Immunochemical characterization of a high molecular weight basic allergen (HMBA) of rye grass (Lolium perenne) pollen.

A high molecular weight basic allergen (HMBA) was isolated from the mixture of non-dialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofocusing, HMBA, a glycoprotein of mol. wt 56,800 (17% carbohydrate) contained all naturally occurring amino acids. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus, it was concluded that HMBA was a unique and highly purified antigen. The allergenicity of HMBA was revealed by its ability to elicit immediate skin reactions in grass allergic patients. Moreover, all patients' sera tested had IgE antibodies to HMBA detectable by direct RAST with HMBA allergosorbent discs. These observations indicated that HMBA was a major allergenic constituent of rye grass pollen. Treatment of HMBA by 6 M guanidine HCl led to a significant reduction in its ability to combine with human IgE antibodies. The treatment also resulted in the complete loss of allergenicity (i.e. inability to elicit PCA reactions with a murine reaginic antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA); hence, it would appear that the allergenic and antigenic determinants of HMBA are 'conformational'.

Partial characterization of an antigenic site of high molecular weight basic antigen, a ryegrass pollen allergen, using a monoclonal antibody.

We have previously reported on the production of murine monoclonal antibodies (Mabs) to the retentate fraction of the aqueous extract of Kentucky bluegrass pollen (KBG-R) [Kisil et al. (1980) Fedn Proc. 39, 3479]. In the present study, the ability of one of these Mabs (Mab 12) to recognize various antigenic components present in KBG-R and the corresponding fraction of ryegrass (rye-R) was evaluated by the Western and immunoblot methods. Thus, KBG-R and rye-R were resolved electrophoretically into a large number of components. Remarkably, the concurrent immunoblot analysis with Mab 12 detected only a single antigenic component in each of the retentate fractions. The position of the antigenic component observed on these immunoblots was identical to that obtained with the rye allergen high mol. wt basic antigen (mol. wt 56,800). To characterize the antigenic site recognized by the Mab, the size of HMBA was reduced by cleavage with CNBr, the resulting fragments separated by high-performance liquid chromatography on a reverse-phase column and their antigenic activity analyzed by immunoblot. Two peptides, CB-1 (mol. wt = 17,400) and CB-2 (mol. wt = 22,000), retained the capacity to react with Mab 12 and also IgE antibodies present in a pool of sera from grass allergic individuals.

Allergenic cross-reactivity of cytochromes c of Kentucky bluegrass and perennial ryegrass pollens.

The allergenic cross-reactivity of cytochromes c isolated from Kentucky bluegrass (KBG) and ryegrass (RG) pollens was demonstrated by the finding that both cytochromes elicited PCA reactions of comparable titers in rats sensitized with murine reaginic antiserum to either KBG or RG cytochrome c. However, allergenic differences were revealed at limiting doses of the cytochromes c; under these conditions RG cytochrome c elicited PCA reactions only with the homologous reaginic serum, whereas KBG cytochrome c elicited PCA reactions with both murine reaginic sera. Moreover, in experiments involving inhibition of PCA, RG cytochrome c failed to neutralize some of the IgE antibodies of the antiserum to KBG cytochrome c, whereas KBG cytochrome c neutralized all IgE antibodies of both murine reaginic antisera. From these results it may be concluded that: (1) the two cytochromes c share common allergenic determinants, and (2) the KBG cytochrome c possesses additional allergenic determinant(s) not present on RG cytochrome c. The radioallergosorbent test, utilizing KBG cytochrome c discs and a pool of sera of individuals allergic to KBG, was inhibited to the extent of 87 or 41% by the addition of 10 micrograms of KBG or RG cytochrome c, respectively. By contrast both cytochromes c at 10 micrograms were equally effective in the inhibition (92%) of the binding of the IgE antibodies in this serum pool to RG cytochrome c allergosorbent discs. These experiments confirmed that the two cytochromes share common allergenic determinants.

Mapping of antibody binding epitopes of a recombinant Poa p IX allergen.

Antibody binding epitopes of a recombinant Poa p IX allergen were delineated using recombinant DNA and solid-phase peptide synthesis procedures. The full-length cDNA clone KBG60 and its four overlapping recombinant fragments, KBG60.1, KBG60.2, KBG8.3 and KBG10 which spanned the entire molecule were synthesized in E. coli with aid of the plasmid expression vector, pWR590.1. The antigenic and allergenic sites of these recombinant proteins were analyzed by ELISA using human IgE and murine IgG antibodies. It was thus demonstrated that although the epitopes were found on all the fragments tested, the majority of these were located on a C-terminal fragment, rKBG8.3. Furthermore, synthetic peptides were also employed to identify the epitopes of rKBG60 protein. The use of antisera raised against native KBG pollen extract and the recombinant KBG8.3 protein to scan a total of 56 overlapping deca-penta peptides, covering the entire rKBG60 protein, revealed that 10 positive peptides involved in the antibody-binding site(s). Taken together, the results of these studies indicate that rKBG60 protein possesses at least 10 antibody binding epitopes.

Determinants of ryegrass pollen cytochrome c recognized by human IgE and murine monoclonal antibodies.

In attempts to produce fragments of an allergenic molecule which would retain allergenic and/or antigenic determinant(s), the cytochrome c of ryegrass (RG) pollen, which had been shown to be an allergenic constituent of this pollen, was digested with trypsin and chymotrypsin and the resulting fragments were separated by high performance liquid chromatography. Several of these fragments were shown, with the aid of the radioallergosorbent test and solid phase radioimmunoassays, to bind IgE antibodies present in a pool of six sera from grass-sensitive patients and three murine monoclonal antibodies, designated as Mab 41, Mab 42 and Mab 43, which had been originally produced against the crossreacting cytochrome c of Kentucky bluegrass (KBG). In summary, (i) fragments C-67 and C-74 reacted with all antibodies, (ii) fragments T-45, T-46 and C-69 bound to human IgE antibodies as well as to Mab 41 and Mab 42, but not to Mab 43, (iii) fragment T-44 reacted only with Mab 41 and Mab 42, and (iv) fragment C-83 bound only Mab 42 and Mab 43. On the basis of these results, it is concluded that (i) immunochemically active fragments of the RG cytochrome c can be readily produced by enzymatic degradation, (ii) there is significant crossreaction between the antigenic determinants of RG and KBG cytochromes c, (iii) whereas all fragments possessed at least two of the original antigenic determinants, fragments C-83 and T-44 were devoid of allergenic determinants, (iv) the antigenic determinants recognized by Mab 41 and Mab 42 were different from those reacting with human IgE antibodies and Mab 43, (v) each of the three monoclonal antibodies recognized a distinct antigenic determinant, (vi) fragments C-67 and C-74 possessed all determinants recognized by the human IgE and mouse antibodies used.

Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody.

A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen.

Antibody responses to allergen Lol pIV are suppressed following adoptive transfer of B lymphocytes from the internal image anti-idiotypic antibody-treated mice.

An internal image anti-idiotypic antibody, designated B1/1, was generated against an idiotope (Id91) of the monoclonal antibody (mAb91) specific for Lol pIV. The administration of B1/1 in PBS, at doses ranging from 100 ng to 100 micrograms/mouse, to syngeneic Balb/c mice resulted in the suppression of the formation of anti-Lol pIV antibodies that possessed the Id91. Spleen cells obtained from the mice 2 weeks after the treatment with B1/1 (25 micrograms/mouse) were adoptively transferred intravenously into the syngeneic recipients which were challenged intraperitoneally with Lol pIV in alum 2 hr after the transfer. The recipients were boosted with Lol pIV 14 days later. It was demonstrated that the transfer of splenic B cells (but not of T cells) from B1/1-treated donors induced a significant suppression of not only the level of IgE and IgG antibodies to Lol pIV, but also the level of antibodies possessing the Id91. Treatment of the B cells with mAb91 plus complement abrogated their ability to transfer the suppression. This study indicates that the treatment with the anti-Id B1/1 generated B cells that were characterized, serologically, as possessing the anti-Id-like antibodies on their surface and were responsible for transferring the suppression of the formation of antibodies to allergen Lol pIV and the expression of Id91.

Allergenic fragments of ryegrass (Lolium perenne) pollen allergen Lol p IV.

To facilitate studies on establishing the nature of structure/function relationships of allergens, ryegrass pollen allergen, Lol p IV, was cleaved into smaller fragments by cyanogen bromide (CNBr) and the resulting peptides were further digested with trypsin. The resulting peptides were then fractionated by high performance liquid chromatography (HPLC) on a C-18 reverse phase column. The allergenic activity of the HPLC fractions was evaluated in terms of their ability to inhibit the binding of 125I-Lol p IV to serum IgE antibodies of a grass-allergic patient. Many of these fractions inhibited the binding between the native allergen and IgE antibodies in a dose-dependent manner. The inhibitions were specific, i.e., the fractions did not inhibit the binding between 125I-Lol p I (a group-I ryegrass pollen allergen) and the IgE antibodies present in the allergic human serum. The possibility that the allergenic peptide fractions were contaminated by the native undegraded allergen, which might have accounted for the observed inhibition, was ruled out by the fact that the native allergen could not be detected by SDS-PAGE and the elution profiles of allergenically active peptides did not coincide with that of native allergen. One of the allergenic sites recognized by monoclonal antibody (Mab) 90, i.e., site A, was located in HPLC fractions 90-100 while another allergenic site B (recognized by Mab 12) appeared to be lost following the sequential digestion of Lol p IV with CNBr and trypsin.

Isolation of a low molecular weight constituent from Kentucky blue grass pollen possessing carrier activity of high molecular weight constituent(s).

An immunologically active fraction, designated as C-I-3 was isolated from the dialysate of the aqueous extract of defatted Kentucky Blue Grass pollen by differential dialysis and gel filtration. This fraction was shown to consist of a glycoprotein with a molecular size corresponding to 4,800 daltons and with a pI value of 3.6, and to contain all amino acids except cysteine. The intraperitoneal administration of C-I-3 in saline into mice or rats prior to their immunization with the nondialyzable constituents of the aqueous extract of Kentucky Blue Grass (i.e., the retentate, R) in presence of Al(OH)3 resulted in the enhanced production of anti-R IgE antibodies. The enhancement of the anti-R murine IgE response was shown in adoptive cell transfer experiments to be due to the generation of R-specific T helper cells. Hence, it is suggested that the structural elucidation of C-I-3 may prove useful in establishing the nature of carrier determinant(s) of the major allergenic components of Kentucky Blue Grass pollen.

Isolation of allergenically active cytochrome c from Kentucky blue grass pollen.

An allergen, designated as Kentucky blue grass KBG-cytochrome c, was isolated from the nondialysable constituents (R) of the aqueous extract of KBG pollen by a combination of gel filtration and preparative isoelectrofocussing techniques. The allergen had an absorption spectrum characteristic of heme protein and had a molecular weight corresponding to 12,000 daltons. It was a basic protein with a pI value of 9.9 and contained all the common amino acids. KBG-cytochrome c elicited immediate hypersensitivity cutaneous reactions in individuals allergic to KBG pollen.

Isolation of a Kentucky Blue grass pollen allergen using a murine monoclonal antibody immunosorbent.

We have previously reported the production of murine monoclonal antibodies to the retentate fraction of Kentucky Blue Grass pollen (KBG-R). In the present study, one of these monoclonal antibodies (Mab 27) was used in a combination of methods employing Western and immunoblot analysis to establish its reactivity to various antigenic components present in KBG-R. Thus, Mab 27 reacted predominantly with an antigen having a MW of 30 kD and to a lesser extent with other antigenic components with MW of 35 and 17 kD. Fractions of KBG-R obtained by preparative isoelectrofocusing, revealed on analysis by ELISA, that Mab 27 reacted with several components differing in charge. From these observations it was concluded that KBG-R contained a group of related antigens detectable with Mab 27 and differing in size and charge. A reverse immunosorbent, prepared by coupling Mab 27 to CNBr-activated Sepharose 4B, was used to absorb KBG-R. Bound material was recovered by acid elution and designated as Ag 27. SDS-PAGE analysis revealed that Ag 27 consisted of at least two components with molecular weights of 30 and 17 kD. Analysis by RAST using sera from humans allergic to KBG indicated that Ag 27 was highly allergenic.

Suppression of the IgE antibody response in mice to kentucky blue grass pollen allergens.

Reagins in murine strains A/HeJ, RF/J C3H/HeJ and B6D2F1 were induced by immunization, in the presence of A1(OH)3, with the retentate (R) obtained by dialysis from the aqueous extract of Kentucky blue grass pollen (KBG-Aq. ext.). Administration of doses of R ranging from 50-200 microgram resulted in IgE antibody responses which were sustained for periods of the order of 6 months. The possibility of suppressing the anti-R IgE antibody response in sensitized A/HeJ mice was tested by the administration into these animals for a period of 14 weeks, biweekly injections of a skin-active, immunogenic fraction (DI) isolated from the dialysate of KBG-Aq. ext. This treatment effectively suppressed the secondary IgE antibody response to R and markedly increased levels of hemagglutinating antibodies. On the basis of the fact that DI comprised only 1.5% of the parent fraction KBG-Aq. ext. and since it was capable of modulating the immune response to R as described, it is suggested that DI may prove to be a useful agent for the immunotherapy of allergies to KBG pollen.

Modulation of the IgE antibody response in rats to Kentucky blue grass pollen allergens.

The low molecular weight dialyzable fraction (D) prepared from the aqueous extract of Kentucky Blue Grass pollen was shown to suppress the formation of IgE antibodies in rats immunized with the nondialyzable fraction (R). In an attempt to establish the nature of the constituents responsible for this suppression, D was fractionated by gel filtration through Sephadex G-25. The first fraction eluted (DI) elicited skin reactions in rats sensitized with a rat reaginic serum to R and also gave a precipitate with a rabbit antiserum to R. A later fraction (DIII) was devoid of these two properties. To investigate the effects of these fractions on the antibody response, rats received either DI or DIII, administered in saline, prior to their immunization with R in presence of aluminum hydroxide. Pretreatment with DI resulted in a reduction of IgE antibody levels as compared with the IgE antibody response in control animals which had received pretreatment only with saline; however, pretreatment with DI did not affect the anti-R-hemagglutinating titers. In contrast, pretreatment with DIII enhanced both the IgE and the hemagglutinating antibodies. Hence, it is concluded that the composite fraction D contains one group of constituents capable of suppressing and another of enhancing the IgE antibody response.

Isolation and characterization of Poa p I allergens of Kentucky bluegrass pollen with a murine monoclonal anti-Lol p I antibody.

The Poa p I allergens were isolated from the retentate fraction of a dialyzed preparation of an aqueous extract of Kentucky bluegrass pollen by means of a reverse immunosorbent prepared with a murine anti-Lol p I monoclonal antibody, Mab 290-A-167. By sodium dodecyl sulphate-polyacrylamide gel electrophoresis and preparative isoelectrofocusing, Poa p I was found to consist of a 35.8-kD component with an isoelectric point of 6.4 and a 33-kD component with one of 9.1 and designated as Poa p Ia (acidic) and Poa p Ib (basic), respectively. The relative protein content of these components was estimated from the intensity of the stained bands following sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, Poa p Ia appeared to be the major protein constituent, and on Western immunoblot it also bound the monoclonal antibody to a greater extent than Poa p Ib. On the other hand, Poa p Ib was shown by Western immunoblot and autoradiographic analysis, to bind to a greater extent the IgE antibodies present in a pool of sera from grass-allergic individuals. Therefore, Poa p Ib was considered as the major allergenic component of Poa p I. By competitive inhibition of the radioallergosorbent test, it was demonstrated that the Mab inhibited the binding of Poa p I allergens to human IgE antibodies to the extent of 70%. Hence, it is suggested that Mab and human IgE antibodies recognize identical or closely related determinants of Poa p I allergens.

Allergens of Kentucky Blue Grass pollen. II. Isolation of hapten-like components from Kentucky Blue Grass pollen by preparative isoelectrofocussing.

Components with hapten-like properties were isolated from the nondialyzable fraction, i.e. the retentate (R) and the dialyzable fractions of the aqueous extract of Kentucky Blue Grass pollen (KBC aq.ext.), by preparative isoelectrofocussing on Sephadex G-100 gel. These haptenic components could not elicit the passive cutaneous anaphylaxis (PCA) reactions in rats passively sensitized with a murine reaginic antiserum to R, but could inhibit completely and specifically the PCA reaction which is normally elicitable with R. It was concluded that the specificity of the murine IgE antibodies was directed to a determinant(s) which was common to either allergenic or haptenic fractions. Moreover, by employing a pool of human sera from individuals allergic to KBG pollen in the radioallergosorbent test procedure, it was apparent that most of the haptenic fractions lacked some of the specificities present on allergenic components of R that were recognized by the human IgE antibodies. Evidence was obtained to suggest that the electrophoretic heterogeneity of allergenic components present in various fractions of KBG aq.ext. may be due primarily to differences in their net charge, rather than to differences in their allergenic specificity.

Isolation of a purified allergen from Kentucky blue grass pollen.

An allergenic fraction, designated as C-I-2-6, was isolated from among the allergenic constituents present in the dialysate of the aqueous extract of defatted Kentucky blue grass (KBG) pollen by a combination of gel filtration, ion-exchange chromatography and preparative isoelectrofocusing. This fraction was shown in consist of glycoprotein with a molecular size corresponding to 10,000 daltons and of two compounds with closely related pI values (4.5--4.9); its protein moiety contained all the amino acids except cysteine. As demonstrated by crossed immunoelectrophoresis with a sheep anti-R serum, fraction C-I-2-6 shared the antigenic determinants of 2 of the 15 antigenic components of the nondialysable components, i.e. retentate (R) of the aqueous extract of KBG pollen. Remarkably, in spite of the markedly lower molecular weight than R, fraction C-I-2-6 possessed all the allergenic determinants of R responsible for inducing murine IgE antibodies.

Human and murine antibodies to rye grass pollen allergen LolpIV share a common idiotope.

The possibility that a murine monoclonal antibody (mAb 12) to Rye grass pollen allergen LolpIV and LolpIV-specific antibodies in the sera of grass allergic individuals share a common idiotope (Id) was investigated. It was first established that mAb 12 and human IgE antibodies recognized the same (or similar) epitope(s) on LolpIV; i.e. mAb 12 could inhibit, to the extent of 35-60%, the binding of 125I-LolpIV to the human IgE antibodies present in the sera of grass pollen-allergic individuals. Subsequently, a rabbit anti-Id antiserum was produced against mAb 12 and rendered Id-specific by appropriate immune absorptions, and its IgG antibody fraction was isolated (Rb-aId). The specificity of Rb-aId was demonstrated by the fact that the antibodies bound only to mAb 12 and not to any other murine monoclonal antibody tested. Observations that Rb-aId inhibited the binding of 125I-LolpIV to mAb 12 indicated that the Id determinants recognized on mAb 12 were located at or near the antibody-combining sites. The Rb-aId also bound specifically to affinity-purified human anti-LolpIV antibodies isolated from human sera, but not to affinity-purified human anti-tetanus toxoid antibodies. This indicated that the human anti-LolpIV antibodies share a cross-reactive Id. The binding of Rb-aId to human anti-LolpIV antibody could also be inhibited by mAb 12. Therefore, it was concluded that the murine and human antibodies to LolpIV share a cross-reactive idiotope.

Recognition of a site of a Kentucky bluegrass pollen allergen by antibodies in the sera of allergic and non-atopic humans and a murine monoclonal antibody.

Allergen 27 was isolated from the aqueous extract of Kentucky Bluegrass pollen (KBG-R) with a reversed immunosorbent prepared by coupling murine monoclonal antibody, Mab 27, to Sepharose 4B. Sera of patients allergic to KBG pollen, as well as serum of nonatopic individuals possessing anti-KBG antibodies, inhibited the binding of Mab 27 to either Ag 27 or KBG-R to the extent of 20 to 35% in ELISA. In contrast, sera devoid of antibodies to KBG-R had no inhibitory capacity. In a radioallergosorbent test, it was demonstrated that Mab 27 could inhibit the binding of human IgE antibodies to Ag 27 to the extent of 52%. From these results, it is concluded that Ag 27 contains a determinant recognized by both human IgE and blocking antibodies and a murine Mab.

Allergenic relationships of two different antigens of Kentucky bluegrass pollen.

Two purified allergens, allergen C and KBG-1, isolated from Kentucky bluegrass (KBG) pollen, were found to be antigenically distinct with respect to rabbit precipitating antisera produced separately to each allergen. On the other hand, allergen C and KBG-1 appeared to be allergenically identical in terms of the specificities evaluated with murine reaginic antisera produced to each of these allergens; whereas they appeared to be only partially identical when evaluated with respect to the IgE antibodies present in a pool of human sera from individuals allergic to KBG pollen. Therefore, not all of the allergenic determinants recognized by the human IgE antibodies were detected by the murine reaginic antisera. This study serves to emphasize that for a meaningful evaluation of allergenic specificities any antisera prepared in animals and intended for the standardization of allergens must recognize allergenic specificities which are similar or identical to those to which the human IgE antibodies are directed.

Isolation and partial characterization of allergen C from Kentucky blue grass pollen.

An allergenic component, designated as Allergen C, was isolated from the retentate (R) of the aqueous extract of defatted Kentucky blue grass (KBG) pollen by a combination of preparative isoelectrofocussing and gel filtration. Allergen C was found to be a glycoprotein with molecular size of the order of 11,000 daltons and had a pI value of approximately 9.7. It possessed all the allergenic determinants collectively present in R. The allergenic activity was found to be associated with the protein moiety of the molecule. Allergen C was one of the 15 antigenic components detectable in R by crossed immunoelectrophoresis with a hyperimmune sheep antiserum to R. Observations based on enzymatic degradation of the molecule suggested that the determinants recognized by murine IgE antibodies were different from those which combined with rabbit precipitating antibodies.