Preparation And Characterization Of Recombinant Human Monoclonal Antibodies Specific To Dermatophagoides Farinae

Allergic diseases especially asthma are public health problems worldwide. In most countries, the mobility and mortality of asthma are increasing continuously. House dust mites (HDMs) including Dermatophagoides farinae and Dermatophagoid.es pteronyssinus are among the most important allergen sources. They are the most potent causes of allergic diseases especially for allergic asthma in children. Genetic predisposition and environmental factors influence the development of allergic diseases together. A program entitled Prevention of Allergy and Asthma was launched by WHO under this situation, at the same time, primary prevention was stressed. Several studies showed that the increase of maternal IgG antibody would reduce subsequent sensitization rate and atopic symptoms in the offsprings. Therefore, specific human Fab antibodies are prepared for further study on prevention of mite allergic diseases.Human peripheral blood lymphocytes were isolated from the allergic asthma patients after a long period of immunotherapy with house dust mites extracts. Human IgG genes library with a titer of 8×108 was then constructed using total RNA of the lymphocytes. Another human IgG genes library was prepared from 300 healthy volunteers without any infectious diseases even common cold in the past 2 months. These two libraries were screened for the production of human monoclonal antibody Fab fragments to Der f 2 by cloning blotting assay and ELISA method. About 1.8×106 clones were screened and only 2 positive Fab fragments specific to rDer f 2 (named AM1 and AM2) were obtained from the IgG library of healthy volunteers. The heavy chains of AM1 and AM2 yield completely homologous. A new clone with higher affinity was got from a reshuffling library which was constructed by the light chain of AM1 and the heavy chain library from asthmatic patients. Sequence analysis of both AM1 and AM2 heavy chain genes revealed the nearest V-segment germline were VH3-30 with 96%homology. The closest V-segment germline of the light chain gene were K2-30 and K3-27 with 87%and 95%homology respectively. And the nearest V-segment germline of AM1L-Hsh was VH5-1 with 93%homology. Affinity of these 3 Fab fragments to recombinant Der f 2 and Der p 2 was 1.27×10-9 to 6.3×10-8. The affinity of AM1L-Hsh was eight times higher than AM1. The immunoassay data indicated that these Fab fragments had high specificity to recombinant and native protein of dust mite. In vitro mastocyte degranulation inhibition test showed that mite allergen specific IgG Fab could significantly inhibit mast cell degranulation. As a result, mite allergen specific Fab fragments may inhibit the specific binding of IgE to rDer f 2 competitively.This is the first report of gene analysis and bacterial expression of human monoclonal antibody Fab fragments to mite group 2 antigens. The combinatorial immunoglobulin gene library derived from human seems to be a potential tool for clinical immunoprophylaxis and treatment of mite allergic diseases.