- Email: [email protected]
STOMACH CANCER AFTER GASTRIC SURGERY
110
SIR,-Dr Frank incorrectly states that we support the routine use of faecal occult blood screening. The aim of our letter (March 8, p 562) was simply to counterbalance your unjustifiably negative editorial of Jan 4 and to estimate the potential risks and benefits. Frank is convinced that we substantially overestimated the gross benefits of screening. We cannot find support for his speculation that some of the screen-detected cancers might have regressed spontaneously if they had never been found. He cites Koss and Spratt,2who referred to benign cervical dysplasia and colorectal adenomas, not to established cancers. Would competing causes of death render the detected slower-growing cancers a false threat to the "older elderlies", as Frank suggests? Perhaps, but most lesions are diagnosed well before age 75 and even at that age a woman’s life expectancy is 10years.3 Time will tell whether we are right that colonoscopists will cure more cancers than the number of colons they perforate, one of the questions raised in the editorial which we tried to address objectively. Frank appears to be more certain than we are that a symptomless screen-positive subject should undergo full work-up. It is curious, though, that he considers this to be irrelevant to the issue; surely it is the outcome of the investigation and treatment of such patients that will constitute the results of the current controlled trials and will therefore be crucial to the formal assessment of the usefulness of faecal occult blood screening. Educating patients about screening will vary with one’s philosophy and "facts". Frank prefers to tell the patient that one-third of cancers will be missed. Is the cup one-third empty or two-thirds full? Moreover, he would tell a 50-year-old man considering screening that he has only a 3% risk for symptomatic colorectal cancer, yet recent US data suggest that the risk for this cancer is twice that estimate.’ If early diagnosis improves outcome, a one-in-two chance of undergoing a once-in-a-lifetime colonoscopy with a 5-10% chance of finding a cancer might be a very acceptable risk. The "larger policy question" is not whether health systems "can afford to invest" in an unproven mass screening programme but rather whether occult blood screening can reduce the mortality and morbidity from the second commonest killing cancer. Before that verdict is produced, screening should not be condemned; rather, research into the validity and practicalities of the approach should continue.5
The 266 gastrectomy patients were grouped by age and compared with 4344 patients who had not had a gastrectomy and who were seen in our endoscopy unit in the same period. Among these 4344 patients we found 157 gastric carcinomas. The prevalence of gastric carcinoma among gastrectomised patients was 3-61 % and that among the non-gastrectomised patients was 3-75%. However, there was an increased prevalence of gastric carcinoma in gastrectomised patients for ages 60-70 (7,1 1 % vs 4.11 °-, ) and 70--80 (25% vs 15%), but these differences were not significant. Among the 10 patients operated on and subsequently found to have gastric carcinoma, 5 had been operated on before and 5 after the age 45. In the first 5 the mean interval between gastrectomy and gastric stump carcinoma was 31-2+1-5 years, while in the 5 patients gastrectomised after the age 45 the interval was 19-41: 35 years (p < 0’02). If interval between gastrectomy and development of gastric stump carcinoma is an important risk factor and if the mean intervals are significantly different in patients operated on before and after the age of 45, there ought to be a significant difference between the mean ages of onset of gastric stump carcinoma in patients operated on before and after age 45. In our small series, the mean age of onset of gastric stump cancer was 68-6+2-8 years in patients gastrectomised before the age 45 and 67-6 ±3 01 years in patients gastrectomised after the age 45. The difference is not significant. Might advanced age, more than a previous gastrectomy or interval from gastrectomy, be a risk factor for gastric stump carcinoma? If so, there would be important practical implications for the management of gastrectomy patients, related to the need for regular endoscopic follow-up in symptomless patients after gastrectomy. Our series is very small; perhaps our calculations should be repeated on larger series. Division of Medicine I, Ospedale di Garbagnate Milanese, USSL 67, Garbagnate Milanese (Milan), Italy
1. Kalina TV, Kivilaakso E. Is the risk of stump cancer increased after partial gastrectomy? Scand J Gastroenterol 1983; 18: 35-36. 2. Ross AHM, et al. Late mortality after surgery for peptic ulcer. N Engl J Med 1982; 307: 519-22. 3. Sandier RS, et al. Risk of stomach cancer after gastric surgery for benign conditions: a case-control study. Dig Dis Sci 1984; 29: 703-08. 4. Schafer L, et al. The risk of gastric carcinoma after surgical treatment for benign ulcer disease. N Engl J Med 1983; 309: 1210.
Department of Epidemiology, London School of Hygiene and Tropical Medicine, London WC1 7HT
JOEL KETTNER
ICRF Colorectal Cancer Unit, St Mark’s Hospital for Diseases of the Rectum and Colon, London EC1
J. M. A. NORTHOVER
1. 2.
3. 4. 5.
Koss LG. The attack on the annual "Pap Smear". Acta Cytol 1980; 24: 181-83. Spratt JS. Gross rates of growth of colonic neoplasms. In: Burdette JW, ed. Carcinoma of the colon and antecedent epithelium. Springfield, Illinois: Charles C Thomas, 1970: 66-77. Life table, 1981-1983. London: Office of Population Censuses and Surveys, England and Wales: table 23, ser DH1, no 14. Seidman H, et al. Probability of eventually developing or dying of cancer-United States, 1985. CA 1985; 35: 36-56. Chamberlain J, et al. UICC workshop of the project on evaluation of screening programmes for gastrointestinal cancer. Int J Cancer 1986; 37: 329-34.
STOMACH CANCER AFTER GASTRIC SURGERY
SIR,-According to Dr Caygill and her colleagues (April 26, p 929) the risk of gastric stump carcinoma increases in Billroth II gastrectomy patients after more than 20 years have elapsed since the operation. The issue is a controversial one and several workers have not found an increased risk of cancer in patients operated on for peptic ulcer. 1-4 Caygill et al did not examine the relation between age at operation and risk of death from gastric cancer. In our small endoscopy unit we saw, between 1976 and 1984, 266 patients who had had a gastrectomy for peptic ulcer more than 10 years earlier. We found 10 cases of gastric stump carcinoma. 8 had had a Billroth II gastrectomy and 2 a gastroenterostomy. The interval between gastrectomy and development of gastric stump carcinoma averaged 25-3 years.
E. COLOMBO M. BOCCHI
SIR,-Dr Caygill and her colleagues’ data confirm our findings 480 necropsies of patients who had undergone gastric resection for gastroduodenal peptic ulcer.1 The only discrepancies are the site of the ulcer and the type of surgery (Billroth I or II), which in our series did not seem to be related to an increased risk of stump cancer. Caygill et al should have stressed age at time of operation since we found that this does influence the relative risk (2-46 in patients operated before 45 years; 0-65 in patients operated at 45 years or later). We agree with Caygill et al about the pathogenesis of gastric stump carcinoma although we think they should have analysed the neoplasms by age at death both in the resected group and in the unresected controls. The distribution in our sample is very similar, with an increasing frequency starting at 55 years. Since we have found a homologous distribution of Lauren’s types of gastric cancer (ie, intestinal, especially correlated with environmental factors, and diffuse, mostly correlated with genetic factors3)-in both resected and unresected patients,4 we still believe that changes induced by gastric resection are risk factors for the development of gastric carcinoma. Either way they do not seem to affect the role of factors customarily claimed to play a part in gastric on
cancer.
Institute of Pathological Anatomy, University of Tneste, Ospedale Maggiore, 34100 Trieste, Italy
L, Melato M, Stanta G, Bucconi S, Manconi R. Gastric resection: a cause of high frequency of gastric carcinoma. Cancer 1983, 52: 1113-16. Laurén P The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification Acta Pathol Microbiol Scand 1965; 64: 31-49.
1. Glarelli 2.
MAURO MELATO SERGIO BUCCONI
111 3. Lethola J. Family study of gastric carcinoma; with special reference to histological types. Scand J Gastroenterol 1978; 13 (suppl): 3-54. 4. Melato M, Bucconi S. Common features of gastric cancer in patients unresected and resected for benign conditions. Ital J Gastroenterol 1985; 17: 221-26.
ENVELOPE GENE-DERIVED RECOMBINANT PEPTIDE IN THE SERODIAGNOSIS OF
HUMAN IMMUNODEFICIENCY VIRUS INFECTION
SIR,-The tests for confirming a positive enzyme-linked immunoassay for antibody to human immunodeficiency virus (HIV) include western blotting, immunofluorescence assays, and radioimmunoprecipitation. Some of these confirmatory tests are expensive and cumbersome, and often require the handling of live virus or virus-infected cells. One alternative might be to use as diagnostic antigens recombinant peptides derived from parts of the HIV (LAV/HTLV-III) genome. Successful expression of such sequences in Escherichia coli has been reported but little is known about how such recombinant peptides, when used as diagnostic antigens, compared with conventional confirmatory tests. The sera were from patients with AIDS or AIDS-related complex, from outpatients with skin diseases but no AIDS-related symptoms, from drug abusers, and from blood donors found to be
seropositive (Abbott ELISA). Three conventional confirmatory tests were used-namely, westem blotting (WB),1,2 immunofluorescence (IF) (dilution 1:40) on H9-III cells, and immunoprecipitation from 35S-cysteine labelled H9-III cells with patient sera and protein A-sepharose3,4 (RIPA). For WB, virus from the H9-III T-cell line was run on a 10% SDS-polyacrylamide gel and blotted to nitrocellulose in a Bio-Rad apparatus. The nitrocellulose filter was blocked for 1 h in 3% bovine serum albumin and 0’1% ’Triton X-100’ in isotonic phosphate-buffered solution. A WB strip was interpreted as positive if at least two virus-specific bands (pl6, p24, p31, p41, p55, p64) were present. WB with recombinant proteins (see below) followed a similar procedure, and full methodological details may be REACTION PATTERNS OF THREE OR FOUR CONSECUTIVE SERA FROM THREE PATIENTS (A, B, AND C)
by SDS-PAGE and western blot, as described above. Two proteins, with apparent molecular weights of 160 000 and 40 000, reacted with anti-HIV
sera
but not with normal
sera.
The 160 kD
protein consists of the HIV env fragment fused to a 117 kD cro-&bgr;-gal fusion protein derived from PEX-2. The 40 kD band is thought to correspond to the isolated env fragment. A protein of identical size was also produced by bacteria transformed with another recombinant plasmid, pcL970-41, in which the lacz gene of the parent plasmid PTL 9706had been replaced by the 1 -4 kb Bgl II fragment of HIV. 251 human sera were tested on WB strips prepared from whole bacterial lysates of cultures harbouring PEX-41 and pCL-970-41 and the results were compared with those obtained with a commercial screening ELISA and with conventional confirmatory tests. 169 sera (67-3%) gave clear-cut positive results in all tests. 62 sera were non-reactive in all confirmatory assays, 44 having been positive by screening ELISA. Conventional WB with 13 sera were difficult to interpret, but the IF results were identical to those obtained on WB prepared with recombinant material, 7 being positive and 6 negative. 7 sera gave rise to discrepant results. A final diagnosis was arrived at by RIPA or by repeatedly positive conventional WB. In all the WB with recombinant p41 produced the correct diagnosis. In 3 sera the recombinant test proved to be more sensitive than one or both conventional confirmatory tests because previous or subsequent sera from the same patient were clearly positive in several or all tests. The comparison is summarised in the figure. The sensitivity of WB with recombinant material is illustrated in ’
the table. We observed no false positives with the recombinant proteins. In a serum from a patient with preterminal AIDS which no longer reacted in IF or conventional WB both recombinant blots were only weakly positive, reflecting the decreased antibody production. In some sera the typical 40 kD band was clearer on blots prepared with pCL970-41 than on PEX-41 blots, probably because the sera were diluted 1:100 for pCL970-41 but 1:1000 for PEX-41. Several groups have successfully expressed HIV genes in bacteria and recorded the reactivity with patients’ sera of the recombinant proteins obtained. For example, in one study 131 of 132 sera from patients with AIDS or AIDS-related complex reacted with a recombinant peptide containing 82 aminoacid residues encoded by a segment in the env region.9 We have expressed an env-derived peptide of 391 aminoacids that includes the complete gp41 glycoprotein. We have also shown that, as a confirmatory test, a WB with recombinant p41 as antigen compares favourably with other tests.
*Weak.
obtained from T. F. S. To obtain recombinant material for western blotting, bacterial clones carrying HIV-expression plasmids were grown in bulk culture at 30°C and induced to produce either cro-lacz-HIV-env (PEX-41) or cro-HTLV-III-env (pCL970) fusion proteins by growing them at 42°C for 90 min. Subsequently bacteria were pelleted and resuspended in 1/10 original volume of 50 mmol/1 "tris" HCl pH 6-8 containing 5% SDS, 1% mercaptoethanol, 10% glycerol. This lysate was used to prepare western blots as described above. For construction of expression plasmids we used vectors PEX-25 and pCL9706 and E coli POP2136, kindly donated by Dr K. K. Stanley (EMBL, Heidelberg). Both vectors contain the Pr-promotor controlling expression of a cro-lac fusion gene. The 3 ’8 kb SalI-SstI fragment containing the env gene of HIV was subcloned from lambda BH10 (kindly provided by Dr R. C. Gallo)It was amplified in PUC18 and the 1-4 kb Bglll fragment (nucleotides 7198-86277) was obtained. The 1-4 kb BglII fragment contains the sequence assumed to code for gp41, the transmembrane envelope glycoprotein of the virus, and additional aminoacids derived from gpl208. This fragment was ligated into the BamHI site of PEX-2. Upon transformation of E coli POP 2136 carrying the cI857 gene colonies were screened for expression of virus-specific proteins by an indirect immunoperoxidase method with antibody positive sera. Positive colonies were grown in bulk culture and the proteins were analysed
gp41 was so immunogenic that it elicited antibodies in all our seropositive patients, as Chang et al9 found for an 82 aminoacid recombinant polypeptide derived from p41 and others have with WB prepared from purified virus.1,2 However, the gp41 band is often difficult to detect on conventional WB, and we find blots prepared with the recombinant material much easier to read. Besides being sensitive the recombinant test seems to be more
Evaluation of 251 sera tested by recombinant pCL-970-41 and PEX-41 in comparison to ELISA and conventional confirmatory tests.
SIR,-Dr Frank incorrectly states that we support the routine use of faecal occult blood screening. The aim of our letter (March 8, p 562) was simply to counterbalance your unjustifiably negative editorial of Jan 4 and to estimate the potential risks and benefits. Frank is convinced that we substantially overestimated the gross benefits of screening. We cannot find support for his speculation that some of the screen-detected cancers might have regressed spontaneously if they had never been found. He cites Koss and Spratt,2who referred to benign cervical dysplasia and colorectal adenomas, not to established cancers. Would competing causes of death render the detected slower-growing cancers a false threat to the "older elderlies", as Frank suggests? Perhaps, but most lesions are diagnosed well before age 75 and even at that age a woman’s life expectancy is 10years.3 Time will tell whether we are right that colonoscopists will cure more cancers than the number of colons they perforate, one of the questions raised in the editorial which we tried to address objectively. Frank appears to be more certain than we are that a symptomless screen-positive subject should undergo full work-up. It is curious, though, that he considers this to be irrelevant to the issue; surely it is the outcome of the investigation and treatment of such patients that will constitute the results of the current controlled trials and will therefore be crucial to the formal assessment of the usefulness of faecal occult blood screening. Educating patients about screening will vary with one’s philosophy and "facts". Frank prefers to tell the patient that one-third of cancers will be missed. Is the cup one-third empty or two-thirds full? Moreover, he would tell a 50-year-old man considering screening that he has only a 3% risk for symptomatic colorectal cancer, yet recent US data suggest that the risk for this cancer is twice that estimate.’ If early diagnosis improves outcome, a one-in-two chance of undergoing a once-in-a-lifetime colonoscopy with a 5-10% chance of finding a cancer might be a very acceptable risk. The "larger policy question" is not whether health systems "can afford to invest" in an unproven mass screening programme but rather whether occult blood screening can reduce the mortality and morbidity from the second commonest killing cancer. Before that verdict is produced, screening should not be condemned; rather, research into the validity and practicalities of the approach should continue.5
The 266 gastrectomy patients were grouped by age and compared with 4344 patients who had not had a gastrectomy and who were seen in our endoscopy unit in the same period. Among these 4344 patients we found 157 gastric carcinomas. The prevalence of gastric carcinoma among gastrectomised patients was 3-61 % and that among the non-gastrectomised patients was 3-75%. However, there was an increased prevalence of gastric carcinoma in gastrectomised patients for ages 60-70 (7,1 1 % vs 4.11 °-, ) and 70--80 (25% vs 15%), but these differences were not significant. Among the 10 patients operated on and subsequently found to have gastric carcinoma, 5 had been operated on before and 5 after the age 45. In the first 5 the mean interval between gastrectomy and gastric stump carcinoma was 31-2+1-5 years, while in the 5 patients gastrectomised after the age 45 the interval was 19-41: 35 years (p < 0’02). If interval between gastrectomy and development of gastric stump carcinoma is an important risk factor and if the mean intervals are significantly different in patients operated on before and after the age of 45, there ought to be a significant difference between the mean ages of onset of gastric stump carcinoma in patients operated on before and after age 45. In our small series, the mean age of onset of gastric stump cancer was 68-6+2-8 years in patients gastrectomised before the age 45 and 67-6 ±3 01 years in patients gastrectomised after the age 45. The difference is not significant. Might advanced age, more than a previous gastrectomy or interval from gastrectomy, be a risk factor for gastric stump carcinoma? If so, there would be important practical implications for the management of gastrectomy patients, related to the need for regular endoscopic follow-up in symptomless patients after gastrectomy. Our series is very small; perhaps our calculations should be repeated on larger series. Division of Medicine I, Ospedale di Garbagnate Milanese, USSL 67, Garbagnate Milanese (Milan), Italy
1. Kalina TV, Kivilaakso E. Is the risk of stump cancer increased after partial gastrectomy? Scand J Gastroenterol 1983; 18: 35-36. 2. Ross AHM, et al. Late mortality after surgery for peptic ulcer. N Engl J Med 1982; 307: 519-22. 3. Sandier RS, et al. Risk of stomach cancer after gastric surgery for benign conditions: a case-control study. Dig Dis Sci 1984; 29: 703-08. 4. Schafer L, et al. The risk of gastric carcinoma after surgical treatment for benign ulcer disease. N Engl J Med 1983; 309: 1210.
Department of Epidemiology, London School of Hygiene and Tropical Medicine, London WC1 7HT
JOEL KETTNER
ICRF Colorectal Cancer Unit, St Mark’s Hospital for Diseases of the Rectum and Colon, London EC1
J. M. A. NORTHOVER
1. 2.
3. 4. 5.
Koss LG. The attack on the annual "Pap Smear". Acta Cytol 1980; 24: 181-83. Spratt JS. Gross rates of growth of colonic neoplasms. In: Burdette JW, ed. Carcinoma of the colon and antecedent epithelium. Springfield, Illinois: Charles C Thomas, 1970: 66-77. Life table, 1981-1983. London: Office of Population Censuses and Surveys, England and Wales: table 23, ser DH1, no 14. Seidman H, et al. Probability of eventually developing or dying of cancer-United States, 1985. CA 1985; 35: 36-56. Chamberlain J, et al. UICC workshop of the project on evaluation of screening programmes for gastrointestinal cancer. Int J Cancer 1986; 37: 329-34.
STOMACH CANCER AFTER GASTRIC SURGERY
SIR,-According to Dr Caygill and her colleagues (April 26, p 929) the risk of gastric stump carcinoma increases in Billroth II gastrectomy patients after more than 20 years have elapsed since the operation. The issue is a controversial one and several workers have not found an increased risk of cancer in patients operated on for peptic ulcer. 1-4 Caygill et al did not examine the relation between age at operation and risk of death from gastric cancer. In our small endoscopy unit we saw, between 1976 and 1984, 266 patients who had had a gastrectomy for peptic ulcer more than 10 years earlier. We found 10 cases of gastric stump carcinoma. 8 had had a Billroth II gastrectomy and 2 a gastroenterostomy. The interval between gastrectomy and development of gastric stump carcinoma averaged 25-3 years.
E. COLOMBO M. BOCCHI
SIR,-Dr Caygill and her colleagues’ data confirm our findings 480 necropsies of patients who had undergone gastric resection for gastroduodenal peptic ulcer.1 The only discrepancies are the site of the ulcer and the type of surgery (Billroth I or II), which in our series did not seem to be related to an increased risk of stump cancer. Caygill et al should have stressed age at time of operation since we found that this does influence the relative risk (2-46 in patients operated before 45 years; 0-65 in patients operated at 45 years or later). We agree with Caygill et al about the pathogenesis of gastric stump carcinoma although we think they should have analysed the neoplasms by age at death both in the resected group and in the unresected controls. The distribution in our sample is very similar, with an increasing frequency starting at 55 years. Since we have found a homologous distribution of Lauren’s types of gastric cancer (ie, intestinal, especially correlated with environmental factors, and diffuse, mostly correlated with genetic factors3)-in both resected and unresected patients,4 we still believe that changes induced by gastric resection are risk factors for the development of gastric carcinoma. Either way they do not seem to affect the role of factors customarily claimed to play a part in gastric on
cancer.
Institute of Pathological Anatomy, University of Tneste, Ospedale Maggiore, 34100 Trieste, Italy
L, Melato M, Stanta G, Bucconi S, Manconi R. Gastric resection: a cause of high frequency of gastric carcinoma. Cancer 1983, 52: 1113-16. Laurén P The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification Acta Pathol Microbiol Scand 1965; 64: 31-49.
1. Glarelli 2.
MAURO MELATO SERGIO BUCCONI
111 3. Lethola J. Family study of gastric carcinoma; with special reference to histological types. Scand J Gastroenterol 1978; 13 (suppl): 3-54. 4. Melato M, Bucconi S. Common features of gastric cancer in patients unresected and resected for benign conditions. Ital J Gastroenterol 1985; 17: 221-26.
ENVELOPE GENE-DERIVED RECOMBINANT PEPTIDE IN THE SERODIAGNOSIS OF
HUMAN IMMUNODEFICIENCY VIRUS INFECTION
SIR,-The tests for confirming a positive enzyme-linked immunoassay for antibody to human immunodeficiency virus (HIV) include western blotting, immunofluorescence assays, and radioimmunoprecipitation. Some of these confirmatory tests are expensive and cumbersome, and often require the handling of live virus or virus-infected cells. One alternative might be to use as diagnostic antigens recombinant peptides derived from parts of the HIV (LAV/HTLV-III) genome. Successful expression of such sequences in Escherichia coli has been reported but little is known about how such recombinant peptides, when used as diagnostic antigens, compared with conventional confirmatory tests. The sera were from patients with AIDS or AIDS-related complex, from outpatients with skin diseases but no AIDS-related symptoms, from drug abusers, and from blood donors found to be
seropositive (Abbott ELISA). Three conventional confirmatory tests were used-namely, westem blotting (WB),1,2 immunofluorescence (IF) (dilution 1:40) on H9-III cells, and immunoprecipitation from 35S-cysteine labelled H9-III cells with patient sera and protein A-sepharose3,4 (RIPA). For WB, virus from the H9-III T-cell line was run on a 10% SDS-polyacrylamide gel and blotted to nitrocellulose in a Bio-Rad apparatus. The nitrocellulose filter was blocked for 1 h in 3% bovine serum albumin and 0’1% ’Triton X-100’ in isotonic phosphate-buffered solution. A WB strip was interpreted as positive if at least two virus-specific bands (pl6, p24, p31, p41, p55, p64) were present. WB with recombinant proteins (see below) followed a similar procedure, and full methodological details may be REACTION PATTERNS OF THREE OR FOUR CONSECUTIVE SERA FROM THREE PATIENTS (A, B, AND C)
by SDS-PAGE and western blot, as described above. Two proteins, with apparent molecular weights of 160 000 and 40 000, reacted with anti-HIV
sera
but not with normal
sera.
The 160 kD
protein consists of the HIV env fragment fused to a 117 kD cro-&bgr;-gal fusion protein derived from PEX-2. The 40 kD band is thought to correspond to the isolated env fragment. A protein of identical size was also produced by bacteria transformed with another recombinant plasmid, pcL970-41, in which the lacz gene of the parent plasmid PTL 9706had been replaced by the 1 -4 kb Bgl II fragment of HIV. 251 human sera were tested on WB strips prepared from whole bacterial lysates of cultures harbouring PEX-41 and pCL-970-41 and the results were compared with those obtained with a commercial screening ELISA and with conventional confirmatory tests. 169 sera (67-3%) gave clear-cut positive results in all tests. 62 sera were non-reactive in all confirmatory assays, 44 having been positive by screening ELISA. Conventional WB with 13 sera were difficult to interpret, but the IF results were identical to those obtained on WB prepared with recombinant material, 7 being positive and 6 negative. 7 sera gave rise to discrepant results. A final diagnosis was arrived at by RIPA or by repeatedly positive conventional WB. In all the WB with recombinant p41 produced the correct diagnosis. In 3 sera the recombinant test proved to be more sensitive than one or both conventional confirmatory tests because previous or subsequent sera from the same patient were clearly positive in several or all tests. The comparison is summarised in the figure. The sensitivity of WB with recombinant material is illustrated in ’
the table. We observed no false positives with the recombinant proteins. In a serum from a patient with preterminal AIDS which no longer reacted in IF or conventional WB both recombinant blots were only weakly positive, reflecting the decreased antibody production. In some sera the typical 40 kD band was clearer on blots prepared with pCL970-41 than on PEX-41 blots, probably because the sera were diluted 1:100 for pCL970-41 but 1:1000 for PEX-41. Several groups have successfully expressed HIV genes in bacteria and recorded the reactivity with patients’ sera of the recombinant proteins obtained. For example, in one study 131 of 132 sera from patients with AIDS or AIDS-related complex reacted with a recombinant peptide containing 82 aminoacid residues encoded by a segment in the env region.9 We have expressed an env-derived peptide of 391 aminoacids that includes the complete gp41 glycoprotein. We have also shown that, as a confirmatory test, a WB with recombinant p41 as antigen compares favourably with other tests.
*Weak.
obtained from T. F. S. To obtain recombinant material for western blotting, bacterial clones carrying HIV-expression plasmids were grown in bulk culture at 30°C and induced to produce either cro-lacz-HIV-env (PEX-41) or cro-HTLV-III-env (pCL970) fusion proteins by growing them at 42°C for 90 min. Subsequently bacteria were pelleted and resuspended in 1/10 original volume of 50 mmol/1 "tris" HCl pH 6-8 containing 5% SDS, 1% mercaptoethanol, 10% glycerol. This lysate was used to prepare western blots as described above. For construction of expression plasmids we used vectors PEX-25 and pCL9706 and E coli POP2136, kindly donated by Dr K. K. Stanley (EMBL, Heidelberg). Both vectors contain the Pr-promotor controlling expression of a cro-lac fusion gene. The 3 ’8 kb SalI-SstI fragment containing the env gene of HIV was subcloned from lambda BH10 (kindly provided by Dr R. C. Gallo)It was amplified in PUC18 and the 1-4 kb Bglll fragment (nucleotides 7198-86277) was obtained. The 1-4 kb BglII fragment contains the sequence assumed to code for gp41, the transmembrane envelope glycoprotein of the virus, and additional aminoacids derived from gpl208. This fragment was ligated into the BamHI site of PEX-2. Upon transformation of E coli POP 2136 carrying the cI857 gene colonies were screened for expression of virus-specific proteins by an indirect immunoperoxidase method with antibody positive sera. Positive colonies were grown in bulk culture and the proteins were analysed
gp41 was so immunogenic that it elicited antibodies in all our seropositive patients, as Chang et al9 found for an 82 aminoacid recombinant polypeptide derived from p41 and others have with WB prepared from purified virus.1,2 However, the gp41 band is often difficult to detect on conventional WB, and we find blots prepared with the recombinant material much easier to read. Besides being sensitive the recombinant test seems to be more
Evaluation of 251 sera tested by recombinant pCL-970-41 and PEX-41 in comparison to ELISA and conventional confirmatory tests.