F.T. Kisil

Isolation and Characterization of a cDNA Clone Encoding an IgE-Binding Protein from Kentucky Bluegrass (Poa pratensis) Pollen

We reported previously on the isolation and characterization of several allergens from Kentucky bluegrass (KBG) (Poa pratensis L.) pollen with the aid of the corresponding murine monoclonal antibodies (Mabs). In the present study, (1) an analysis of various tissues of this grass revealed that the allergenic components recognized by these Mabs were confined to the pollen; (2) intact translatable mRNA was isolated from the KBG pollen, and (3) a cDNA library was constructed with this mRNA in the lambda gt11 expression vector. Screening of this library with a pool of six sera from KBG-allergic patients, in combination with enzyme-labeled antibodies to human IgE, led to the isolation of a cDNA clone, referred to as KBG7.2. The nick-translated cDNA probe of KBG7.2 hybridized to a 1.5-kbp RNA transcript from KBG pollen. Moreover, transcripts corresponding to KBG7.2 were found in pollens of eight other grasses, indicating that the proteins similar to the one encoded by this cDNA may be present in these grasses. The nucleotide sequence of KBG7.2 was determined; interestingly, the corresponding derived amino acid sequence did not match any other sequence recorded in the protein data banks. The peptide encoded by KBG7.2 was expressed as a fusion protein utilizing the plasmid vector pWR590.1. Whereas none of the above allergen-specific Mabs bound to the fusion protein, all the 15 individual sera from grass pollen allergic patients recognized the fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Allergenic and Antigenic Cross-Reactivities of Group IX Grass Pollen Allergens

The allergenic and antigenic cross-reactivities between a major recombinant Poa pratensis (Poa p) IX allergen, rKBG8.3, and its corresponding proteins of different grass pollens were examined. Immunoblotting of the proteins of thirteen different grass pollens using anti-rKBG8.3 antibodies indicated that Poa p IX-like proteins are present in ten other grass pollens, albeit in variable amounts and polymorphic forms. These proteins ranged in size from 20 to 88 kDa in different grass pollens. The percent relative binding determined for each grass pollen extract using allergic human sera showed a significant correlation (r = 0.891) with that of anti-rKBG8.3 antiserum. Moreover, there was a strong association (r = 0.901) between the Kentucky bluegrass extract and rKBG8.3 with respect to their inhibition of the binding of human IgE antibodies to allergens in grass pollen extracts. Taken together, these results suggest that the allergenic and antigenic epitopes of the Poa p IX-related proteins in some but not all grass pollens are similar in structure and specificities. It is concluded that the group IX allergens constitute a major family of homologous proteins in several grass pollens.

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.

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.