Monoclonal antibodies to group II Dermatophagoides spp. allergens: Murine immune response, epitope analysis, and development of a two-site ELISA (1)
Dermatophagoides pteronyssinus and D. farinae: Group I (25 kd cysteine proteases), group II (14 kd proteins) are the most significant cause of sensitization, inducing IgG and IgE antibody responses in 80% to 95% of patients allergic to mites, and each of these allergens (Der p I, Der f I, Der p II, and Der f II) has been cloned and sequenced. All responding mouse strains immunized with Der p I showed a predominantly species-specific response, and in most cases IgG anti-Der f I antibody was less than 20% of the anti-Der p I antibody. IgG antibody responses to Der p II were strongly cross-reactive with Der f II in all responding strains. BALB.B, BALB.K, and BALB.A10 mice produced high levels of IgG anti-group II antibody when immunized in CFA or alum. BALB/c mice had weak antibody responses after immunization with CFA and significantly lower titers than the other strains when immunized with Der p II in alum.
The RIA results showed that most mAbs (11 of 16) bound to cross-reactive epitopes on both Der p II and Der f II. Polyclonal mouse IgG anti-Der p II antibodies also bound equally well to both allergens. Three clones showed five to 10 times greater binding to Der p II than to Der f II (3G5-A2, 3G5-H11, and 2B12), and two clones showed significantly higher binding to Der f II (15E11 and 4E5).
When the allergen was presented by mAb 7A1, the 1D8 and 4G7 clones showed 70% to 98% cross inhibition, suggesting that they were directed against the same epitope. In contrast, several other mAbs (e.g., 4E5, 3G5, and 2B12) showed less than 30% inhibition and appeared to be directed against different epitopes. Similar results were obtained with mAb αDpX on the solid phase, although the levels of inhibition tended to be lower with this mAb.
Some combinations (e.g. mAb αDpX, 7A1, or 3G5) on the solid phase showed strong binding (20,000 to 50,000 cpm bound) with each 125I-mAb. Other mAbs (e.g. 6D6, 1D8, 4G7, or 13A4) showed binding when used in combination with 125I-αDpX but not with the other five 125I-mAbs. Although several combinations of solid-phase mAbs coupled to CNBr-activated disks were effective in two-site RIA for the detection Der p II and Der f II, only three of 16 mAbs (7A1, aDpX, 13A4) bound to plastic microtiter wells and could be used to develop an ELISA.
These mAbs which can attach to ELISA plates were used in combination with biotinylated mAbs 1D8 or 4G7, and ELISA control curves for either Der p II or Der f II were compared. The combination of mAb 7A1 on the solid phase and biotinylated 1D8 was selected for use. This assay produces parallel control curves for both group II allergens and is approximately two times more sensitive to DerflI than to Derp II (limits of sensitivity 2 to 4 ng/ml). Forty house dust extracts showed good correlation between group II levels obtained by ELISA and those obtained by RIA.
Studies on IgE antibody responses showed that CBA and C57B1 were high responders to Derp I, whereas BALB/c, C3H, and AKR were poor responders. With T-cell responses to Derp II, H-2 a mice had poor T-cell responses to Der p II or Der p II peptides; whereas H-2 b mice had high responses, and H-2 k mice had intermediate responses. The mAbs defined four non-overlapping epitopes in both Der p II and Derf II which are consistent with the high degree of amino acid sequence homology between the two allergens (88%). The group II allergens assay will be of practical value in assessing the effects of chemical treatments to reduce mite allergen levels, particularly treatments involving allergen denaturation, because it has been established that group II allergens are more resistant to denaturation than are group I allergens. Extracts prepared from whole-mite cultures usually have high ratio of group I to group II (often > 10:1), whereas extract of pure mite bodies usually have ratios of less than 2:1.