A.K.M. Ekramoddoullah

Detection of Cross-Reactive Allergens in Kentucky Bluegrass Pollen and Six Other Grasses by Crossed Radioimmunoelectrophoresis

Using crossed immunoelectrophoretic analysis of an aqueous extract of Kentucky bluegrass (KBG)pollen employing hyperimmune rabbit and sheep precipitating antibodies to KBG and various grasses we detected at least 33 antigenic components. The majority of these antigens were extractable from the pollen within a period of 14 min and possessed anodal electrophoretic mobility. The extensive recognition of antigens in the KBG extract by antibodies raised to false oat, and to the combined extracts of timothy, orchard, meadow, velvet and rye grasses indicate that these grasses contain many cross-reactive antigens. One of the KBG antigens with cathodal mobility--the major component of the previously isolated allergen C--was identified as immunologically identical to timothy Ag 30. Crossed radioimmunoelectrophoretic analysis revealed differences in the extent to which IgE antibodies present in sera of individuals allergic to KBG bound to various antigens of KBG pollen. Those antigens that were recognized as allergens by all of the allergic sera examined were regarded as candidates for future isolation and characterization.

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 Epitopes on Poa p I and Lol p I Allergens with Monoclonal Antibodies

Allergen Poa p I isolated from the dialysed aqueous extract of Kentucky blue grass pollen by affinity chromatography with an anti-Lol p I murine monoclonal antibody (MAb) 290A-167 was previously shown to consist of a 35.8-kilodalton (kD) component with a pI of 6.4, designated as Poa p Ia, and a 33-kD component with a pI of 9.1, designated as Poa p Ib. The present study reports on the comparative antigenic analyses of these two components, using MAbs produced separately against Poa p I and Lol p I. Thus, anti-Poa p I MAbs 60 and 61 and anti-Lol p I MAb 290A-167 recognized Poa p Ia and Poa p Ib whereas anti-Poa p I MAbs 62, 63 and 64 and anti-Lol p I MAb 348A-6 recognized only Poa p Ia. The specificities of the MAbs were further resolved by comparing their respective abilities to inhibit the binding of 125I-Poa p I or 125I-Lol p I to the different MAbs prepared in the form of solid phase. These studies revealed that at least 4 distinct epitopes (designated as E1, E2, E3 and E4) were shared by both Poa p I and Lol p I. All 4 epitopes were present on Poa p Ia whereas only E1 and E3 were detected on Poa p Ib. E1 was recognized by MAbs 60 and 61, E2 by MAbs 62, 63 and 64, E3 by MAb 290A-167 and E4 by MAb 348A-6.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Two-dimensional gel electrophoretic analyses of Kentucky bluegrass and rye grass pollen allergens. Detection with a murine monoclonal anti-Poa p I antibody and amino terminal amino acid sequence of Poa p I allergen.

Allergenic extracts of Kentucky bluegrass (Poa pratensis) and rye grass (Lolium perenne) pollen were shown by 2-dimensional (2-D) gel electrophoresis to consist of several hundred protein components. The pollen extracts of the two related grasses had unique 2-D gel patterns. Two major grass pollen allergens, Poa p I and Lol p I, and their isoforms (i.e. isoallergens) were detected and localized on 2-D gels by immunoblotting with anti-Poa p I antibody mAb 60. Poa p I isoallergens were less acidic than Lol p I isoallergens. The relative proportion of four Poa p I isoallergens was (in decreasing pI): A, 13%; B, 37%; and D, 15%. The amino terminal amino acid sequences of the two major isoallergens of Poa p I were identical. However, the amino acid composition of these isoallergens showed enough differences to account for their charge differences. The amino terminal amino acid sequence of two major Poa p I isoallergens had a 70% homology in 20 amino acid overlap with the previously published amino terminal amino acid sequence of rye grass pollen allergen R-7.

Standardization of Rye-Grass Pollen (Lolium perenne) Extract

Six candidate extracts of Lolium perenne (rye-grass) pollen have been studied in 6 laboratories using a variety of immunochemical and physicochemical techniques. Radioallergosorbent test inhibition, crossed immunoelectrophoresis, crossed radio-immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with immunoblot, thin-layer isoelectric focusing and enzyme-linked immunosorbent assay inhibition were used to evaluate each of the coded extracts. The source materials were also studied for identity and possible contamination by light microscopy. On the basis of these data, the Rye-Grass Working Party recommended to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies that the extract coded C be chosen as the candidate international reference preparation.

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 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 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 (Ma