Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1.
BACKGROUND
Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts.


METHODS
After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay.


RESULTS
The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition.


CONCLUSIONS
The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.
Duffort
O
O
Research and Development Department, ALK-ABELLO, Madrid, Spain.
Calabozo
B
B
González
R
R
Carpizo
J A
JA
Barber
D
D
Polo
F
F
D000485:Allergens / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D052179:Antigens, Plant; D015153:Blotting, Western; D002846:Chromatography, Affinity; D031744:Cynodon / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010940:Plant Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D011058:Pollen / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D011852:Radioallergosorbent Test; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C071778:CYND1 protein, Cynodon dactylon; D010940:Plant Proteins