Analysis of ragweed allergens recognized by monoclonal antibody 8-5 using two-dimensional gel electrophoresis and immunoblotting.

A series of MoAbs against major allergens (Amb a I and Amb a II) of the short ragweed pollen is reported by this laboratory. One of these MoAbs, 8-5, showed strong reactivity to Amb a II, weak reactivity to Amb a I-B and I-C, and no reactivity to Amb a I-B2 of the short ragweed pollen by SDS-PAGE and immunoblotting. In the present study, ragweed allergens recognized by MoAb 8-5 were further analyzed by two-dimensional gel electrophoresis and immunoblotting. The results showed that MoAb 36-6, which has been identified to be reactive with Amb a I only, reacted mainly with components with MW of about 39 kD, which corresponded to creatine kinase charge standards from -14 to -20 in both the Amb a I-B and I-C preparation and the crude extract of the short ragweed pollen. However, MoAb 8-5 showed strong reactivity to components with about the same MW but charge standards from -8 to -13 in Amb a II and the crude extract of the short ragweed pollen. Furthermore, a weak positive reactivity of MoAb 8-5 to components with MW of about 39 kD and charge standards from -8 to -14 in Amb a I-B and I-C was also observed. It was thus concluded that MoAb 8-5 reacted with Amb a II only.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of allergenic components of Bermuda grass pollen by monoclonal antibodies.

A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens (Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP-allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP-allergic patients. Among those human IgE-binding molecules, Cyn d Bd35K reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purification of these allergens.

Isolation and partial characterization of Bermuda grass pollen allergen, BG-60a.

In an earlier study we showed that Bermuda grass (Cynodon dactylon) pollen contains at least 12 IgE-binding proteins that can be analysed by immunoblot technique. One of the active components (BG-60) proved to be a basic protein of glycoprotein nature. It contained about 28% carbohydrate as determined from the dry weight and consisted of four molecules. One of the components was purified from the pollen extract by a combination of ammonium sulphate precipitation, ion-exchange chromatography on carboxymethyl-TSK, gel filtration on Ultrogel AcA 44 and chromatofocusing. Its molecular weight was approximately 60 kD by SDS-PAGE and 34 kD by gel filtration chromatography. The isoelectric point of the antigen was about 9.7. The homogeneity of the antigen BG-60a was assessed by one single arc of immunoprecipitation both in immunodiffusion and crossed immunoelectrophoresis and by one single band after SDS-PAGE. Its allergenicity was demonstrated by direct intradermal skin test on allergic patients and by examining IgE-binding reactivity with allergic patients' serum.

Isolation and partial characterization of Bermuda grass-pollen allergen: BG-60b.

Our earlier studies have shown that the pollen of Bermuda grass (Cynodon dactylon)-pollen contain at least 12 IgE-binding proteins which can be analyzed by the immunoblot technique. One of the highly active components was found to be a basic protein with a molecular weight of 60,000 daltons, designated as BG-60. This component was showed to consist of a group of proteins. One of them, BG-60a (pI 9.7), has been isolated and characterized. In this study, we have further isolated and characterized the second component of the antigen, designated as BG-60b. Its purity was demonstrated by gel electrophoresis experiment and antigen-antibody precipitation studies. The antigen is of glycoprotein nature with a pI of 10.0. It exhibits IgE-binding activity and shows cross-reactivity to antigen BG-60a in double diffusion. Its chemical and physical properties are similar to antigen BG-60a.

Identification of allergens and antigens of Bermuda grass (Cynodon dactylon) pollen by immunoblot analysis.

Allergens and antigens of Bermuda grass pollen fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes were identified using twenty-one sera of Bermuda grass pollen-allergic patients. The IgE- and IgG-binding pollen components transferred to nitrocellulose were detected by reaction with enzyme-labelled anti-human IgE and anti-human IgG, respectively. There was heterogeneity in both IgE- and IgG-binding patterns of the allergic sera tested. Fourteen pollen components, ranging in molecular weight from 16,000 to 88,000 daltons, bound to IgE antibodies. Only two of the fourteen allergens identified reacted with IgE antibodies of more than 50% of the twenty-one allergic sera. The pollen component with a molecular weight of 32,000 daltons showed by far the highest frequency of IgE binding, being recognized by sixteen (76%) of the twenty-one sera examined. Fifteen (71%) of the twenty-one sera tested had IgE antibodies that reacted with more than one of the fourteen allergenic components identified. Pollen components recognized by IgE antibodies also reacted with IgG antibodies, and there were components only recognized by IgG antibodies. Results obtained from this study should be useful both clinically and in research.

Characteristics of five monoclonal antibodies to major allergens of the short ragweed pollen.

Monoclonal antibodies against major allergens of the short ragweed pollen were produced by fusion of NS-1 cells with splenic cells from BaLB/C mice that had been immunized separately with the major allergens, AgE-B + E-C and AgK, of the short ragweed pollen. These monoclonal antibodies were detected by enzyme-linked immunosorbent assay and further characterized by immunoblot analysis using the crude extract and highly purified allergens of the ragweed pollen. Three monoclonal antibodies obtained by immunization with AgE-B + E-C, designated as 36-6, 27-2 and 48-5, reacted mainly with the beta (36-6) and alpha (27-2) subunit of AgE and both AgE and AgK (48-5), respectively. Two monoclonal antibodies obtained by immunization with AgK, 4-7 and 8-5, had a similar reactivity with AgE-B + E-C, AgK, AgK-A and AgK-B. In addition, however, antibody 4-7 also reacted with AgE-B2 as well as the 36- and the 24- to 22-kilodalton antigens of the crude extract. All 5 monoclonal antibodies were characterized as IgGl subclass. Besides, monoclonal antibody 48-5 also showed cross-reactivity to components of sage pollen. Beyond academic interests, the monoclonal antibodies described here may also be useful in clinical allergy.

[Isolation and partial characterization of allergen from Bermuda grass pollen].

Allergens of bermuda grass pollen extract have been studied and identified. Immunoblotting studies revealed that at least 12 SDS-denatured polypeptide showed IgE-binding activity. Molecular weight was estimated between 10,000 to 90,000 daltons. An allergenic component was isolated by a combination of ion exchange chromatography and gel filtration chromatography. The allergen preparation was shown to be homogeneous by PAGE and SDS-PAGE studies. Allergen was found to be an acidic protein with a molecular size of the order of 16,300 daltons. Ultraviolet spectrum scanning showed weak absorption at 280 nm. Amino acid analysis revealed that the purified allergen contained no tyrosine, proline and cysteine residues but contained a high percentage of glutamic acid and aspartic acid residues. The results of amino analysis indicate that tyrosine, proline and cysteine residues are not involved in the allergenic determinant site.

[A monoclonal antibody against ragweed pollen cross-reacting with yellow dock pollen].

Using monoclonal antibodies with different specificity against the major allergenic components of ragweed pollen, we analyzed their cross-reactivity with two tree pollens, two grass pollens and five other weed pollens which are common in Taiwan by the immunoblot method. It was found that besides reacting with AgE and AgK of the ragweed pollen, the monoclonal antibody 48-5 also reacted with antigens of yellow dock pollen with molecular weights of 40K, 38K, 24K, and 21K. In a preliminary study, sera of two patients containing IgE antibodies to ragweed pollen antigens also reacted to the 40K component of the yellow dock pollen. Furthermore, from the results of allergenic skin testings on 109 patients with bronchial asthma, we found that of 22 patients who had a positive reaction to a crude extract of ragweed pollen, 18(81.8%) also reacted to the crude extract of yellow dock pollen. In conclusion, our results suggest that there exists a common allergenic determinant between pollens of ragweed and yellow dock. It may play an important role in the expression of the sensitivity of patients to these two kinds of pollens.