- Email: [email protected]
Standardization of allergenic extracts
4. Kadin, M. E. I980. Ia-like (HLA-DR) antigens in the diagnosis of lymphoma and undifferentiated tumors. Arch. PathoI. Lab. Med. I04:503-508. 5. Rappaport, H. 1966. Tumors of the hematopoietie system. In Atlas of tumor pathology, section III, Fascicle 8. Armed Forces Institute of Pathology, Washington, D. C.
6. Sallan, S. E., et al. 1980. Cell surface antigens: Prognostic implications in childhood acute lymphoblastic leukemia. Blood 55:395-402. 7. Salmon, S. E., and M. Seligmann. 1974. B-cell neoplasia in man. Lancet 2:1230-1233. 8. Siegal, F. P., D. A. Filippa, and B. Koziner. 1978. Surface markers in leu-
kemias and lymphomas. Am. J. Pathol. 90:451-460. 9. Warnke, R., et al. 1980. Immunologic phenotype in 30 patients with diffuse large-cell lymphoma. N. Engl. J. Med. 303:293-300.
Federal Forum Standardization of Allergenic Extracts
Harold Baer, P h . D . Director, Allergenic Products Branch Bureau o f Biologics Food and Drug A dministration 8800 Rockville Pike Bethesda, MD 20205 The standardization o f allergenic extracts will permit the availability o f extracts with a k n o w n quantity of allergenic activity and reference materials with lot-to-lot consistency. Standardizing an extract requires standards to which new lots o f extract can be c o m p a r e d and implies a methodology for making the comparison. The science o f allergy is now reaching this stage. Achieving the goal rapidly is difficult because o f the large dumber of extracts currently used in the practice o f allergy. The total is in excess of a thousand when consideration is given to the climatic and geographic diversity of the U. S., and consequently, there is a wide range o f biologic sources f r o m which extracts are made. Since extracts have been in use for m a n y decades and seem to have been successfully used by allergists, why is there a need to engage in a complex and difficult p r o g r a m that may increase the cost o f extracts? Examination o f commercially prepared extracts over the last decade has demonstrated their allergenic variability as well as the problems associated with interpreting the results obtained using them. The first study o f commercially produced short ragweed pollen extracts attempted to correlate allergenic activity, as measured by quantita|
94
tive skin testing and histamine release f r o m peripheral leukocytes with laboratory analysis o f extracts (5); there was no correlation between protein nitrogen units (PNU) or weightto-volume and allergenic activity, but there was a good correlation between allergenic activity and antigen E content, showing the importance o f measuring the defined allergenic substance. The study also demonstrated that commercially prepared extracts m a y vary 500- fold in allergenic activity. A subsequent study of short ragweed pollen extracts showed a thousand-fold variability in the allergenic activity o f commercially available short ragweed pollen extracts (10); the situation was no different for grass pollen extracts (6), Alternaria extracts (25), mite extracts (23), etc. Currently available extracts are
probably just as variable from manufacturer to m a n u f a c t u r e r as they were in 1970 (23). The effect o f this variability is both economically and clinically important for the practice o f allergy. The price o f all extracts is roughly the same, even though some may contain as little as 0.1 °70 of the aIlergenically active material of another extract. The impact of such variability on the science and practice o f allergy is not necessarily apparent to an individual allergist who is not making regular comparisons a m o n g extracts. It was recently demonstrated, using skin prick testing, that m a n y individuals are positive to one extract but negative to others (23). This results in confusion in diagnosis. The effect of this variability on immunotherapy is difficult to assess.
Editors: H e r m a n Friedman, Mario Escobar, and Noel Rose Editorial Committee: Charles D. Graber, Ph.D., Medical University of South Carolina; John R. Kateley, Ph.D., Edward W. Sparrow Hospital Association; Bruce S. Rabin, M.D., Ph.D., University of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York State Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina. Subscription Rates in U.S. and Canada: one year $54.00; two years $103.00; three years $146.00. All other countries: one year $62.00; two years $118.50; three years $168.00. Single copies are $2.50 in U.S. and Canada; $2.90 in all other countries. Single issues are available in quantity (prices available upon request). Volume ! (1980), 24 issues $46.50. All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada.
Address correspondence rega.t'ding subscription to: Clinical Immunology Newsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clinical bnmunology Newsletter is published twice monthly. All rights, including that of translation into other languages, reserved, Photomechanical reproduction (photocopy, microcopy) of this newslener or parts thereof without special permission of the publisher is prohibited.
With few exceptions (i.e., insect venoms and the short ragweed pollen extract of one manufacturer), extracts are labeled in weight-to-volume or PNU, which serve as the current measures of potency. The weight-tovolume unit is a record-keeping unit and cannot be confirmed by independent analysis. PNU, although frequently referred to as a protein nitrogen unit, is, in reality, a phosphotungstic acid precipitable nitrogen unit. Thus, any basic nitrogenous compounds will appear in the precipitate, including certain amino acids such as lysine. Furthermore, the value is not linear with concentration. The measurement of PNU and its analytic problems have been examined in detail (14). References for allergen standardization require appropriate source material from which extracts can be made. This is relatively simple for pollen since the grains are the cause of allergy and the allergenically important components are extracted rapidly (13, 16). For allergy caused by animals, the source is less evident. Although frequently referred to as epidermal, the allergens may not always be in or on the epidermis. As one example, the allergens from mice and rats have been the subject of several recent investigations and are almost certainly of urinary origin; extracts of pelts are likely to be low or even lacking in allergenic activity (21, 22). The mold powders from which extracts are made are also poorly characterized. Studies of commercially prepared extracts of Alternaria using radioallergosorbent tests (RAST) showed that some of these extracts had very different specificities from others (25). Ten strains of Cladosporium grown in one laboratory produced a variety of different allergens (3). Food allergens have been the subject of even fewer investigations but where they have been studied seem to be similar to other allergens, having relatively low molecular weights and inducing skin reactions in allergic individuals (15). For example, the allergens of codfish have been isolated and well characterized (8), and
peanut allergens are under investigation (20). The confusion that exists in the subject of food allergy is almost certainly the result of poor extracts and failure to differentiate those individuals with IgE-mediated disease from those with other food problems (e.g., metabolic disturbances). Standardization of all of these allergens awaits the proper definition of source material. The best studied and standardized allergens are the venoms of certain stinging insects: honeybee, wasp, yellow hornet, white-faced hornet, and yellow jacket. These are available only as standardized extracts. The source materials for the vespid venoms are the venom sacs removed from insects and identified to genus and species by a trained entomologist. The extraction of the sacs with buffer results in a solution of the venom including all of the venom proteins, which are the allergens, with very small quantities of other proteins. The source material for honeybee venom is obtained by electrically stimulating the bees and collecting the pure venom. The vials of final container are labeled with the protein content, which permits the use of measured doses. Since all of the insect venoms contain hyaluronidase, a known allergen in some venoms and almost certainly for all venoms, lotto-lot consistency is established by measuring the hyaluronidase content by a simple method employing hyaluronic acid agar (19). Each lot is also shown to contain phospholipase, an allergenically important component of all venoms. The reference hyaluronidase is a honeybee venom product prepared at the Bureau of Biologics. For the standardization of pollen extracts, the Bureau of Biologics has prepared reference extracts, either freeze-dried or in 50°70 glycerol, from some of the common pollens such as short and giant ragweed, grasses, and oak. Standardization of short ragweed pollen extract can be accomplished by the analysis of the antigen E content using radial immunodiffusion. Although the minimal acceptable level for a 1: 10
wt/vol extract is 135/ag/ml, the usual fresh extract should contain 250-400 /~g/ml. A short ragweed pollen extract standardized by this procedure is currently available from one manufacturer. Other pollen extracts may be standardized by RAST inhibition (2). Such extracts are not yet commercially available. One of the most useful procedures for examining the quality of pollen extracts is isoelectric focusing (IEF); its utility for other kinds of extracts has not been investigated. Pollen extracts that have been studied give distinctive IEF patterns; consequently, it is possible to distinguish pollen extracts from each other. This may be the only currently available method for distinguishing such closely related extracts as those derived from grass pollens and is the only simple, objective method for establishing their identity. All pollen extracts thus far studied by the author and his colleagues give sharply defined patterns that change as the extracts deteriorate until no bands at all are observed. Extracts with poor IEF patterns have low allergenic activity as determined by RAST or skin testing (1). IEF has the advantage of requiring no immunologic or isotopic reagents. By the analysis of individual bands, it is possible to determine their relationship to the allergenic activity of the extract (7, 24). A more powerful and much more complex method for evaluating allergenic extracts is crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis (4, 12). These techniques are useful for determining which proteins of any extract are allergens. Interpretation of the data is based on the assumption that the immunized rabbits have produced precipitating antibody to all of the aUergenically important proteins. In addition to having extracts manufactured with relatively constant allergenic activity, stability studies must be carried out to determine how long such extracts remain stable and to identify the best diluents to effect such stabilization of properties. Stability studies must be carried out for all recommended dilutions. Thus
95
rll
far, this has been accomplished for the venom products where it has been demonstrated that the most dilute extracts start losing their activity after one day. The stability o f pollen extracts has been studied extensively by laboratory analyses and b y skin-testing methods (9, 11, 17). Extracts that are freeze-dried and those in 50°70 glycerol-saline appear to be very stable for years at refrigerator temperatures. Some freeze-dried extracts increase in moisture content during storage, which slowly results in loss o f allergenic activity. Extracts in saline or buffer without glycerol maintain most of their allergenic activity even for a year if constantly kept cold, even during office use. Such extracts are very temperature-sensitive and m a y lose hal f o f their allergenic activity in less than a week at 35°C (11). The changes in such extracts can be visualized by isoelectric focusing and in crossed immunoelectrophoresis (1). Extracts used at high dilution for intradermal skin testing or for the initial i m m u n o t h e r a p y injection lose activity due to surface adsorption, and stability is improved by adding extra protein such as h u m a n serum albumin (l 8). Even dilute extracts maintain most o f their skin test reactivity for at least a year ~f maintained continuously cold (9). Short but frequent increases to r o o m temperatures, a c o m m o n office practice, result in rapid loss in allergenic activity, even in the presence o f stabilizers (17). Potency and stability studies o f very dilute extracts can be done by RAST inhibition or by quantitative skin testing.
96
References 1. Anderson, M. C., and H. Baer. 1980. Physiochemical and biologic changes in pollen extracts during storage. J. Allergy Clin. Immunol. 65:211. 2. Anderson, M. C., and H. Baer. 1981. RAST inhibition procedure. Technical bulletin. Bureau of Biologics, FDA, Bethesda, Md. 3. Ankrust, L. 1979. Crossed radioimmunoelectrophoretic studies of distinct allergens in two extracts of Cladosporium herbarum. Int. Arch. Allergy Appl. lmmunoI. 58:371-386. 4. Axelson, N. H., J. Kroll, and B. Weeke. 1973. Quantitative immunoelectrophoresis. Scand. J. Immunol. [Suppl.] 2:71-103. 5. Baer, H., et al. 1970. The potency and antigen E content of commercially prepared ragweed extracts. J. Allergy Clin. Immunol. 45:347-354. 6. Baer, H., et al. 1974. The potency and group I antigen content of six commercially prepared grass pollen extracts. J. Allergy Clin. Immunol. 54:157-164. 7. Brighton, W. D. 1975. Profiles of allergen extract components by isoelectric focusing and radioimmunoassay. Dev. Biol. Stand. 29:362-367. 8. Elsayed, S., et al. 1980. A synthetic hexadecapeptide from allergen M imposing allergenic and antigenic reactivity. Scand. J. Immunol. 12:171-175. 9. Franklin, R., et al. 1976. The stability of short ragweed pollen extract as measured by skin test and antigen E. J. Allergy Ciin. lmmunol. 58:51-59. I0. Gleich, G. J., el al. 1974. Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test. J. Allergy Clin. Immunol. 53:158-169. 11. Hiatt, C. W., H. Baer, and M. L. Hooton. 1977. Kinetics of thermal decay of antigen E from short ragweed pollen. J. Biol. Stand. 5:39-44. 12. Lowenstein, H. 1978. Quantitative immunoelectrophoretic methods as a tool for the analysis and isolation of allergens. Prog. Allergy 25:1-62. 13. Marsh, D. G., et al. 1981. Rapidly released allergens from short ragweed pollen I. Kinetics of release of known allergens in relation to biologic activ-
ity. J. Allergy Ciin. Immunol. 67:206-216. 14. May, C . D , and S. A. Block. 1978. A modern clinical approach to food hypersensitivity. Allergy 33:166-188. 15. May, J. 1979. Optimization of parameters in protein nitrogen unit precipitation procedure for allergenic extracts. J. Allergy Clin. Immunol. 63:87-97. 16. Morrow-Brown, H., and M. Thantrey. 1976. Rapid extraction of grass pollen allergens and separation of their active fractions. Acta AIlergologica 31:22-30. 17. Nelson, H. S. 1979. The effect of preservatives and dilution on the deterioration of Russian thistle (Salsola pestifer), a pollen extract. J. Allergy Clin. Immunol. 63:417-425. 18. Norman, P. S., and D. G. Marsh. 1978. Human serum albumin and tween 80 as stabilizers of allergen solutions. J. Allergy Clin. Immunol. 62:314-319. 19. Richman, P. G., and H. Baer. 1981. A convenient plate assay for the quantitation of hyaluronidase in Hymenoptera venoms. Anal. Biochem. 109:376-381. 20. Sachs, M. I., R. T. Jones, and J. W. Yunginger. 1981. Isolation and partial characterization of a major peanut allergen. J. Allergy Clin. Immunol. 67:27-34. 21. Schumacher, M. J. 1980. Characterization of allergens from urine and pelts of laboratory mice. Mol. lmmunol. 17:1087-1091. 22. Taylor, A. N., J. L. Longbottom, and J. Pepys. 1977. Respiratory allergy to urine proteins of rats and mice. Lancet ii:847. 23. Turner, K. J., el al. 1980. Standardization of allergen extracts by inhibition of RAST, skin test and chemical composition. Clin. Allergy 10:441-450. 24. Varga, J. M., and M. Ceska. 1972. Characterization of allergen extracts by gel isoelectric focusing and radioimmunosorbent assay. J. Allergy Clin. lmmunol. 49:274-284. 25. Yunginger, J. W., G. D. Roberts, and G. J. Gleich. 1976. Studies on Alternaria allergens. J. Allergy Clin. Immunol. 57:293-301.
6. Sallan, S. E., et al. 1980. Cell surface antigens: Prognostic implications in childhood acute lymphoblastic leukemia. Blood 55:395-402. 7. Salmon, S. E., and M. Seligmann. 1974. B-cell neoplasia in man. Lancet 2:1230-1233. 8. Siegal, F. P., D. A. Filippa, and B. Koziner. 1978. Surface markers in leu-
kemias and lymphomas. Am. J. Pathol. 90:451-460. 9. Warnke, R., et al. 1980. Immunologic phenotype in 30 patients with diffuse large-cell lymphoma. N. Engl. J. Med. 303:293-300.
Federal Forum Standardization of Allergenic Extracts
Harold Baer, P h . D . Director, Allergenic Products Branch Bureau o f Biologics Food and Drug A dministration 8800 Rockville Pike Bethesda, MD 20205 The standardization o f allergenic extracts will permit the availability o f extracts with a k n o w n quantity of allergenic activity and reference materials with lot-to-lot consistency. Standardizing an extract requires standards to which new lots o f extract can be c o m p a r e d and implies a methodology for making the comparison. The science o f allergy is now reaching this stage. Achieving the goal rapidly is difficult because o f the large dumber of extracts currently used in the practice o f allergy. The total is in excess of a thousand when consideration is given to the climatic and geographic diversity of the U. S., and consequently, there is a wide range o f biologic sources f r o m which extracts are made. Since extracts have been in use for m a n y decades and seem to have been successfully used by allergists, why is there a need to engage in a complex and difficult p r o g r a m that may increase the cost o f extracts? Examination o f commercially prepared extracts over the last decade has demonstrated their allergenic variability as well as the problems associated with interpreting the results obtained using them. The first study o f commercially produced short ragweed pollen extracts attempted to correlate allergenic activity, as measured by quantita|
94
tive skin testing and histamine release f r o m peripheral leukocytes with laboratory analysis o f extracts (5); there was no correlation between protein nitrogen units (PNU) or weightto-volume and allergenic activity, but there was a good correlation between allergenic activity and antigen E content, showing the importance o f measuring the defined allergenic substance. The study also demonstrated that commercially prepared extracts m a y vary 500- fold in allergenic activity. A subsequent study of short ragweed pollen extracts showed a thousand-fold variability in the allergenic activity o f commercially available short ragweed pollen extracts (10); the situation was no different for grass pollen extracts (6), Alternaria extracts (25), mite extracts (23), etc. Currently available extracts are
probably just as variable from manufacturer to m a n u f a c t u r e r as they were in 1970 (23). The effect o f this variability is both economically and clinically important for the practice o f allergy. The price o f all extracts is roughly the same, even though some may contain as little as 0.1 °70 of the aIlergenically active material of another extract. The impact of such variability on the science and practice o f allergy is not necessarily apparent to an individual allergist who is not making regular comparisons a m o n g extracts. It was recently demonstrated, using skin prick testing, that m a n y individuals are positive to one extract but negative to others (23). This results in confusion in diagnosis. The effect of this variability on immunotherapy is difficult to assess.
Editors: H e r m a n Friedman, Mario Escobar, and Noel Rose Editorial Committee: Charles D. Graber, Ph.D., Medical University of South Carolina; John R. Kateley, Ph.D., Edward W. Sparrow Hospital Association; Bruce S. Rabin, M.D., Ph.D., University of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York State Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina. Subscription Rates in U.S. and Canada: one year $54.00; two years $103.00; three years $146.00. All other countries: one year $62.00; two years $118.50; three years $168.00. Single copies are $2.50 in U.S. and Canada; $2.90 in all other countries. Single issues are available in quantity (prices available upon request). Volume ! (1980), 24 issues $46.50. All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada.
Address correspondence rega.t'ding subscription to: Clinical Immunology Newsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clinical bnmunology Newsletter is published twice monthly. All rights, including that of translation into other languages, reserved, Photomechanical reproduction (photocopy, microcopy) of this newslener or parts thereof without special permission of the publisher is prohibited.
With few exceptions (i.e., insect venoms and the short ragweed pollen extract of one manufacturer), extracts are labeled in weight-to-volume or PNU, which serve as the current measures of potency. The weight-tovolume unit is a record-keeping unit and cannot be confirmed by independent analysis. PNU, although frequently referred to as a protein nitrogen unit, is, in reality, a phosphotungstic acid precipitable nitrogen unit. Thus, any basic nitrogenous compounds will appear in the precipitate, including certain amino acids such as lysine. Furthermore, the value is not linear with concentration. The measurement of PNU and its analytic problems have been examined in detail (14). References for allergen standardization require appropriate source material from which extracts can be made. This is relatively simple for pollen since the grains are the cause of allergy and the allergenically important components are extracted rapidly (13, 16). For allergy caused by animals, the source is less evident. Although frequently referred to as epidermal, the allergens may not always be in or on the epidermis. As one example, the allergens from mice and rats have been the subject of several recent investigations and are almost certainly of urinary origin; extracts of pelts are likely to be low or even lacking in allergenic activity (21, 22). The mold powders from which extracts are made are also poorly characterized. Studies of commercially prepared extracts of Alternaria using radioallergosorbent tests (RAST) showed that some of these extracts had very different specificities from others (25). Ten strains of Cladosporium grown in one laboratory produced a variety of different allergens (3). Food allergens have been the subject of even fewer investigations but where they have been studied seem to be similar to other allergens, having relatively low molecular weights and inducing skin reactions in allergic individuals (15). For example, the allergens of codfish have been isolated and well characterized (8), and
peanut allergens are under investigation (20). The confusion that exists in the subject of food allergy is almost certainly the result of poor extracts and failure to differentiate those individuals with IgE-mediated disease from those with other food problems (e.g., metabolic disturbances). Standardization of all of these allergens awaits the proper definition of source material. The best studied and standardized allergens are the venoms of certain stinging insects: honeybee, wasp, yellow hornet, white-faced hornet, and yellow jacket. These are available only as standardized extracts. The source materials for the vespid venoms are the venom sacs removed from insects and identified to genus and species by a trained entomologist. The extraction of the sacs with buffer results in a solution of the venom including all of the venom proteins, which are the allergens, with very small quantities of other proteins. The source material for honeybee venom is obtained by electrically stimulating the bees and collecting the pure venom. The vials of final container are labeled with the protein content, which permits the use of measured doses. Since all of the insect venoms contain hyaluronidase, a known allergen in some venoms and almost certainly for all venoms, lotto-lot consistency is established by measuring the hyaluronidase content by a simple method employing hyaluronic acid agar (19). Each lot is also shown to contain phospholipase, an allergenically important component of all venoms. The reference hyaluronidase is a honeybee venom product prepared at the Bureau of Biologics. For the standardization of pollen extracts, the Bureau of Biologics has prepared reference extracts, either freeze-dried or in 50°70 glycerol, from some of the common pollens such as short and giant ragweed, grasses, and oak. Standardization of short ragweed pollen extract can be accomplished by the analysis of the antigen E content using radial immunodiffusion. Although the minimal acceptable level for a 1: 10
wt/vol extract is 135/ag/ml, the usual fresh extract should contain 250-400 /~g/ml. A short ragweed pollen extract standardized by this procedure is currently available from one manufacturer. Other pollen extracts may be standardized by RAST inhibition (2). Such extracts are not yet commercially available. One of the most useful procedures for examining the quality of pollen extracts is isoelectric focusing (IEF); its utility for other kinds of extracts has not been investigated. Pollen extracts that have been studied give distinctive IEF patterns; consequently, it is possible to distinguish pollen extracts from each other. This may be the only currently available method for distinguishing such closely related extracts as those derived from grass pollens and is the only simple, objective method for establishing their identity. All pollen extracts thus far studied by the author and his colleagues give sharply defined patterns that change as the extracts deteriorate until no bands at all are observed. Extracts with poor IEF patterns have low allergenic activity as determined by RAST or skin testing (1). IEF has the advantage of requiring no immunologic or isotopic reagents. By the analysis of individual bands, it is possible to determine their relationship to the allergenic activity of the extract (7, 24). A more powerful and much more complex method for evaluating allergenic extracts is crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis (4, 12). These techniques are useful for determining which proteins of any extract are allergens. Interpretation of the data is based on the assumption that the immunized rabbits have produced precipitating antibody to all of the aUergenically important proteins. In addition to having extracts manufactured with relatively constant allergenic activity, stability studies must be carried out to determine how long such extracts remain stable and to identify the best diluents to effect such stabilization of properties. Stability studies must be carried out for all recommended dilutions. Thus
95
rll
far, this has been accomplished for the venom products where it has been demonstrated that the most dilute extracts start losing their activity after one day. The stability o f pollen extracts has been studied extensively by laboratory analyses and b y skin-testing methods (9, 11, 17). Extracts that are freeze-dried and those in 50°70 glycerol-saline appear to be very stable for years at refrigerator temperatures. Some freeze-dried extracts increase in moisture content during storage, which slowly results in loss o f allergenic activity. Extracts in saline or buffer without glycerol maintain most of their allergenic activity even for a year if constantly kept cold, even during office use. Such extracts are very temperature-sensitive and m a y lose hal f o f their allergenic activity in less than a week at 35°C (11). The changes in such extracts can be visualized by isoelectric focusing and in crossed immunoelectrophoresis (1). Extracts used at high dilution for intradermal skin testing or for the initial i m m u n o t h e r a p y injection lose activity due to surface adsorption, and stability is improved by adding extra protein such as h u m a n serum albumin (l 8). Even dilute extracts maintain most o f their skin test reactivity for at least a year ~f maintained continuously cold (9). Short but frequent increases to r o o m temperatures, a c o m m o n office practice, result in rapid loss in allergenic activity, even in the presence o f stabilizers (17). Potency and stability studies o f very dilute extracts can be done by RAST inhibition or by quantitative skin testing.
96
References 1. Anderson, M. C., and H. Baer. 1980. Physiochemical and biologic changes in pollen extracts during storage. J. Allergy Clin. Immunol. 65:211. 2. Anderson, M. C., and H. Baer. 1981. RAST inhibition procedure. Technical bulletin. Bureau of Biologics, FDA, Bethesda, Md. 3. Ankrust, L. 1979. Crossed radioimmunoelectrophoretic studies of distinct allergens in two extracts of Cladosporium herbarum. Int. Arch. Allergy Appl. lmmunoI. 58:371-386. 4. Axelson, N. H., J. Kroll, and B. Weeke. 1973. Quantitative immunoelectrophoresis. Scand. J. Immunol. [Suppl.] 2:71-103. 5. Baer, H., et al. 1970. The potency and antigen E content of commercially prepared ragweed extracts. J. Allergy Clin. Immunol. 45:347-354. 6. Baer, H., et al. 1974. The potency and group I antigen content of six commercially prepared grass pollen extracts. J. Allergy Clin. Immunol. 54:157-164. 7. Brighton, W. D. 1975. Profiles of allergen extract components by isoelectric focusing and radioimmunoassay. Dev. Biol. Stand. 29:362-367. 8. Elsayed, S., et al. 1980. A synthetic hexadecapeptide from allergen M imposing allergenic and antigenic reactivity. Scand. J. Immunol. 12:171-175. 9. Franklin, R., et al. 1976. The stability of short ragweed pollen extract as measured by skin test and antigen E. J. Allergy Ciin. lmmunol. 58:51-59. I0. Gleich, G. J., el al. 1974. Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test. J. Allergy Clin. Immunol. 53:158-169. 11. Hiatt, C. W., H. Baer, and M. L. Hooton. 1977. Kinetics of thermal decay of antigen E from short ragweed pollen. J. Biol. Stand. 5:39-44. 12. Lowenstein, H. 1978. Quantitative immunoelectrophoretic methods as a tool for the analysis and isolation of allergens. Prog. Allergy 25:1-62. 13. Marsh, D. G., et al. 1981. Rapidly released allergens from short ragweed pollen I. Kinetics of release of known allergens in relation to biologic activ-
ity. J. Allergy Ciin. Immunol. 67:206-216. 14. May, C . D , and S. A. Block. 1978. A modern clinical approach to food hypersensitivity. Allergy 33:166-188. 15. May, J. 1979. Optimization of parameters in protein nitrogen unit precipitation procedure for allergenic extracts. J. Allergy Clin. Immunol. 63:87-97. 16. Morrow-Brown, H., and M. Thantrey. 1976. Rapid extraction of grass pollen allergens and separation of their active fractions. Acta AIlergologica 31:22-30. 17. Nelson, H. S. 1979. The effect of preservatives and dilution on the deterioration of Russian thistle (Salsola pestifer), a pollen extract. J. Allergy Clin. Immunol. 63:417-425. 18. Norman, P. S., and D. G. Marsh. 1978. Human serum albumin and tween 80 as stabilizers of allergen solutions. J. Allergy Clin. Immunol. 62:314-319. 19. Richman, P. G., and H. Baer. 1981. A convenient plate assay for the quantitation of hyaluronidase in Hymenoptera venoms. Anal. Biochem. 109:376-381. 20. Sachs, M. I., R. T. Jones, and J. W. Yunginger. 1981. Isolation and partial characterization of a major peanut allergen. J. Allergy Clin. Immunol. 67:27-34. 21. Schumacher, M. J. 1980. Characterization of allergens from urine and pelts of laboratory mice. Mol. lmmunol. 17:1087-1091. 22. Taylor, A. N., J. L. Longbottom, and J. Pepys. 1977. Respiratory allergy to urine proteins of rats and mice. Lancet ii:847. 23. Turner, K. J., el al. 1980. Standardization of allergen extracts by inhibition of RAST, skin test and chemical composition. Clin. Allergy 10:441-450. 24. Varga, J. M., and M. Ceska. 1972. Characterization of allergen extracts by gel isoelectric focusing and radioimmunosorbent assay. J. Allergy Clin. lmmunol. 49:274-284. 25. Yunginger, J. W., G. D. Roberts, and G. J. Gleich. 1976. Studies on Alternaria allergens. J. Allergy Clin. Immunol. 57:293-301.