INFECTION AND IMMUNITY, Nov. 1977, p. 352-355
Copyright © 1977 American Society for Microbiology
Vol. 18, No. 2 Printed in U.S.A.
New Test for Endotoxin Potency Based upon Histamine Sensitization in Mice ROBERT K. BERGMAN,* KELSEY C. MILNER, AND JOHN J. MUNOZ National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840 Received for publication 20 May 1977
The results of a test of endotoxic potency based upon the development of histamine hypersensitivity in mice were compared with the results obtained by testing the same materials for pyrogenicity in rabbits and lethality for chicken embryos (CELD50). The results of the histamine hypersensitization test (HHT) correlated well with those of the other two tests. The sensitivity of the HHT was about the same as that of the CELD5o assay. The HHT may provide a relatively inexpensive, fast, and reliable assay method for endotoxin laboratories that do not have the facilities for the more elaborate assays. The identification and quantitation of endotoxin is almost wholly dependent on the effects it produces in biological systems, because chemical analyses do not give any measure of endotoxic potency. Most of the bioassays in common use are expensive and/or time consuming. Endotoxins, in sub-microgram doses, from gram-negative bacteria will induce a transient histamine hypersensitivity in mice (2). This observation led us to test this ability of endotoxin as a quick, simple, and reasonably inexpensive method for detecting and quantitating endotoxin. For this purpose, we tested the ability of a number of biological preparations to induce histamine hypersensitivity in mice and compared these results with those obtained from other bioassays for endotoxin potency, i.e., the rabbit pyrogenicity and chicken embryo lethality (CELD50) tests.
mine sensitivity 90 min after administration. Human serum albumin, bovine immunoglobulin, hen egg albumin, sucrose, soluble starch, glycogen, and deoxyribonucleic acid were all administered in 10-fLg doses given intravenously (i.v.) in 0.2 ml of saline. Suspensions of cephalin, lecithin, and bovine cerebrosides were made in water by sonic treatment for 30 s with a Biosonik IV sonic oscillator (Bronwill, VWR Scientific). Mice were inoculated intraperitoneally (i.p.) with 10 itg of the lipids given in 0.2 ml of water. Endotoxin was given in the same manner to serve as a positive control. Animals for bioassay tests. Three strains of mice, all reared in our laboratory, were tested for use in these studies. The sources of these strains were as follows: the C57BL strain originated from stock at the National Institutes of Health; the CFW strain' was purchased from Carworth Farms, New City, N.Y.; and the RML strain has been raised at the Rocky Mountain Laboratory for many years. The age and sex of the mice were as follows: C57BL, 8- to 12-week-old males; CFW, 7- to 9-week-old males and females; and RML, 8- to 11-week-old males and females. Adult, MATERIALS AND METHODS white male and female New Zealand rabbits were Study plan. Thirteen biological preparations se- purchased from a local supplier. Eleven-day-old lected to cover a broad range of endotoxic activity chicken embryos were produced by incubating fertile were tested in standard assays for pyrogenicity to eggs of White Leghorn chickens from the flock of a rabbits, for lethality to chicken embryos, and for abil- local supplier. ity to produce histamine hypersensitivity in mice. Histamine hypersensitivity test (HHT). Mice Data from the three assays were collated, ranked, and were housed in glass jars (five mice per jar) on wood interpreted. shavings and allowed feed and water ad libitum. When Endotoxic preparations. All preparations were male mice were used, mice from different lots were aqueous solutions or stabilized suspensions, freshly not mixed. Mice in groups of 10 were inoculated with made or diluted from stock solutions that had been various dilutions of the endotoxin preparations given preserved with Formalin (0.2% concentration) and i.v. in 0.2 ml of physiological saline. The maximum kept at 2 to 5°C for months or even years. Concentra- dose of any preparation was 16 ,g, and the least was tions of stock solutions ranged from 500 to 5,000 ,g/ 0.000125,g. At least three doses in fourfold dilutions ml, and for this study all were diluted in pyrogen-free were given in any one test. Ninety minutes after water to contain 500 ug/ml. Table 1 lists general receiving the endotoxin preparation, the mice were inoculated i.p. with 0.5 mg of histamine base (given information on the nature of the preparations. Other substances tested. Several other sub- as histamine diphosphate; Sigma Chemical Co.) in 0.2 stances were tested for their ability to produce hista- ml of physiological saline. Deaths among the chal352
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353
TABLE 1. General description ofpreparations tested for endotoxic properties General nature
Designation code
S-E-390 St 2 and 3 Reference ET-EC St 2 and 3, sonic S-E-388 SR-11 RNA B. pertussis ET
Poly(I C) B. abortus Fr. 5 Ec 91/92 NH Ec 178 NH
Poly(I) Arabinogalactan
Aqueous ether extract (12) of Salmonella enteritidis strain S-795 Hot phenol-water extract (17) of Salmonella typhi strain 0901 Hot phenol-water extract of Escherichia coli 0113:H10:K-negative; from large pool of endotoxin prepared as a national standard and designated "Reference" endotoxin EC (13) Same as St 2 and 3 except that method of dispersion in water included vibration in an ultrasonic cleaning bath for 30 s Aqueous ether extract of S. enteritidis Nikaido strain 11, wild type (10) Ribonucleic acid preparation by M. R. Venneman from Salmonella typhimurium strain SR-11 (16) Hot phenol-water extract (17) of Bordetella pertussis strain 3779B12S4 Synthetic double-stranded polyriboinosinic-polyribocytidylic acid, purchased in dry form (sodium salt) from P-L Biochemicals, Inc., Milwaukee, Wis. "Fraction 5" (7). Mildly endotoxic material obtained from the phenol phase of a hot phenol-water extraction of whole cells of virulent (strain 2308) Brucella abortus. "Native hapten" (1) or "NPP" (14). 0-specific substance isolated from the soluble non-cell wall fraction of disrupted E. coli. Different preparations may be slightly, but variably, contaminated with endotoxin. Same type of preparation as Ec 91/92 NH Polyriboinosinic acid, purchased in dry form from K & K Laboratories, Inc., Irvine, Calif. STRactan-2, from Larix occidentalis, St. Regis Paper Co., Libby, Mont.
lenged mice were recorded 2 h later. Insofar as possible, the 50% sensitizing dose (SD5o) was calculated for each preparation by the method of Reed and Muench (11) as a means of expressing relative potencies. With preparations in which 16 ,ug was not sufficient to produce lethal sensitivity in half the tested mice, it was not possible to calculate an SDso value. Pyrogenicity test. Preconditioned white New Zealand rabbits, with rectally implanted thermocouples, were inoculated with graded doses of the endotoxin preparations in a predetermined range as described elsewhere (8) and were observed for 1 h before and 6 h after inoculation. Rectal temperatures were recorded automatically every 12 min, and the temperature curves were plotted on rectangular coordinate paper. The areas between temperature curves and base lines (fever index [FI]) were determined in square centimeters with a Hewlett-Packard 9810A calculator, and the average Fl of seven or eight rabbits per dose level was used to obtain the FI40, or that dose [in micrograms] that produced an FI of 40 cm2, as recommended by Keene et al. (6). Values expressed as "greater than" were, in each case, the highest dose tested. CELD.. test. Fertile eggs from a single flock of White Leghorn chickens were incubated at 38°C for 11 days in a humidified, self-turning commercial apparatus as previously described (8). After 11 days of development, the embryos were inoculated i.v. with graded doses contained in 0.1-rnl volumes. At least four dose levels, differing by fivefold, were injected into 10 embryos, and 50% lethal dose values (designated as CELD5o) were calculated from the 24-h mortality data by the Spearman-Karber method as described by Finney (5). The test stems from the studies of Smith and Thomas (15) and was conducted essentially as modified by Finkelstein (4).
RESULTS Of the three strains of mice tested (CFW, RML, and C57BL), only the CFW strain at 7 weeks or older became consistently hypersensitive to histamine after receiving endotoxin. All the tests reported here were performed with 7to 9-week-old CFW male mice; however, female mice of this strain will also become sensitive. The results of the three different assay procedures to obtain measurements of endotoxic potency in the 13 different preparations are shown in Table 2. Values of potency are expressed as (i) SD50, (ii) FI40, and (iii) CELD5o. The ranking of the first eight preparations in Table 2 was based on the SD50 determinations, from most to least potent. The last five preparations, which were of very low or nil endotoxin content, could not be ranked by this test because the largest doses given (16,ug) were insufficient to sensitize mice. The ranking of these last five preparations was based on their potency determined by the other two tests. In general, there was very good agreement among the three tests for the relative endotoxic potency of the various preparations tested, considering the observed variability within any one assay for endotoxin and the known lack of correlation between certain pairs of assay (3, 8, 9). For example, preparation S-E-388 was anomalous in that it was strongly pyrogenic, but only weakly lethal to chicken embryos. In this case, the SD5o and FLI4o values were the most closely correlated. In general, however, the HHT in mice had about the
354
BERGMAN, MILNER, AND MUNOZ
INFECT. IMMUN.
(SDs])
(FI4h)
TABLE 2. Comparison of sensitization of mice to histamine with pyrogenicity and lethality to chicken embryos (CELD5s) as measures of endotoxic potency Prepn Rank Rank SD5, (/Ag) FL,4 (ug) Rank CELDJ, (Ag) 1 (1) 0.002 (0.005)a S-E-390 0.064 1 1 0.0028 0.003 (0.011) 2 (4) St 2 and 3 0.13 3 0.015 3 0.005 (0.008) 4 Reference ET-EC 0.19 3 (2 or 3) 0.0086 2 0.006 (0.089) 4 (7) 2 St 2 and 3, sonic 0.12 0.019 4 0.007 (0.008) 5 (2 or 3) S-E-388 0.20 5 =70 9 0.022 (0.020) 6 (5) 0.60 7 SR-11 RNA 0.021 5-6 0.035 (0.058) 7 (6) B. pertussis ET 0.50 6 0.021 5-6 3.4 8 (8) 8 Poly(I C) 216 0.73 7 (4.6) >16 9-13 (9-13) =200 B. abortus Fr. 5 9 >100 (>16) 11 >16 9-13 (9-13) >200 10 Ec 91/92 NH 15 8 (>16) >16 9-13 (9-13) >500 11 190 Ec 178 NH 10 (>16) >16 9-13 (9-13) >500 12 Poly(I) >500 (>16) 12-13 9-13 (9-13) >16 >1000 13 >200 12-13 Arabinogalactan (>16) a Values in parentheses represent the results obtained from a duplicate test to determine reproducibility of SD5o values. TABLE 3. Test to determine the specificity of the HHT for endotoxin Test substance
Dose (Lg)
Route
D/Ta
Human serum albumin 10 iv. 3/10 10 Bovine immunoglobulin i.v. 0/10 10 Hen egg albumin i.v. 2/10 10 Sucrose . ........ i.v. 0/10 10 Starch .... i.v. 2/10 10 Glycogen i.v. 0/10 10 Deoxyribonucleic acid i.v. 0/10 0.5 Endotoxin i.v. 10/10 10 Lecithin 4/20 i.p. 10 Cephalin ...... i.p. 4/20 Cerebrosides 10 3/20 i.p. Endotoxin .... ...... 0.5 i.p. 14/20 a Deaths per total number of mice tested after mice were challenged i.p. with 0.5 mg of histamine base given 90 min after administration of the test substance.
same ability to detect endotoxin as the CELD50 test. To check the reproducibility of the HHT, a second test was run on the preparations and a second SD50 value was calculated for each. A comparison of the results obtained in the two tests showed good reproducibility (Table 2, cf. two columns of data under SD50). "St 2 and 3, sonic" was the only anomalous preparation in that it had an SD50 value of 0.006 in the first test and a value of 0.089 in the second. The relative ranking of the preparations did not change much between the two tests. The highpotency and low-potency fractions remained the same, and no preparation shifted in rank by more than three positions. A number of other substances were tested for their ability to induce hypersensitivity to histamine at a dose level of 10 ,g per mouse given i.v. or i.p. 90 min before an i.p. challenge with 0.5 mg of histamine (Table 3). None of the substances induced a significant degree of hypersensitivity to histamine.
DISCUSSION These data show that the ability of the HHT to identify and quantitate endotoxin activity compares well with that of the rabbit pyrogenicity and CELD50 tests. The sensitivity of the HHT was equal to or greater than that of the CELD50 test (cf. results in Table 2). The results obtained by the HHT on the endotoxic potency of the various preparations correlated well with the results obtained by the other two tests. A comparison of the rankings obtained by the three different test methods on the tested preparations shows a high degree of correspondence among the three methods. The most potent endotoxin preparation by all three tests was SE-390. As would be expected in such tests, the correlation was not perfect. One anomalous preparation, S-E-388, was not highly lethal to chicken embryos but was pyrogenic and induced histamine hypersensitivity. It is interesting that the double-stranded mixture of polyinosinic and polycytidylic acids [poly (I C)] was active by the HHT. This preparation is probably slightly contaminated with endotoxin, as judged from samples of singlestranded synthetic polynucleotides examined in this laboratory. However, the level of activity shown in Table 2 cannot be accounted for by such contamination. Double stranding of poly(I) and poly(C) always produces a marked increase in endotoxin-like activity. None of the other substances tested by HHT, i.e., carbohydrates, proteins, or lipids, which were not endotoxic in nature, induced a significant degree of hypersensitivity to histamine. The HHT provides a test for endotoxin potency that is sensitive and reproducible and that has many advantages over other endotoxin tests presently in use: (i) it is very simple to perform; (ii) it is relatively inexpensive; (iii) it requires
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VOL. 18, 1977
no elaborate equipment; (iv) it detects sub-microgram amounts of endotoxin; (v) it takes only about 3 h to complete; and (vi) since the results are measured by death or survival of mice, it is simple to interpret. One of the drawbacks of the HHT may be that it requires -7-week-old CFW mice. Other strains tested (C57BL and RML) have not been satisfactory because they fail to sensitize to histamine. There are probably other strains that will prove suitable for this test, but we have not made an extensive survey. The number of mice required is relatively large because at least 10 mice per dose of endotoxin are required to obtain reliable results. The test is not absolutely specific because poly(I C) and pertussigen (2) sensitize mice to histamine within 90 to 120 min after administration. The effect of pertussigen can be differentiated from that of endotoxin because its activity is destroyed by heating at 80°C for 30 min. We believe that this HHT may prove to be a practical test for detecting and quantitating the activity of endotoxin.
5. 6.
7. 8.
9.
10. 11. 12.
13.
ACKNOWLEDGMENTS We wish to thank our technicians, Joe C. Ayers and Elsworth R. Pfeifer, for their competent assistance in these studies. 1.
2. 3.
4.
LITERATURE CITED Anacker, R. L., R. A. Finkelstein, W. T. Haskins, M. L. Landy, K. C. Milner, E. Ribi, and P. W. Stashak. 1964. Origin and properties of naturally occurring hapten from Escherichia coli. J. Bacteriol. 88:1705-1720. Bergman, R. K., and J. J. Munoz. 1977. Increased histamine sensitivity in mice after administration of endotoxin. Infect. Immun. 15:72-77. Cundy, K. R., and A. Nowotny. 1968. Comparisons of five toxicity parameters of Serratia marcescens endotoxins. Proc. Soc. Exp. Biol. Med. 127:999-1003. Finkelstein, R. A. 1964. Observations on the mode of
14.
15. 16.
17.
355
action of endotoxin in chick embryos. Proc. Soc. Exp. Biol. Med. 115:702-707. Finney, D. J. 1964. Statistical method in biological assay, 2nd ed., p. 524-530. Charles Griffin and Co., London. Keene, W. R., H. R. Silberman, and M. Landy. 1961. Observations on the pyrogenic response and its application to the bioassay of endotoxin. J. Clin. Invest. 40:295-301. Leong, D., R. Diaz, K. Milner, J. Rudbach, and J. B. Wilson. 1970. Some structural and biological properties of Brucella endotoxin. Infect. Immun. 1:174-182. Milner, K. C., and R. A. Finkelstein. 1966. Bioassay of endotoxin: correlation between pyrogenicity for rabbits and lethality for chick embryos. J. Infect. Dis. 116:529-536. Milner, K. C., J. A. Rudbach, and E. Ribi. 1971. General characteristics, p. 33-35. In S. J. Ajl, A. Ciegler, S. Kadis, T. C. Montie, and G. Weinbaum (ed.), Microbial toxins, vol. 4. Academic Press Inc., New York and London. Nikaido, H. 1961. Galactose-sensitive mutants of Salmonella. I. Metabolism of galactose. Biochim. Biophys. Acta 48:460-469. Reed, L. J., and H. Muench. 1938. A simple method for estimating fifty percent endpoints. Am. J. Hyg. 27:493-497. Ribi, E., K. C. Milner, and T. D. Perrine. 1959. Endotoxic and antigenic fractions from the cell wall of Salmonella enteritidis. Methods for separation and some biologic activities. J. Immunol. 82:75-84. Rudbach, J. A., F. I. Akiya, R. J. Elin, H. D. Hochstein, M. K. Luoma, E. C. B. Milner, K. C. Milner, and K. R. Thomas. 1976. Preparation and properties of a national reference endotoxin. J. Clin. Microbiol. 3:21-25. Rudbach, J. A., R. L. Anacker, W. T. Haskins, K. C. Milner, and E. Ribi. 1967. Physical structure of a native protoplasmic polysaccharide from Escherichia coli. J. Immunol. 98:1-7. Smith, R. T., and L. Thomas. 1956. The lethal effect of endotoxins on the chick embryo. J. Exp. Med. 104:217-231. Venneman, M. R., and N. J. Bigley. 1969. Isolation and partial characterization of an immunogenic moiety obtained from Salmonella typhimurium. J. Bacteriol. 100:140-148. Westphal, O., 0. Luderitz, and F. Bister. 1952. Uber die Extraktion von Bacterien mit Phenol/Wasser. Z. Naturforsch. 7:148-155.
Copyright © 1977 American Society for Microbiology
Vol. 18, No. 2 Printed in U.S.A.
New Test for Endotoxin Potency Based upon Histamine Sensitization in Mice ROBERT K. BERGMAN,* KELSEY C. MILNER, AND JOHN J. MUNOZ National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840 Received for publication 20 May 1977
The results of a test of endotoxic potency based upon the development of histamine hypersensitivity in mice were compared with the results obtained by testing the same materials for pyrogenicity in rabbits and lethality for chicken embryos (CELD50). The results of the histamine hypersensitization test (HHT) correlated well with those of the other two tests. The sensitivity of the HHT was about the same as that of the CELD5o assay. The HHT may provide a relatively inexpensive, fast, and reliable assay method for endotoxin laboratories that do not have the facilities for the more elaborate assays. The identification and quantitation of endotoxin is almost wholly dependent on the effects it produces in biological systems, because chemical analyses do not give any measure of endotoxic potency. Most of the bioassays in common use are expensive and/or time consuming. Endotoxins, in sub-microgram doses, from gram-negative bacteria will induce a transient histamine hypersensitivity in mice (2). This observation led us to test this ability of endotoxin as a quick, simple, and reasonably inexpensive method for detecting and quantitating endotoxin. For this purpose, we tested the ability of a number of biological preparations to induce histamine hypersensitivity in mice and compared these results with those obtained from other bioassays for endotoxin potency, i.e., the rabbit pyrogenicity and chicken embryo lethality (CELD50) tests.
mine sensitivity 90 min after administration. Human serum albumin, bovine immunoglobulin, hen egg albumin, sucrose, soluble starch, glycogen, and deoxyribonucleic acid were all administered in 10-fLg doses given intravenously (i.v.) in 0.2 ml of saline. Suspensions of cephalin, lecithin, and bovine cerebrosides were made in water by sonic treatment for 30 s with a Biosonik IV sonic oscillator (Bronwill, VWR Scientific). Mice were inoculated intraperitoneally (i.p.) with 10 itg of the lipids given in 0.2 ml of water. Endotoxin was given in the same manner to serve as a positive control. Animals for bioassay tests. Three strains of mice, all reared in our laboratory, were tested for use in these studies. The sources of these strains were as follows: the C57BL strain originated from stock at the National Institutes of Health; the CFW strain' was purchased from Carworth Farms, New City, N.Y.; and the RML strain has been raised at the Rocky Mountain Laboratory for many years. The age and sex of the mice were as follows: C57BL, 8- to 12-week-old males; CFW, 7- to 9-week-old males and females; and RML, 8- to 11-week-old males and females. Adult, MATERIALS AND METHODS white male and female New Zealand rabbits were Study plan. Thirteen biological preparations se- purchased from a local supplier. Eleven-day-old lected to cover a broad range of endotoxic activity chicken embryos were produced by incubating fertile were tested in standard assays for pyrogenicity to eggs of White Leghorn chickens from the flock of a rabbits, for lethality to chicken embryos, and for abil- local supplier. ity to produce histamine hypersensitivity in mice. Histamine hypersensitivity test (HHT). Mice Data from the three assays were collated, ranked, and were housed in glass jars (five mice per jar) on wood interpreted. shavings and allowed feed and water ad libitum. When Endotoxic preparations. All preparations were male mice were used, mice from different lots were aqueous solutions or stabilized suspensions, freshly not mixed. Mice in groups of 10 were inoculated with made or diluted from stock solutions that had been various dilutions of the endotoxin preparations given preserved with Formalin (0.2% concentration) and i.v. in 0.2 ml of physiological saline. The maximum kept at 2 to 5°C for months or even years. Concentra- dose of any preparation was 16 ,g, and the least was tions of stock solutions ranged from 500 to 5,000 ,g/ 0.000125,g. At least three doses in fourfold dilutions ml, and for this study all were diluted in pyrogen-free were given in any one test. Ninety minutes after water to contain 500 ug/ml. Table 1 lists general receiving the endotoxin preparation, the mice were inoculated i.p. with 0.5 mg of histamine base (given information on the nature of the preparations. Other substances tested. Several other sub- as histamine diphosphate; Sigma Chemical Co.) in 0.2 stances were tested for their ability to produce hista- ml of physiological saline. Deaths among the chal352
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353
TABLE 1. General description ofpreparations tested for endotoxic properties General nature
Designation code
S-E-390 St 2 and 3 Reference ET-EC St 2 and 3, sonic S-E-388 SR-11 RNA B. pertussis ET
Poly(I C) B. abortus Fr. 5 Ec 91/92 NH Ec 178 NH
Poly(I) Arabinogalactan
Aqueous ether extract (12) of Salmonella enteritidis strain S-795 Hot phenol-water extract (17) of Salmonella typhi strain 0901 Hot phenol-water extract of Escherichia coli 0113:H10:K-negative; from large pool of endotoxin prepared as a national standard and designated "Reference" endotoxin EC (13) Same as St 2 and 3 except that method of dispersion in water included vibration in an ultrasonic cleaning bath for 30 s Aqueous ether extract of S. enteritidis Nikaido strain 11, wild type (10) Ribonucleic acid preparation by M. R. Venneman from Salmonella typhimurium strain SR-11 (16) Hot phenol-water extract (17) of Bordetella pertussis strain 3779B12S4 Synthetic double-stranded polyriboinosinic-polyribocytidylic acid, purchased in dry form (sodium salt) from P-L Biochemicals, Inc., Milwaukee, Wis. "Fraction 5" (7). Mildly endotoxic material obtained from the phenol phase of a hot phenol-water extraction of whole cells of virulent (strain 2308) Brucella abortus. "Native hapten" (1) or "NPP" (14). 0-specific substance isolated from the soluble non-cell wall fraction of disrupted E. coli. Different preparations may be slightly, but variably, contaminated with endotoxin. Same type of preparation as Ec 91/92 NH Polyriboinosinic acid, purchased in dry form from K & K Laboratories, Inc., Irvine, Calif. STRactan-2, from Larix occidentalis, St. Regis Paper Co., Libby, Mont.
lenged mice were recorded 2 h later. Insofar as possible, the 50% sensitizing dose (SD5o) was calculated for each preparation by the method of Reed and Muench (11) as a means of expressing relative potencies. With preparations in which 16 ,ug was not sufficient to produce lethal sensitivity in half the tested mice, it was not possible to calculate an SDso value. Pyrogenicity test. Preconditioned white New Zealand rabbits, with rectally implanted thermocouples, were inoculated with graded doses of the endotoxin preparations in a predetermined range as described elsewhere (8) and were observed for 1 h before and 6 h after inoculation. Rectal temperatures were recorded automatically every 12 min, and the temperature curves were plotted on rectangular coordinate paper. The areas between temperature curves and base lines (fever index [FI]) were determined in square centimeters with a Hewlett-Packard 9810A calculator, and the average Fl of seven or eight rabbits per dose level was used to obtain the FI40, or that dose [in micrograms] that produced an FI of 40 cm2, as recommended by Keene et al. (6). Values expressed as "greater than" were, in each case, the highest dose tested. CELD.. test. Fertile eggs from a single flock of White Leghorn chickens were incubated at 38°C for 11 days in a humidified, self-turning commercial apparatus as previously described (8). After 11 days of development, the embryos were inoculated i.v. with graded doses contained in 0.1-rnl volumes. At least four dose levels, differing by fivefold, were injected into 10 embryos, and 50% lethal dose values (designated as CELD5o) were calculated from the 24-h mortality data by the Spearman-Karber method as described by Finney (5). The test stems from the studies of Smith and Thomas (15) and was conducted essentially as modified by Finkelstein (4).
RESULTS Of the three strains of mice tested (CFW, RML, and C57BL), only the CFW strain at 7 weeks or older became consistently hypersensitive to histamine after receiving endotoxin. All the tests reported here were performed with 7to 9-week-old CFW male mice; however, female mice of this strain will also become sensitive. The results of the three different assay procedures to obtain measurements of endotoxic potency in the 13 different preparations are shown in Table 2. Values of potency are expressed as (i) SD50, (ii) FI40, and (iii) CELD5o. The ranking of the first eight preparations in Table 2 was based on the SD50 determinations, from most to least potent. The last five preparations, which were of very low or nil endotoxin content, could not be ranked by this test because the largest doses given (16,ug) were insufficient to sensitize mice. The ranking of these last five preparations was based on their potency determined by the other two tests. In general, there was very good agreement among the three tests for the relative endotoxic potency of the various preparations tested, considering the observed variability within any one assay for endotoxin and the known lack of correlation between certain pairs of assay (3, 8, 9). For example, preparation S-E-388 was anomalous in that it was strongly pyrogenic, but only weakly lethal to chicken embryos. In this case, the SD5o and FLI4o values were the most closely correlated. In general, however, the HHT in mice had about the
354
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INFECT. IMMUN.
(SDs])
(FI4h)
TABLE 2. Comparison of sensitization of mice to histamine with pyrogenicity and lethality to chicken embryos (CELD5s) as measures of endotoxic potency Prepn Rank Rank SD5, (/Ag) FL,4 (ug) Rank CELDJ, (Ag) 1 (1) 0.002 (0.005)a S-E-390 0.064 1 1 0.0028 0.003 (0.011) 2 (4) St 2 and 3 0.13 3 0.015 3 0.005 (0.008) 4 Reference ET-EC 0.19 3 (2 or 3) 0.0086 2 0.006 (0.089) 4 (7) 2 St 2 and 3, sonic 0.12 0.019 4 0.007 (0.008) 5 (2 or 3) S-E-388 0.20 5 =70 9 0.022 (0.020) 6 (5) 0.60 7 SR-11 RNA 0.021 5-6 0.035 (0.058) 7 (6) B. pertussis ET 0.50 6 0.021 5-6 3.4 8 (8) 8 Poly(I C) 216 0.73 7 (4.6) >16 9-13 (9-13) =200 B. abortus Fr. 5 9 >100 (>16) 11 >16 9-13 (9-13) >200 10 Ec 91/92 NH 15 8 (>16) >16 9-13 (9-13) >500 11 190 Ec 178 NH 10 (>16) >16 9-13 (9-13) >500 12 Poly(I) >500 (>16) 12-13 9-13 (9-13) >16 >1000 13 >200 12-13 Arabinogalactan (>16) a Values in parentheses represent the results obtained from a duplicate test to determine reproducibility of SD5o values. TABLE 3. Test to determine the specificity of the HHT for endotoxin Test substance
Dose (Lg)
Route
D/Ta
Human serum albumin 10 iv. 3/10 10 Bovine immunoglobulin i.v. 0/10 10 Hen egg albumin i.v. 2/10 10 Sucrose . ........ i.v. 0/10 10 Starch .... i.v. 2/10 10 Glycogen i.v. 0/10 10 Deoxyribonucleic acid i.v. 0/10 0.5 Endotoxin i.v. 10/10 10 Lecithin 4/20 i.p. 10 Cephalin ...... i.p. 4/20 Cerebrosides 10 3/20 i.p. Endotoxin .... ...... 0.5 i.p. 14/20 a Deaths per total number of mice tested after mice were challenged i.p. with 0.5 mg of histamine base given 90 min after administration of the test substance.
same ability to detect endotoxin as the CELD50 test. To check the reproducibility of the HHT, a second test was run on the preparations and a second SD50 value was calculated for each. A comparison of the results obtained in the two tests showed good reproducibility (Table 2, cf. two columns of data under SD50). "St 2 and 3, sonic" was the only anomalous preparation in that it had an SD50 value of 0.006 in the first test and a value of 0.089 in the second. The relative ranking of the preparations did not change much between the two tests. The highpotency and low-potency fractions remained the same, and no preparation shifted in rank by more than three positions. A number of other substances were tested for their ability to induce hypersensitivity to histamine at a dose level of 10 ,g per mouse given i.v. or i.p. 90 min before an i.p. challenge with 0.5 mg of histamine (Table 3). None of the substances induced a significant degree of hypersensitivity to histamine.
DISCUSSION These data show that the ability of the HHT to identify and quantitate endotoxin activity compares well with that of the rabbit pyrogenicity and CELD50 tests. The sensitivity of the HHT was equal to or greater than that of the CELD50 test (cf. results in Table 2). The results obtained by the HHT on the endotoxic potency of the various preparations correlated well with the results obtained by the other two tests. A comparison of the rankings obtained by the three different test methods on the tested preparations shows a high degree of correspondence among the three methods. The most potent endotoxin preparation by all three tests was SE-390. As would be expected in such tests, the correlation was not perfect. One anomalous preparation, S-E-388, was not highly lethal to chicken embryos but was pyrogenic and induced histamine hypersensitivity. It is interesting that the double-stranded mixture of polyinosinic and polycytidylic acids [poly (I C)] was active by the HHT. This preparation is probably slightly contaminated with endotoxin, as judged from samples of singlestranded synthetic polynucleotides examined in this laboratory. However, the level of activity shown in Table 2 cannot be accounted for by such contamination. Double stranding of poly(I) and poly(C) always produces a marked increase in endotoxin-like activity. None of the other substances tested by HHT, i.e., carbohydrates, proteins, or lipids, which were not endotoxic in nature, induced a significant degree of hypersensitivity to histamine. The HHT provides a test for endotoxin potency that is sensitive and reproducible and that has many advantages over other endotoxin tests presently in use: (i) it is very simple to perform; (ii) it is relatively inexpensive; (iii) it requires
TEST FOR ENDOTOXIN POTENCY
VOL. 18, 1977
no elaborate equipment; (iv) it detects sub-microgram amounts of endotoxin; (v) it takes only about 3 h to complete; and (vi) since the results are measured by death or survival of mice, it is simple to interpret. One of the drawbacks of the HHT may be that it requires -7-week-old CFW mice. Other strains tested (C57BL and RML) have not been satisfactory because they fail to sensitize to histamine. There are probably other strains that will prove suitable for this test, but we have not made an extensive survey. The number of mice required is relatively large because at least 10 mice per dose of endotoxin are required to obtain reliable results. The test is not absolutely specific because poly(I C) and pertussigen (2) sensitize mice to histamine within 90 to 120 min after administration. The effect of pertussigen can be differentiated from that of endotoxin because its activity is destroyed by heating at 80°C for 30 min. We believe that this HHT may prove to be a practical test for detecting and quantitating the activity of endotoxin.
5. 6.
7. 8.
9.
10. 11. 12.
13.
ACKNOWLEDGMENTS We wish to thank our technicians, Joe C. Ayers and Elsworth R. Pfeifer, for their competent assistance in these studies. 1.
2. 3.
4.
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