- Email: [email protected]
Isolation and preparation of antigens
VOLUME NUMBER
61 4
Antigens in hypersensitivity
mannan described by Hasenclever and Mitchell, reacts in humans with two classes of antibody to give immediate reactions, IgE, and short-term sensitizing IgG antibody. There are also precipitins to the mannan antigen which can mediate type III reactions. Mannan does not give type IV skin test reactions and we do not know if it can stimulate lymphoid cells in culture. In contrast, the purified somatic proteins of Candida albicans which have no mannan in them react with specific IgE antibody to give immediate reac-
ts
km and pmparatim
pneumonitis
203
tions. They give type III-like reactions for which we have not been able to find precipitins and also typical type IV reactions involving lymphoid cells. Thus, we have in one yeast cell a variety of antigens capable of doing many different things, showing us that, once we have satisfied ourselves about the sources of relevant allergens in the form of spores or whatever else they may be, it is necessary to proceed with careful detailed antigenic analysis and analysis of the immunopathogenetic effects of thcw individual antigens.
of a
8
S. Lehrer, Ph.D. New Orleans, La.
The nature of inhaled material is of profound importance in the production of hypersensitivity pneumonitis (HP). Therefore, the isolation and separation of antigens present in such material is critical in order to analyze immune pathologic mechanisms in disease and to establish immunologic techniques useful as diagnostic assays. The first step in isolation of an antigen is to establish a quantitative or semiquantitative biological assay. For antigens active in HP this is no easy task, since evidence suggests that pulmonary infiltrates in animal models and in man may be associated with several biological mechanisms. These include immunologic reactions such as cellular immune complex or cytotoxic allergic tissue injury. It is also possible that particulate organic dust antigens may possess adjuvant, irritant, alternate complement pathway activating or toxic properties, all of which could be important in production of pulmonary lesions. Obviously no single biological assay can adequately cover all of these activities and therefore several assays should be employed in isolating antigens. There are two obvious approaches to assay of antigens in HPthat is the use of patients or animal models. A patient’s response to antigens, obviously the most direct assay, is generally difficult to manipulate and quantitate. One can challenge or skin test a patient with antigen, but it is not practical for initial assays. In vitro tests such as the precipitin reaction with patients’ sera or antigen stimulation of patients’ lymphocytes are easier to perform but are usually limited by the small quantities of material available.
Many investigators use animal models since they are much easier to manipulate. One can assess functional or histologic changes induced by antigen, but these are time consuming and difficult to quantitate. The skin test can always be used for in vitro tests such as antigen reaction with serum or cells of immune animals. These are easily done and readily quantitated. Once a reliable quantitative assay of antigen is established, extraction can begin. The main objective of any extraction procedure is to solubilize as much material as possible while minimizing artifactual changes. Although crude material (such as motdy hay or bagasse) is usually a rich source of antigen, it is best to attempt to grow any offending microorganism in vitro. In such cases culture fluids can provide excellent sources of soluble antigens. If homogenization, of which there are several methods available, is necessary, it must be carried out under conditions which do not inactivate the material under investigation. Mechanical disintegration can be achieved by a variety of methods. Chemical disintegration can be performed with different chemicals or biochemicals such as treatment with detergents, high salts, high concentrations of salts, or enzymatic digestion. Once a soluble extract of antigens has been obtained, purification can proceed. It is stressed that during any fractionation procedure one must demonstrate where activity resides and what the recovery i?;. There are many techniques available for fractionation and analysis of antigen based on their physical-chemical properties.
204
NIAID Workshop
Gel filtration and ion-exchange column chromatography are two popularly used methods: the first, separation of molecules based on their shape and size; the second, fractionation of molecules based on their net electric charge. Adsorption and partition chromatography are infrequently used methods but can provide elegant separation of antigens. These methods separate molecules based on their affinity or solubility in different substances. Ultracentrifugation and electrophoresis are methods frequently used. Recently, immunochemical methods have been used. These combine immunologic principles with other methods of separation such as immunoelectrophoresis or immunoabsorption. After obtaining the maximal activity unit per dry weight, it is necessary to demonstrate homogeneity of an antigen before additional characterization can be done. Generally, any of the techniques can be used to characterize homogeneity, but it should be stressed that one must use more than one assay when determining homogeneity of a substance. Also, when using these assays one should demonstrate the biological activity contained in the single peak or single band demonstrated. As an example of a specific isolation procedure, I refer to my own work with isolation of the histaminesensitizing factor from Bordetella pertussis. Although this conference deals with antigens active in HP, I believe the following will serve not only as an example of an isolation procedure but also a demonstration of microbial substance which can profoundly alter the immune response of an animal, a situation which may occur with other microbial factors in HP. Pertussis induces a mononuclear cell infiltrate in the lungs of animals treated with it. This may have relevance to those changes observed in HP. It is well established that Bordetella pertussis can alter the normal responses of laboratory animals or man to a number of stimuli. One of the changes most significant for immunology is that pertussis can alter the immune response of an antigen both quantitatively and qualitatively. Whole cells or extracts of pertussis can induce a profound leukocytosis due primarily to an increase in circulating lymphocytes. In addition to these changes, pertussis induces a hypersensitivity in experimental animals to a variety of agents such as pharmacologic mediators and bacterial toxins. The most frequently investigated of these hypersensitivities induced by pertussis is sensitization to histamine. Usually 20 to 40 mg of histamine base is a lethal dose in a normal mouse. However, if the mouse is pretreated with whole cells of Bordetella pertussis, it will be easily killed by less than 1 mg from what appears to be an anaphylaxis-like reaction. The bac-
J. ALLERGY
CLIN. IMMUNOL. APRIL 1978
terial component responsible for sensitization is called the histamine-sensitizing factor (HSF). HSF activity is quantitated by the sensitizing dose-50 determination (SD,,). This is the functional assay for HSF. Mice are sensitized by an intravenous injection containing different concentrations of test material. After a latent period of 3 to 4 days the animals are challenged with 1 mg histamine base and deaths recorded for the next hour. From these results one can calculate the SDS0in test material. The total number of SDS,,values can be estimated by dividing test material weight by its SD,,. In the past, soluble preparations of HSF have been obtained from culture supernates and bacterial cells treated by a variety of methods. All of these were contaminated with other bacterial components. Further purification results in decreased solubility of HSF and loss of bacterial activity. Since none of these procedures yielded optimal results, we chose to develop our own extraction and purification procedure. In order to extract HSF from bacterial cells, different concentrations and combinations of solvents were tested and the number of SDS0 values released determined and expressed as percentage of the total activity. The different combinations of urea, sodium chloride, sodium deoxycholate, and triton X-100 is different concentrations were used in attempts to release histamine-sensitizing factor. Clearly the greatest amount of activity was extracted with a combination of 4 M urea and 1 M sodium chloride which extracted approximately 78% of the total activity. All other solvents appear to be less effective. However, biological activity was not stable in urea sodium chloride solution and after several days at 4” C was completely destroyed. We found, though, if urea was removed from the extract, biological activity was relatively stable at 4” for several weeks. However, this led to other problems. Upon concentration of HSF extract in 1 M sodium chloride, more than half of the activity became insoluble. The remaining activity was polydispersed when analyzed by sucrose density-gradient ultracentrifugation. This represents a 5% to 20% sucrose density gradient in 1 M sodium chloride. All the gradient fractions were analyzed for ultraviolet absorption at 280, 260, and 230 nm, and fractions were also assayed for histamine-sensitizing activity expressed as SDS,, per fraction. There was a 260 absorbing peak at approximately 50% of the gradient, and a 230 absorbing peak at approximately 80% of the gradient. Small amounts of histamine-sensitizing activity were demonstrated in the heavier portion of the gradient, but, clearly, most of the histamine-sensitizing activity was dispersed
VOLUME NUMBER
61 4
over a broad region of the lighter portion of the gradient. We concluded that the removal of urea led to the insolubility and heterogeneity of HSF, and this was supported in the following experiment. First, the problem of lability of HSF in urea sodium chloride was resolved by buffering the solution at a pH range of 4 to 6.4. This stabilized HSF activity for several weeks. If urea remained in the extract now, all of the extracted activity remained soluble after concentration. After sucrose density-gradient analysis in buffered urea sodium chloride solution the HSF activity was more homogeneous and sedimented in the region of the bovine serum albumin (BSA) marker. Pertussis vaccine was centrifuged and the pelleted bacterial cells extracted with urea sodium chloride solution. This was done by sonication. Sonicate was mixed overnight and centrifuged and the resulting supernate concentrated and then centrifuged a second time. The final extract contained more than 50% of the initial histamine-sensitizing activity and had an SDjo of 1.045 pg which represents approximately a 16-fold increase in specific activity compared with the bacterial vaccine. In addition to histamine-sensitizing activity this extract also produced a leukocytosis in mice and had adjuvant activity. Since the soluble relatively homogeneous HSF extract could now be obtained, this preparation was purified by gel filtration on a biogel A 0.5 M column, equilibrated with urea sodium chloride buffer. At 230, gel filtration of the pertussis extract demonstrated five peaks of ultraviolet absorbing material. Column fractions are pooled and assayed for histamine-sensitizing activity. Minimal amounts of activity were associated with the first peak, but most of the histamine-sensitizing activity appeared to be associated with a third peak and came off the column just prior to the BSA marker. The portion of the column eluate that contained maximal histamine-sensitizing activity also contained leukocytosis-promoting activity. Column fractions were also assayed for adjuvant activity for hemagglutinating antibody. In a control mouse which was immunized only with ovalbumin, maximal passive hemagglutination antibody titers of 40 were obtained. In animals treated with column fractions in addition to immunization with ovalbumin there was enhancement of antibody response of phytohemagglutinin (PHA) titers up to 640. There were clearly three regions of maximal adjuvant activity for hemagglutinating antibody, the second of which also contained histamine-sensitizing and leukocytosis-promoting activity. Adjuvant activity for reaginic antibody was assayed by enhanced reaginic antibody response determined by passive cutaneous anaphylaxis (PCA)
Antigens in hypersensitivity
pneumonitis
205
titer. There were two regions of column eluate which contained adjuvant activity for reaginic antibody. The second one also contained histamine-sensitizing, leukocytosis-promoting, and adjuvant activities for reaginic and hemagglutinating antibody. This portion of the column eluate was pooled and rechromatographed on a Sephadex G- 100 column. The ultraviolet absorption profile demonstrated the gel filtration on G-100 revealing four peaks. Histamine-sensitizing activity appeared to be associated with the descending portion of the second peak and corresponded to a molecular weight of 90,000 daltons. This fraction also contained the leukocytosis-promoting activity. Adjuvant activity for hemagglutinating antibody again appeared to be more dispersed. However, adjuvant activity for reaginic antibody was more restricted and present in the same fractions that contained histamine-sensitizing activity and leukocytosis-promoting activity. This portion of the column eluate was pooled and designated G-100 HSF. It had an SD,, of 6 pg which represents a 280-fold purification of histamine-sensitizing activity compared with crude bacterial vaccine. Further purification was achieved through use of a cationic exchanger sulfopropyl sulfadex. A sample was equilibrated with 4 M urea, 0.2 M sodium chloride buffered with citrate at pH 4. UltravioIet absorption at 280 nm demonstrated at least five different components with little affinity for the resin. None of these components contained any biological activity. A pH gradient from 4 ro 4.8 eluted a component which appeared to contain all the histamine-sensitizing activity adjuvant of leukocytosis-promoting activity and adjuvant activities for hemagglutinating and reaginic antibodies. A further pH and sodium chloride gradient eluted a component which contained considerable amounts of protein but did not contain biological activity. Chemical analysis of partially purified HSF demonstrated that mainly protein and lipid were present, suggesting that HSF may be a lipoprotein. This was supported in part by the fact that histamine-sensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibodies were not altered by treatment of extracts with DNase or KNase but were significantly reduced by treatment with proteolytic enzymes. In addition to biochemical studies, immunochemical analysis of HSF was performed. Antisera from rabbits immunized with HSF isolated from the G-100 column detected several antigens by immunoelectrophoresis. All the antigens demonstrated a slight movement toward the cathode. In order to determine if biological activity might coin-
206
J. ALLERGY
NIAID Workshop
tide in electrophoretic mobility with these antigens, slides were cut in small segments and eluted, and the eluate was tested for biological activity. The results showed that maximal histamine-sensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibody were present in fraction 6. Adjuvant activity for hemagglutinating antibody appeared to be rather dispersed. Since biological activities were present in regions in which precipitating antigens were detected, experiments were performed to determine if antisera could react with these activities. When pertussis extracts were treated with rabbit antiserum, in vitro antigen-antibody precipitates developed. After removal of these precipitates it was demonstrated that the supernates with reduced antigen content contained minimal biological activity compared with extracts treated with normal rabbit serum. Furthermore, in vivo neutralization of histaminesensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibody was achieved by passive immunization of mice with rabbit antiserum to HSF prior to sensitization. From these results it was concluded that HSF is a pertussis antigen. Evidence suggests that it is a lipoprotein of approximately 90,000 daltons in size. In addition to histamine-sensitizing activity it contains leukocyto-
Standardization
CLIN.
IMMUNOL. APRIL 1979
sis-promoting activity and adjuvant activity for reaginic antibody. By extraction and sucrose density-gradient analysis, optimal solvent conditions for HSF were determined. Further studies with the use of gel filtration and ion-exchange column chromatography demonstrated the size and charge of HSF and suggested that it contained other biological activities since they all were of identical physical-chemical properties. These chromatographic procedures also yielded partially purified preparation of HSF on which meaningful chemical and biochemical analysis could be done. In summary, the important steps in antigen isolation have been reviewed. The first is to establish a reliable quantitative or at least semiquantitative biological assay. Once this is done solubilization of antigens can be achieved. After we have a soluble extract of antigens there are a variety of methods which can be used to fractionate and analyze antigens. As an example of a microbial antigen isolation, I have reviewed the extraction and purification of histamine-sensitizing factor of Bordetella pertussis. Although this is not directly related to HP, HSF may be similar to other microbial antigens in this disease. Analysis of such antigens is essential before we can examine immunopathologic mechanisms in HP or devise meaningful diagnostic assays.
of antigens
H. Baer, Ph.D. Bethesda, Md.
Standardization of antigens or allergens in areas such as hypersensitivity pneumonitis (HP) and hay fever-type allergy presents a number of problems. Dr. Norman of John Hopkins Allergy Clinic, Dr. Gleich of the Mayo Foundation, and I started our investigation of these problems by determining the activity of commercially available antigens. Extracts from these sources had very diverse activities, which were definable by a variety of tests. Measurement of the reactivity of these extracts is complicated by variability in the tests used. Reactivity of a group of extracts may appear to differ by lo- to 100-fold in one test, but the same extracts in another test may vary in reactivity by only 2- or 3-fold. Depending on the test used, the same extracts may be very different or only slightly different.
The enormous number of extracts available makes standardization particularly difficult. Most of the companies that produce allergenic extracts have listings of approximately 1,000 such extracts; they also produce extracts for HP. Therefore, we decided to concentrate on those antigens which were most frequently implicated as causing disease. In the pollen field we decided to concentrate on ragweed and certain grasses which are rather widespread and cause allergy quite frequently in the United States. Geographic variations, such as deserts and forests, might change these choices for allergists in some areas, but these allergen selections are still valid for the most part. There are two aspects in any standardization program of this type. The scientific aspect demands the
61 4
Antigens in hypersensitivity
mannan described by Hasenclever and Mitchell, reacts in humans with two classes of antibody to give immediate reactions, IgE, and short-term sensitizing IgG antibody. There are also precipitins to the mannan antigen which can mediate type III reactions. Mannan does not give type IV skin test reactions and we do not know if it can stimulate lymphoid cells in culture. In contrast, the purified somatic proteins of Candida albicans which have no mannan in them react with specific IgE antibody to give immediate reac-
ts
km and pmparatim
pneumonitis
203
tions. They give type III-like reactions for which we have not been able to find precipitins and also typical type IV reactions involving lymphoid cells. Thus, we have in one yeast cell a variety of antigens capable of doing many different things, showing us that, once we have satisfied ourselves about the sources of relevant allergens in the form of spores or whatever else they may be, it is necessary to proceed with careful detailed antigenic analysis and analysis of the immunopathogenetic effects of thcw individual antigens.
of a
8
S. Lehrer, Ph.D. New Orleans, La.
The nature of inhaled material is of profound importance in the production of hypersensitivity pneumonitis (HP). Therefore, the isolation and separation of antigens present in such material is critical in order to analyze immune pathologic mechanisms in disease and to establish immunologic techniques useful as diagnostic assays. The first step in isolation of an antigen is to establish a quantitative or semiquantitative biological assay. For antigens active in HP this is no easy task, since evidence suggests that pulmonary infiltrates in animal models and in man may be associated with several biological mechanisms. These include immunologic reactions such as cellular immune complex or cytotoxic allergic tissue injury. It is also possible that particulate organic dust antigens may possess adjuvant, irritant, alternate complement pathway activating or toxic properties, all of which could be important in production of pulmonary lesions. Obviously no single biological assay can adequately cover all of these activities and therefore several assays should be employed in isolating antigens. There are two obvious approaches to assay of antigens in HPthat is the use of patients or animal models. A patient’s response to antigens, obviously the most direct assay, is generally difficult to manipulate and quantitate. One can challenge or skin test a patient with antigen, but it is not practical for initial assays. In vitro tests such as the precipitin reaction with patients’ sera or antigen stimulation of patients’ lymphocytes are easier to perform but are usually limited by the small quantities of material available.
Many investigators use animal models since they are much easier to manipulate. One can assess functional or histologic changes induced by antigen, but these are time consuming and difficult to quantitate. The skin test can always be used for in vitro tests such as antigen reaction with serum or cells of immune animals. These are easily done and readily quantitated. Once a reliable quantitative assay of antigen is established, extraction can begin. The main objective of any extraction procedure is to solubilize as much material as possible while minimizing artifactual changes. Although crude material (such as motdy hay or bagasse) is usually a rich source of antigen, it is best to attempt to grow any offending microorganism in vitro. In such cases culture fluids can provide excellent sources of soluble antigens. If homogenization, of which there are several methods available, is necessary, it must be carried out under conditions which do not inactivate the material under investigation. Mechanical disintegration can be achieved by a variety of methods. Chemical disintegration can be performed with different chemicals or biochemicals such as treatment with detergents, high salts, high concentrations of salts, or enzymatic digestion. Once a soluble extract of antigens has been obtained, purification can proceed. It is stressed that during any fractionation procedure one must demonstrate where activity resides and what the recovery i?;. There are many techniques available for fractionation and analysis of antigen based on their physical-chemical properties.
204
NIAID Workshop
Gel filtration and ion-exchange column chromatography are two popularly used methods: the first, separation of molecules based on their shape and size; the second, fractionation of molecules based on their net electric charge. Adsorption and partition chromatography are infrequently used methods but can provide elegant separation of antigens. These methods separate molecules based on their affinity or solubility in different substances. Ultracentrifugation and electrophoresis are methods frequently used. Recently, immunochemical methods have been used. These combine immunologic principles with other methods of separation such as immunoelectrophoresis or immunoabsorption. After obtaining the maximal activity unit per dry weight, it is necessary to demonstrate homogeneity of an antigen before additional characterization can be done. Generally, any of the techniques can be used to characterize homogeneity, but it should be stressed that one must use more than one assay when determining homogeneity of a substance. Also, when using these assays one should demonstrate the biological activity contained in the single peak or single band demonstrated. As an example of a specific isolation procedure, I refer to my own work with isolation of the histaminesensitizing factor from Bordetella pertussis. Although this conference deals with antigens active in HP, I believe the following will serve not only as an example of an isolation procedure but also a demonstration of microbial substance which can profoundly alter the immune response of an animal, a situation which may occur with other microbial factors in HP. Pertussis induces a mononuclear cell infiltrate in the lungs of animals treated with it. This may have relevance to those changes observed in HP. It is well established that Bordetella pertussis can alter the normal responses of laboratory animals or man to a number of stimuli. One of the changes most significant for immunology is that pertussis can alter the immune response of an antigen both quantitatively and qualitatively. Whole cells or extracts of pertussis can induce a profound leukocytosis due primarily to an increase in circulating lymphocytes. In addition to these changes, pertussis induces a hypersensitivity in experimental animals to a variety of agents such as pharmacologic mediators and bacterial toxins. The most frequently investigated of these hypersensitivities induced by pertussis is sensitization to histamine. Usually 20 to 40 mg of histamine base is a lethal dose in a normal mouse. However, if the mouse is pretreated with whole cells of Bordetella pertussis, it will be easily killed by less than 1 mg from what appears to be an anaphylaxis-like reaction. The bac-
J. ALLERGY
CLIN. IMMUNOL. APRIL 1978
terial component responsible for sensitization is called the histamine-sensitizing factor (HSF). HSF activity is quantitated by the sensitizing dose-50 determination (SD,,). This is the functional assay for HSF. Mice are sensitized by an intravenous injection containing different concentrations of test material. After a latent period of 3 to 4 days the animals are challenged with 1 mg histamine base and deaths recorded for the next hour. From these results one can calculate the SDS0in test material. The total number of SDS,,values can be estimated by dividing test material weight by its SD,,. In the past, soluble preparations of HSF have been obtained from culture supernates and bacterial cells treated by a variety of methods. All of these were contaminated with other bacterial components. Further purification results in decreased solubility of HSF and loss of bacterial activity. Since none of these procedures yielded optimal results, we chose to develop our own extraction and purification procedure. In order to extract HSF from bacterial cells, different concentrations and combinations of solvents were tested and the number of SDS0 values released determined and expressed as percentage of the total activity. The different combinations of urea, sodium chloride, sodium deoxycholate, and triton X-100 is different concentrations were used in attempts to release histamine-sensitizing factor. Clearly the greatest amount of activity was extracted with a combination of 4 M urea and 1 M sodium chloride which extracted approximately 78% of the total activity. All other solvents appear to be less effective. However, biological activity was not stable in urea sodium chloride solution and after several days at 4” C was completely destroyed. We found, though, if urea was removed from the extract, biological activity was relatively stable at 4” for several weeks. However, this led to other problems. Upon concentration of HSF extract in 1 M sodium chloride, more than half of the activity became insoluble. The remaining activity was polydispersed when analyzed by sucrose density-gradient ultracentrifugation. This represents a 5% to 20% sucrose density gradient in 1 M sodium chloride. All the gradient fractions were analyzed for ultraviolet absorption at 280, 260, and 230 nm, and fractions were also assayed for histamine-sensitizing activity expressed as SDS,, per fraction. There was a 260 absorbing peak at approximately 50% of the gradient, and a 230 absorbing peak at approximately 80% of the gradient. Small amounts of histamine-sensitizing activity were demonstrated in the heavier portion of the gradient, but, clearly, most of the histamine-sensitizing activity was dispersed
VOLUME NUMBER
61 4
over a broad region of the lighter portion of the gradient. We concluded that the removal of urea led to the insolubility and heterogeneity of HSF, and this was supported in the following experiment. First, the problem of lability of HSF in urea sodium chloride was resolved by buffering the solution at a pH range of 4 to 6.4. This stabilized HSF activity for several weeks. If urea remained in the extract now, all of the extracted activity remained soluble after concentration. After sucrose density-gradient analysis in buffered urea sodium chloride solution the HSF activity was more homogeneous and sedimented in the region of the bovine serum albumin (BSA) marker. Pertussis vaccine was centrifuged and the pelleted bacterial cells extracted with urea sodium chloride solution. This was done by sonication. Sonicate was mixed overnight and centrifuged and the resulting supernate concentrated and then centrifuged a second time. The final extract contained more than 50% of the initial histamine-sensitizing activity and had an SDjo of 1.045 pg which represents approximately a 16-fold increase in specific activity compared with the bacterial vaccine. In addition to histamine-sensitizing activity this extract also produced a leukocytosis in mice and had adjuvant activity. Since the soluble relatively homogeneous HSF extract could now be obtained, this preparation was purified by gel filtration on a biogel A 0.5 M column, equilibrated with urea sodium chloride buffer. At 230, gel filtration of the pertussis extract demonstrated five peaks of ultraviolet absorbing material. Column fractions are pooled and assayed for histamine-sensitizing activity. Minimal amounts of activity were associated with the first peak, but most of the histamine-sensitizing activity appeared to be associated with a third peak and came off the column just prior to the BSA marker. The portion of the column eluate that contained maximal histamine-sensitizing activity also contained leukocytosis-promoting activity. Column fractions were also assayed for adjuvant activity for hemagglutinating antibody. In a control mouse which was immunized only with ovalbumin, maximal passive hemagglutination antibody titers of 40 were obtained. In animals treated with column fractions in addition to immunization with ovalbumin there was enhancement of antibody response of phytohemagglutinin (PHA) titers up to 640. There were clearly three regions of maximal adjuvant activity for hemagglutinating antibody, the second of which also contained histamine-sensitizing and leukocytosis-promoting activity. Adjuvant activity for reaginic antibody was assayed by enhanced reaginic antibody response determined by passive cutaneous anaphylaxis (PCA)
Antigens in hypersensitivity
pneumonitis
205
titer. There were two regions of column eluate which contained adjuvant activity for reaginic antibody. The second one also contained histamine-sensitizing, leukocytosis-promoting, and adjuvant activities for reaginic and hemagglutinating antibody. This portion of the column eluate was pooled and rechromatographed on a Sephadex G- 100 column. The ultraviolet absorption profile demonstrated the gel filtration on G-100 revealing four peaks. Histamine-sensitizing activity appeared to be associated with the descending portion of the second peak and corresponded to a molecular weight of 90,000 daltons. This fraction also contained the leukocytosis-promoting activity. Adjuvant activity for hemagglutinating antibody again appeared to be more dispersed. However, adjuvant activity for reaginic antibody was more restricted and present in the same fractions that contained histamine-sensitizing activity and leukocytosis-promoting activity. This portion of the column eluate was pooled and designated G-100 HSF. It had an SD,, of 6 pg which represents a 280-fold purification of histamine-sensitizing activity compared with crude bacterial vaccine. Further purification was achieved through use of a cationic exchanger sulfopropyl sulfadex. A sample was equilibrated with 4 M urea, 0.2 M sodium chloride buffered with citrate at pH 4. UltravioIet absorption at 280 nm demonstrated at least five different components with little affinity for the resin. None of these components contained any biological activity. A pH gradient from 4 ro 4.8 eluted a component which appeared to contain all the histamine-sensitizing activity adjuvant of leukocytosis-promoting activity and adjuvant activities for hemagglutinating and reaginic antibodies. A further pH and sodium chloride gradient eluted a component which contained considerable amounts of protein but did not contain biological activity. Chemical analysis of partially purified HSF demonstrated that mainly protein and lipid were present, suggesting that HSF may be a lipoprotein. This was supported in part by the fact that histamine-sensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibodies were not altered by treatment of extracts with DNase or KNase but were significantly reduced by treatment with proteolytic enzymes. In addition to biochemical studies, immunochemical analysis of HSF was performed. Antisera from rabbits immunized with HSF isolated from the G-100 column detected several antigens by immunoelectrophoresis. All the antigens demonstrated a slight movement toward the cathode. In order to determine if biological activity might coin-
206
J. ALLERGY
NIAID Workshop
tide in electrophoretic mobility with these antigens, slides were cut in small segments and eluted, and the eluate was tested for biological activity. The results showed that maximal histamine-sensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibody were present in fraction 6. Adjuvant activity for hemagglutinating antibody appeared to be rather dispersed. Since biological activities were present in regions in which precipitating antigens were detected, experiments were performed to determine if antisera could react with these activities. When pertussis extracts were treated with rabbit antiserum, in vitro antigen-antibody precipitates developed. After removal of these precipitates it was demonstrated that the supernates with reduced antigen content contained minimal biological activity compared with extracts treated with normal rabbit serum. Furthermore, in vivo neutralization of histaminesensitizing activity, leukocytosis-promoting activity, and adjuvant activity for reaginic antibody was achieved by passive immunization of mice with rabbit antiserum to HSF prior to sensitization. From these results it was concluded that HSF is a pertussis antigen. Evidence suggests that it is a lipoprotein of approximately 90,000 daltons in size. In addition to histamine-sensitizing activity it contains leukocyto-
Standardization
CLIN.
IMMUNOL. APRIL 1979
sis-promoting activity and adjuvant activity for reaginic antibody. By extraction and sucrose density-gradient analysis, optimal solvent conditions for HSF were determined. Further studies with the use of gel filtration and ion-exchange column chromatography demonstrated the size and charge of HSF and suggested that it contained other biological activities since they all were of identical physical-chemical properties. These chromatographic procedures also yielded partially purified preparation of HSF on which meaningful chemical and biochemical analysis could be done. In summary, the important steps in antigen isolation have been reviewed. The first is to establish a reliable quantitative or at least semiquantitative biological assay. Once this is done solubilization of antigens can be achieved. After we have a soluble extract of antigens there are a variety of methods which can be used to fractionate and analyze antigens. As an example of a microbial antigen isolation, I have reviewed the extraction and purification of histamine-sensitizing factor of Bordetella pertussis. Although this is not directly related to HP, HSF may be similar to other microbial antigens in this disease. Analysis of such antigens is essential before we can examine immunopathologic mechanisms in HP or devise meaningful diagnostic assays.
of antigens
H. Baer, Ph.D. Bethesda, Md.
Standardization of antigens or allergens in areas such as hypersensitivity pneumonitis (HP) and hay fever-type allergy presents a number of problems. Dr. Norman of John Hopkins Allergy Clinic, Dr. Gleich of the Mayo Foundation, and I started our investigation of these problems by determining the activity of commercially available antigens. Extracts from these sources had very diverse activities, which were definable by a variety of tests. Measurement of the reactivity of these extracts is complicated by variability in the tests used. Reactivity of a group of extracts may appear to differ by lo- to 100-fold in one test, but the same extracts in another test may vary in reactivity by only 2- or 3-fold. Depending on the test used, the same extracts may be very different or only slightly different.
The enormous number of extracts available makes standardization particularly difficult. Most of the companies that produce allergenic extracts have listings of approximately 1,000 such extracts; they also produce extracts for HP. Therefore, we decided to concentrate on those antigens which were most frequently implicated as causing disease. In the pollen field we decided to concentrate on ragweed and certain grasses which are rather widespread and cause allergy quite frequently in the United States. Geographic variations, such as deserts and forests, might change these choices for allergists in some areas, but these allergen selections are still valid for the most part. There are two aspects in any standardization program of this type. The scientific aspect demands the