg/kg daily, associated with fluconazole 50 mg per day
616
because of
oral candidiasis. 6 weeks later the patient of blurred vision and his visual acuity was only 3/10 in complained both eyes. Goldman perimetry revealed scotoma and visual evoked potentials were consistent with bilateral optic retrobulbar neuritis. The optic fundi and a computerised tomographic scan of the brain were normal. CSF examination showed no cells, normal glucose, protein 70 mg/dl, and HIV p24 antigen (80 pg/ml). Tests for cytomegalovirus, syphilis, toxoplasma, cryptococcal antigen, and mycobacteria were negative. ddl therapy was interrupted and the patient’s visual acuity stabilised. 6 weeks later, because of a drop in CD4 count (8/1), the patient asked for further anti-HIV therapy and ddI was introduced as before. After 3 weeks of therapy his visual acuity was 1/20 in both eyes and examination of optic fundi revealed atrophy of the optic disks. Neurological examination was otherwise normal. Amblyopia, diplopia, optic atrophy, and blindness in patients on ddl have occasionally been reported to the manufacturers (BristolMyers Squibb). The neurotropic nature of HIV, the neuropathological manifestations of some opportunistic infections, and the large range of medications prescribed at the same time in AIDS patients make it difficult to sort out the cause of these events. Optic nerve disease in HIV infection may be due to a wide variety of complications, but opportunistic infections are the most frequent.3 In our patient no opportunist could be implicated. The role of HIV itself in direct optic nerve injury cannot be ruled out, but the clinical history is more consistent with toxicity. Clinicians should be aware of this possibility when treating AIDS patients who have visual defects.
persistent
A. LAFEUILLADE L. AUBERT P. CHAFFANJON R. QUILICHINI
Internal Medicine Service,
Hôpital Chalucet, 83000 Toulon, France 1. McGowan
JJ, Tomaszewski JE, Cradok J, et al. Overview of the preclinical development of an antiretroviral drug, 2’,3’-dideoxynosine. Rev Infect Dis 1990;
12: S513-21. 2. Yarchoan R, Pluda
JM, Thomas RV,
2’3’-dideoxyinosine
et al. Long-term toxicity/activity profile of in AIDS and AIDS-related complex. Lancet 1990; 336:
526-29. 3. Winward KE, Hamed LM, Glaser JS. The spectrum of optic nerve disease in human immunodeficiency virus infection. Am J Ophthalmol 1989; 107: 373-80.
Misidentification of HIV-2 proteins by western blots SiR,—The US Food and Drug Administration has approved a screening assay for HIV-2 (HIV-2 EIA; Genetic Systems) but no licensed supplementary test (eg, western blot [WB]) is yet available. We report here the misidentification of protein bands on unlicensed commercial HIV-2 WBs. This had prompted us to re-examine World Health Organisation criteria1 for a positive HIV-2 WB. We have generated eleven monospecific antibodies from rabbits and a murine monoclonal antibody, using synthetic peptides derived from the genomic sequences of HIV-2 (HIV -2RODY and purified HIV-2 proteins as immunogens: Protein Antibody Sequence* E1007 env 23-37 ext E1008 env 473-487 ext E1036 env 633-650 transm E1042 env 846-859 transm P1035 endo pol 947-964 P1037 RT pol 386-403 G1013 gag 107-126 p16 G1020 gag 149-164 p26 G1031 gag 215-229 p26 RabX rgpl20 ext rgpl20 (HIV-2ROD) RabX p26 purified p26 (HIV-2 p26 MablH5 (mouse) transm HIV-2jj lysate *Peptide sequences according to HIV-2ROD (ref 2). Peptides were conjugated to keyhole lympet haemocyanin as immunogen. RT = reverse trans=transmembrane; ext=external ; transcriptase; endo=endonuclease.
Using these
antibodies and a commercial monoclonal antibody Abbott Laboratories) directed against the transmembrane protein of HIV-2, we tested three commercial
(3Al8-73;
Immunoblots of monospecific rabbit sera and monoclonal antibodies with commercial HIV-2 antigen strips from Biotech Research Laboratories (A), Diagnostic Pasteur (B), and Genetic Systems (C). Lane 1=HIV-2-positive human serum provided by individual manufacturer. Lane2= RabX rgp120 ; lane 3= E1007; lane 4= E1008; lane 5=E1042; !ane6=E1036. Lane 7=Mab1H5; lane 8=Mab 3A18-73; lane 9=P1037; lane 10 = P1035; lane 11 RabX p26. Lane 12=G1020; lane 13=G1031; lane 14=G1013, All rabbit antisera diluted 1 in 50 in milk buffer and incubated for 2 h at room temperature; Mab 1 H5 (culture supernatant) was diluted 1 in 2 and Mab 3A18-17 was diluted 1 in 100. Bound antibodies were detected by alkaline phosphatase conjugated anti-rabbit IgG or anti-mouse IgG and BCIP/nitroblue-tetrazolium chromogenic substrates. =
limiting step in the degradation of haem to bilirubin. Bilirubin is a potent antioxidant. Haem oxygenase is active only in the presence of oxygen and so would be activated during the ensuing reperfusion events; this accords with in-vitro data showing that different stress proteins are released in hypoxia and following reperfusion.7 Thus in the hypoxic joint the 32 kD protein could have an important scavenging role. The anti-rheumatic drug auranofin induces synthesis of the 32 kD protein in vitro 8 This is a direct effect, dependent on auranofin crossing the cell membrane; nevertheless, both D-penicillamine and gold(I) thiomalate can induce the formation of ROS and might indirectly induce synthesis of the 32 kD protein in vivo. A target of hypoxic/reperfusion injury is the endothelial cell. Angiogenesis, which follows myocardial infarction, is also a feature of rheumatoid synovia.1 In the presence of tumour necrosis factor alpha, which is found in rheumatoid synovial fluid, the 28 kD stress protein is phosphorylated and can activate endothelial cells,9 possibly initiating proliferation or the release of angiogenic factors. Cytoskeletal derangements occur in hypoxic conditions and the 28 kD protein associates with actin microfilaments (B. Geiger, personal communication), causing changes in morphology and perhaps in vascular permeability. The 28 kD protein may be another focus for drug development. Other cytoskeletal components may have a role in vascular permeability. The intermediate filament vimentin is much increased in synovial cells and is very sensitive to oxidative damage. ROS, like heat shock, cause collapse of the vimentin cytoskeleton around the nucleus,1O where it becomes associated with a 70 kD stress protein. Disodium aurothiomalate at 01 mmol/1 induces a similar rearrangement of intermediate filaments in fibrolasts (K. R. Rogers, unpublished). The highly conserved stress proteins control the basic biology of cellular interactions, and the complex nature of the biochemical processes involved deserves careful exploration. Moreover, apparently diverse therapeutic approaches to suppress rheumatoid disease may have in common the ability to induce stress protein synthesis. Bone and Joint Research Unit, ARC Building, London Hospital Medical College, London E1 2AD, UK
V. R. WINROW D. R. BLAKE
CR, Williams RB, Farrell AJ, Blake DR. Hypoxia and inflammatory synovitis: observations and speculation. Ann Rheum Dis 1991; 50: 124-32. 2. Allen RE, Blake DR, Nazhat NB, Jones P. Superoxide radical generation by inflamed human synovium after hypoxia. Lancet 1989; ii: 282-83. 3. Ananthan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986; 232: 1. Stevens
522-24. 4. Winrow
VR, Mojdehi G, Mapp PI, Rampton DS, Blake DR. Immunohistological localisation of stress proteins in inflammatory tissue. In: Burdon R, Rice-Evans C, Blake D, Winrow V, eds. Stress proteins in inflammation. London: Richelieu Press,
1990: 237-51.
5.de Graeff-Meeder ER, Voorhorst M, van Eden W, et al. Antibodies to the mycobacterial 65-kd heat-shock protein are reactive with synovial tissue of adjuvant arthritic rats and patients with rheumatoid arthritis and osteoarthritis. Am J Pathol 1990; 137: 1013-17. 6. Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-KDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci USA 1989; 86: 99-103. 7. Sciandra JJ, Subjeck JR, Hughes CS. Induction of glucose-regulated proteins after anaerobic exposure and of heat-shock proteins after reoxygenation. Proc Natl Acad Sci USA 1984; 81: 4843-47. 8. Caltabiano MM, Poste G, Greig RG. Induction of the 32-kD human stress protein by auranofin and related triethylphosphine gold analogs. Biochem Pharmacol 1988; 37: 4089-93. 9. Darbon JM, Issandou M, Tournier JF, Bayard F. The respective 27kDa and 28kDa protein kinase C substrates in vascular endothelial and MCF-7 cells are most probably heat shock proteins. Biochem Biophys Res Commun 1990; 168: 527-36. 10. Rogers KR, Morris CJ, Blake DR. Cytoskeletal rearrangment by oxidative stress. Int J Tiss React 1989; 9: 309-14.
Can disposables make matters worse? SiR,—Dr Rudin and colleagues’ letter on HIV, hepatitis, and measles in Romanian children (Dec 22/29, p 1592-93) leaves us wavering between sadness and despair. The situation is terrible: 12 of 20 orphans in hospital in Pascani, north-eastern Romania were HIV positive and most had evidence of exposure to hepatitis B virus. Rudin et al blame the "lack of disposable needles and medical
supplies and the policy of transfusion of untested blood" for this spread of bloodborne diseases among children, and they suggest an international effort to supply disposable needles and syringes. But the situation is more complex than this: a closer look reveals that this "solution" could have an effect opposite to that which might be expected. In Romania the distribution of disposable injecting equipment was a top priority of donor countries and agencies and some hospitals were quickly well supplied with such items, while in others people fought hard to obtain them. The disposable materials were enthusiastically adopted, but the lack of safe disposal points and inadequate education of the public resulted in the presence of large numbers of used, uncleaned needles and syringes in the environment. Moreover, the abundance of disposables favoured the spread of parenteral treatment outside hospitals. The discontinuity in availability of disposable needles and syringes meant frequent switching to the former practice of making do with reusable materials, but now there was antipathy to the extra work of cleaning and sterilisation. Former routines of needle and syringe sterilisation were sometimes forgotten; the media cast doubt on the efficacy of these procedures and as a result the public have tended to reject old-fashioned glass syringes. Thus pressure to re-use non-re-usable materials is at work. When people’s behaviour and the social infrastructure are the culprits, as here, a measure such as the distribution of needles and syringes should be supplemented by a programme of re-education to ensure that every type of needle and syringe is used properly and that instructions are adhered to. Whenever the focus of health intervention is an object, rather than people with human feelings, an apparently simple measure such as distributing needles and syringes will have the capacity to create explosive epidemics. As has been argued in another context, development aid may add only yet more disturbance when the ecological foundation of health is
neglected.’1 This letter could not have been written in the Ceausescu era, when it was an offence punishable by imprisonment to receive a western medical journal? Today we are fortunate to have complimentary subscriptions offered by journals, such as The Lancet and its readers3 or received by exchange agreements between our local medical journal Revista Medico-Chirurgicala and similar western publications. To cite a British opinion, "there are resources that may rescue Romania from total disaster: the intelligence, inventiveness, and wit of its people".2 This feedback is part of that
package. Pulmonary Diseases Clinic, Institute of Medicine and Pharmacy, lasi 6600, Romania
T. MIHAESCU L. VERES
1. Rijpma S, Barnes J, Meegan MK, Taylor CE. The "demographic trap". Lancet 1991; 337: 50-51. 2. Anon. Medicine in Romania. Br Med J 1990; 300: 699. 3. Heley MM. Journals for Romania. Lancet 1990; 336: 1013.
Optic neuritis associated with dideoxyinosine SIR,-2’,3’-dideoxyinosine (ddl) is being studied in patients with AIDS or AIDS-related complex who do not tolerate or who do not respond to zidovudine. The major adverse reactions to ddl are peripheral neuropathy and pancreatitis.2 We report a case of optic neuritis 6 weeks after the introduction of ddI. Although the relation between ddl and optic neuritis cannot be asserted, the stabilisation of visual loss after interruption of ddl and its deterioration when this therapy was resumed are consistent with a toxic role of the drug. A 40-year-old homosexual man with HIV infection (CDC class IVC2 [hairy leukoplakia of the tongue]) had been treated with zidovudine 1200 mg daily since 1988 because of a CD4 count of 190/ul.In June, 1990, oesophageal candidiasis developed and the CD4 count was only 70/tit. 1 month later a herpes zoster eruption was treated with acyclovir. The CD4 count was 20/ul. Zidovudine was discontinued, and in October, 1990, ddl was introduced at a dose of 8 mg/kg daily, associated with fluconazole 50 mg per day
616
because of
oral candidiasis. 6 weeks later the patient of blurred vision and his visual acuity was only 3/10 in complained both eyes. Goldman perimetry revealed scotoma and visual evoked potentials were consistent with bilateral optic retrobulbar neuritis. The optic fundi and a computerised tomographic scan of the brain were normal. CSF examination showed no cells, normal glucose, protein 70 mg/dl, and HIV p24 antigen (80 pg/ml). Tests for cytomegalovirus, syphilis, toxoplasma, cryptococcal antigen, and mycobacteria were negative. ddl therapy was interrupted and the patient’s visual acuity stabilised. 6 weeks later, because of a drop in CD4 count (8/1), the patient asked for further anti-HIV therapy and ddI was introduced as before. After 3 weeks of therapy his visual acuity was 1/20 in both eyes and examination of optic fundi revealed atrophy of the optic disks. Neurological examination was otherwise normal. Amblyopia, diplopia, optic atrophy, and blindness in patients on ddl have occasionally been reported to the manufacturers (BristolMyers Squibb). The neurotropic nature of HIV, the neuropathological manifestations of some opportunistic infections, and the large range of medications prescribed at the same time in AIDS patients make it difficult to sort out the cause of these events. Optic nerve disease in HIV infection may be due to a wide variety of complications, but opportunistic infections are the most frequent.3 In our patient no opportunist could be implicated. The role of HIV itself in direct optic nerve injury cannot be ruled out, but the clinical history is more consistent with toxicity. Clinicians should be aware of this possibility when treating AIDS patients who have visual defects.
persistent
A. LAFEUILLADE L. AUBERT P. CHAFFANJON R. QUILICHINI
Internal Medicine Service,
Hôpital Chalucet, 83000 Toulon, France 1. McGowan
JJ, Tomaszewski JE, Cradok J, et al. Overview of the preclinical development of an antiretroviral drug, 2’,3’-dideoxynosine. Rev Infect Dis 1990;
12: S513-21. 2. Yarchoan R, Pluda
JM, Thomas RV,
2’3’-dideoxyinosine
et al. Long-term toxicity/activity profile of in AIDS and AIDS-related complex. Lancet 1990; 336:
526-29. 3. Winward KE, Hamed LM, Glaser JS. The spectrum of optic nerve disease in human immunodeficiency virus infection. Am J Ophthalmol 1989; 107: 373-80.
Misidentification of HIV-2 proteins by western blots SiR,—The US Food and Drug Administration has approved a screening assay for HIV-2 (HIV-2 EIA; Genetic Systems) but no licensed supplementary test (eg, western blot [WB]) is yet available. We report here the misidentification of protein bands on unlicensed commercial HIV-2 WBs. This had prompted us to re-examine World Health Organisation criteria1 for a positive HIV-2 WB. We have generated eleven monospecific antibodies from rabbits and a murine monoclonal antibody, using synthetic peptides derived from the genomic sequences of HIV-2 (HIV -2RODY and purified HIV-2 proteins as immunogens: Protein Antibody Sequence* E1007 env 23-37 ext E1008 env 473-487 ext E1036 env 633-650 transm E1042 env 846-859 transm P1035 endo pol 947-964 P1037 RT pol 386-403 G1013 gag 107-126 p16 G1020 gag 149-164 p26 G1031 gag 215-229 p26 RabX rgpl20 ext rgpl20 (HIV-2ROD) RabX p26 purified p26 (HIV-2 p26 MablH5 (mouse) transm HIV-2jj lysate *Peptide sequences according to HIV-2ROD (ref 2). Peptides were conjugated to keyhole lympet haemocyanin as immunogen. RT = reverse trans=transmembrane; ext=external ; transcriptase; endo=endonuclease.
Using these
antibodies and a commercial monoclonal antibody Abbott Laboratories) directed against the transmembrane protein of HIV-2, we tested three commercial
(3Al8-73;
Immunoblots of monospecific rabbit sera and monoclonal antibodies with commercial HIV-2 antigen strips from Biotech Research Laboratories (A), Diagnostic Pasteur (B), and Genetic Systems (C). Lane 1=HIV-2-positive human serum provided by individual manufacturer. Lane2= RabX rgp120 ; lane 3= E1007; lane 4= E1008; lane 5=E1042; !ane6=E1036. Lane 7=Mab1H5; lane 8=Mab 3A18-73; lane 9=P1037; lane 10 = P1035; lane 11 RabX p26. Lane 12=G1020; lane 13=G1031; lane 14=G1013, All rabbit antisera diluted 1 in 50 in milk buffer and incubated for 2 h at room temperature; Mab 1 H5 (culture supernatant) was diluted 1 in 2 and Mab 3A18-17 was diluted 1 in 100. Bound antibodies were detected by alkaline phosphatase conjugated anti-rabbit IgG or anti-mouse IgG and BCIP/nitroblue-tetrazolium chromogenic substrates. =
