Grass Pollen Extract Research Articles

The characterization of allergens

Clinical & Experimental AllergyVolume 26, Issue 8 p. 865-867 Free Access The characterization of allergens A.W. Frankland, A.W. Frankland Department of Allergy and Respiratory Medicine 4th Floor, Hunt's House Guy's Hospital London SE1 9RT UKSearch for more papers by this author A.W. Frankland, A.W. Frankland Department of Allergy and Respiratory Medicine 4th Floor, Hunt's House Guy's Hospital London SE1 9RT UKSearch for more papers by this author First published: August 1996 https://doi.org/10.1111/j.1365-2222.1996.tb00620.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Dolen WK. Allergen extract standardization: reality, myth or dream? Ann Allergy 1995; 75: 81– 2. 2 Berrens L. What is a ‘major’ allergen. Clin Exp Allergy 1994; 24: 606– 9. 3 Marsh DG, Goodfriend L, King JP et al. Allergen Nomenclature. WHO Bull 64 1986; 5: 761– 70. 4 Dreborg S, Bousquet J, Lowenstein H, Frew AJ. Response to ‘What is a “major” allergen’ by Berrens. Clin Exp Allergy 1994; 24: 610– 11. 5 Dreborg S, Einarsson R. The major allergen content of allergenic preparations reflects their biological activity. Allergy 1992; 47: 418– 23. 6 Frankland AW, Warner JO. In L Berrens, JA. Leezenberg RThR. Wentges Selection of allergic patients for immuno-therapy. Proceedings of the Second BRL Kurhaus Workshop on Allergy. Amstelveen Beecham Research 1988; 36. 7 Van der Zee JS, Van Swieten P, Jansen HM, Aalberse RC. Skin tests and histamine release with P1-depleted Dernuilophagoides pteronysinus body extracts and purified PI. J Allergy Clin Immunol 1988; 87: 884– 94. 8 Noon L. Prophylactic innoculation against hayfever. Lancet 1911 i: 1572– 3. 9 Bernstein IL, Storms WW. Practice parameters for allergy diagnostic testing. Ann Allergy Asthma Immunol 1995; Part II 75: 543– 615. 10 Methods of the Laboratory of Allergic Products. Laboratory of Allergic Products OBRR, 8800 Rockville Pike, Bethesda, MD 20892. 11 Nelson HS. Diagnostic procedures in Allergy 1. Allergy skin testing. Ann Allergy 1983; 51: 411– 7. 12 Basomba A, Sastre A, Pelaez A et al. Standardization of the prick test. Allergy 1985; 40: 395– 9. 13 Dreborg S. Skin tests used in type I allergy today. Position paper prepared by the subcommittee on skin tests of the European Academy of Allergology and Clinical Immunology. Allergy 1989; 44 (Suppl.): 22– 59. 14 Sehou C. Defining allergens of mammalion origin. Clin Exp Allergy 1993; 23: 7– 14. 15 Dreborg S, Frew A. EAACI position paper on allergen standardization and skin tests. Allergy 1993; 48 (S14): 49– 82. 16 Kordash TR, Freshwater LL, Amend MJ. Standardization of allergenic extracts by basophil histamine release. Ann Allergy 1995; 75: 101– 6. 17 Wheeler AW, Jessberger B, Drachenberg J, Rakoski I. Design of an optimally-diagnostic skin test solution for diagnosis of sensitivity to Timothy grass (phleum pratense) pollen. Clin Exp Allergy 1996. 18 Ownby DR, Petersohn EL. Why mix ROC with RAST. Ann Allergy 1992; 68: 3– 4. 19 Basomba A, Sastre A, Pelaez A, et al. Standardization of the prick test. Allergy 1985; 40: 395– 9. 20 Turkeltaub PC. In vivo methods of standardization. Clin Rev Allergy 1986; 4: 371– 87. 21 Lockey RF, Benedict LM, Tukeltaub PC, Bukantz SC. Fatalities from immunotherapy and skin testing. J Allergy Clin Immunol. 1987; 79: 660– 77. 22 Baur X, Azuppon AB, Sander I. Heating inactivates enzymic antibody and partially inactivates the allergenic activity of Asp 02 Allergy 1996; 26: 232– 4. 23 Malanin K, Lundbergh M, Johansson SGO. Anaphylactic reaction caused by neo allergens in heated pecan nut. Allergy 1995; 50: 988– 91. 24 Prausnitz C, Kustner H. Studies in hypersensitivity. Centralbl f Bacteriol 1921; 86: 160– 9. 25 Aukrust L. Cross radioimniunoelectrophoretic studies of distinct allergens in two extracts of Cladosporium herbarum. Int Arch Allergy App Immunol 1979; 58: 371. 26 Hoffman DR, Kozak PP. Shared specific allergens in mould extracts. J All Clin Immunol 1979; 63: 213. 27 Zhang L, Curron I, Muradia G, Rode H, Vijay HM. Two-dimensional immunoblot analysis of allergens of Cladosporiiim herbarum. Clin Exp Allergy 1994; 24: 263– 9. 28 Zhang L, Muradia G, De Vorge MW, Rode H, Vijay HM. An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA library of Cladosporium herharum. Clin Exp Allergy 1996; 26: 88– 95. 29 Shou C. Defining allergens of mammalian origin. Clin Exp Allergy 1993; 23: 7– 14. 30 Cadot P, Lejoly M, Stevens EAM. The effect of sucrose on quality of rye grass (Lolium peree) pollen extracts. Allergy 1995; 50: 941– 51. Citing Literature Volume26, Issue8August 1996Pages 865-867 ReferencesRelatedInformation

Characterization of Phl p 4, a major timothy grass (Phleum pratense)pollen allergen

Background:Group 4 grass pollen allergens represent glycoproteins with a molecular weight of 50 to 60 kd, which are present in many grass species. Almost 75% of patients allergic to grass pollen display IgE reactivity to group 4 allergens, which hence can be regarded as major grass pollen allergens. Objective:In this study attempts were made to obtain information regarding the immunologic properties, localization, and occurrence of Phl p 4 and related allergens. Methods:Phl p 4 was detected in timothy grass pollen extracts by immunoblotting with serum IgE and monoclonal antibodies and was localized in pollen by immunelectron microscopy. A peptide sequence from Phl p 4 was obtained by amino acid sequencing. The resistance of Phl p 4 against trypsin was analyzed after trypsin treatment of timothy grass pollen extracts with serum IgE and monoclonal antibodies. Cross-reactivities between Phl p 4 and Amb a 1, the major allergen of ragweed, were studied by using monoclonal antibodies and by IgE-inhibition studies. Results:Phl p 4 was characterized as a trypsin-resistant major timothy grass pollen allergen. By immunelectron microscopy Phl p 4 was localized in the exine, cytoplasm, and amyloplast of timothy grass pollen. Significant sequence similarities of a Phl p 4 10 amino acid peptide with Amb a 1, the major ragweed allergen, could be found. The immunologic similarity of Phl p 4 and Amb a 1 was confirmed by cross-reactivity of monoclonal antibodies and patients’ IgE. Conclusion:Phl p 4 represents a trypsin-resistant major timothy grass pollen allergen with immunologic similarities to the major ragweed allergen Amb a 1 and therefore must be considered an important cross-reactive component in grass pollen and weed pollen allergy. (J A LLERGY C LIN I MMUNOL 1996;98:189-98.)

Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass(Lolium perenne)pollen

T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T- cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II–restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR–restricted; this clone expressed a T helper cell phenotype (CD3 +, CD4 +) and the T-cell receptor αb. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease. (J A LLERGY C LIN I MMUNOL 1996;98:124-32.)

A double-blind, placebo-controlled study of immunotherapy with grass-pollen extract Alutard SQ during a 3-year period with initial rush immunotherapy

A double-blind, placebo-controlled study of immunotherapy with grass-pollen extract Alutard SQ during a 3-year period with initial rush immunotherapy

Antihistamine premedication in specific cluster immunotherapy: A double-blind, placebo-controlled study

BACKGROUND: Specific immunotherapy treatment in allergic diseases involves a risk of systemic side effects. A double-blind, placebo-controlled study was performed in 45 patients allergic to pollen to determine whether pretreatment with loratadine could reduce the number and severity of systemic reactions during the dose-increase phase of cluster immunotherapy. METHODS: The patients received cluster immunotherapy with a standardized birch (Betula verrucosa) or grass (Phleum pratense) pollen extract adsorbed to aluminum hydroxide. The immunotherapy schedule involved seven visits and 14 injections to reach a maintenance dose of 100,000 standardized quality units. Loratadine, 10 mg, or placebo tablets were administered 2 hours before the first injection at each visit. RESULTS: A total of 720 injections were given (309 injections in 21 patients receiving loratadine and 411 injections in 24 patients receiving placebo). The median numbers of injections to reach maintenance dose were 15 (range, 14 to 18) in the loratadine group and 16 (range, 14 to 23) in the placebo group (p = 0.037). The numbers of patients with systemic reactions were seven (33%) and 19 (79%) in the loratadine and placebo groups, respectively (p = 0.002). Twenty-five reductions caused by systemic reactions were observed in the placebo group in contrast to nine in the loratadine group (p = 0.047). No life-threatening systemic reactions were observed in either group. Systemic reactions were, however, more severe in the placebo group, mainly because of a significantly higher incidence of urticaria (10 vs 1, p = 0.022). CONCLUSION: Pretreatment with loratadine seems to reduce both the number and severity of systemic reactions in specific cluster immunotherapy. (J Allergy Clin Immunol 1996;97:1207-13.)

Identification and characterization of the major allergens of velvet grass (Holcus lanatus), Hol l 1 and Hol l 5.

Although extract of velvet grass pollen is an important ingredient of commercial allergen extracts for immunotherapy, it is not defined concerning its major allergenic components. We have investigated the extract of velvet grass pollen by Western blotting using the sera of 97 patients and identified two major allergens of 34 and 30 kD. By two-dimensional immunoblotting with monoclonal antibodies and lectins, and by biochemical analysis the 34-kD protein was identified as a group I grass pollen allergen and the 30-kD protein as a group V grass pollen allergen. According to the nomenclature for grass pollen allergens we designated these allergens Hol l 1 and Hol l 5, respectively. Fragmentation of the velvet grass pollen extract by CNBr provided the first information on the localization of IgE binding epitopes on these two allergens. With Hol l 1 and Hol l 5 we identified two important components of commercial allergen extracts.

Detection of allergen-specific IgE in tears of grass pollen-allergic patients with allergic rhinoconjunctivitis.

Allergic conjunctivitis is a common symptom amongst Type I (IgE-mediated) allergic diseases; and most frequently seen as rhinoconjunctivitis. However, the site of production and the significance of allergen specific IgE needs further elucidation. We investigated whether the presence of IgE in tears of grass pollen allergic patients correlated with disease and clinical symptoms, whether the IgE binding pattern to the different grass pollen antigens was different in sera and tears, and whether IgA antibodies to grass pollen allergens were present in tears. Finally, we looked whether specific IgE was produced locally or was exudated from serum. Sera from 44 grass pollen allergic patients suffering from either allergic rhinitis (n = 11) or allergic rhinoconjunctivitis (n = 33) and from healthy controls (n = 7) were used for the experiments. Binding of specific IgE and IgA antibodies to the different groups of grass pollen allergens (Phleum pratense) was evaluated by means of immunoblotting. Specific IgE was detected in sera as well as in tears of allergic patients, whereby tear-derived allergen-specific IgE exerted similar specificities to the corresponding IgE from serum. The correlation between symptoms of ocular allergy and the presence of allergen-specific IgE in tears was highly significant (P < 0.0001). In contrast, only a poor correlation was found between specific and/or total IgE in sera and the manifestation of ocular allergy (P = 0.73). Allergen-specific IgE antibodies in tears seem to be produced locally rather than exudated from serum. IgE in tears seems to be responsible for allergic conjunctivitis. IgA in tears cannot exert a protective function since the IgA antibodies recognize different antigens in a grass pollen (Phleum pratense) extract than IgE antibodies. The highly significant correlation between allergic conjunctivitis and the presence of specific tear IgE emphasizes the diagnostic value of immunoblots with tear IgE, especially in cases in which serum provides inconclusive results.

101 Manufacture and analysis of 100,000 BAU/mL and 10,000 BAU/mL standardized grass pollen extracts

101 Manufacture and analysis of 100,000 BAU/mL and 10,000 BAU/mL standardized grass pollen extracts

T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense): Evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens

Background: The use of peptides representing T-cell epitopes of allergens is a modern concept for improvement of specific immunotherapy. A prerequisite for this approach is the identification of T-cell epitopes of atopic allergens. Methods: T-cell lines and 40 T-cell clones (TCC) specific for Phl p 1, the group I allergen of timothy grass (Phleum pratense), were established from the peripheral blood of nine patients allergic to grass pollen and mapped for epitope specificity by using overlapping dodecapeptides. Phenotype and cytokine production profile of TCC were investigated. Representative TCC were analyzed for HLA-restriction, T-cell receptor Vβ gene usage, and crossreactivity with grass pollen extracts from Dactylis glomerata, Poa pratensis, Lolium perenne, Secale cereale, and selected amino acid sequence-derived peptides. Results: Patients displayed IgE binding to all grass species investigated. Forty TCC were established. Fifteen T-cell epitopes could be identified on Phl p 1. Of 40 TCC, 39 displayed the helper cell (Th) phenotype; one clone was CD8 +. Specific stimulation induced a Th2-like type of cytokine production in 20 of 39 TCC. Crossreactivity studies revealed crossreacting and non-crossreacting T-cell epitopes. Conclusion: Phl p 1, a major grass pollen allergen, harbors multiple T-cell epitopes. Species-specific and crossreacting T-cell epitopes exist among group I allergens of grasses. Epitope recognition patterns could not be correlated with particular HLA haplotypes. A restricted T-cell receptor Vβ gene usage was not observed. (J A LLERGY C LIN I MMUNOL 1995;96:986-96.)

Evaluation of a composite scoring system in conjunctival challenge with allergen: reproducibility and evolution during loratadine treatment

The reproducibility of any challenge and its sensitivity to change during therapeutic interventions should always be examined before any drug trial is conducted. Conjunctival challenge has been widely used to assess objectively the efficacy of antiallergic treatments. A double-blind, placebo-controlled, crossover study was carried out in 26 patients allergic to grass pollen to test the reproducibility of a composite symptom score during placebo and control days, and to check the ability of loratadine to increase the provocative dose inducing a positive challenge. Conjunctival challenge was carried out with increasing concentrations of a standardized orchard grass pollen extract until a composite symptom score of 5 was reached. The provocative dose inducing a positive challenge was similar for the baseline (6.94 +/- 8.7 index of reactivity (IR)) and placebo days (20.0 +/- 32.5 IR) and was significantly increased (P < 0.01 and P < 0.001) when patients received loratadine (117.3 +/- 136.8 IR). This study shows that the composite score used was reproducible and sensitive to change, making it possible to use in drug trials.

Comparison of IgE and IgG antibody responses of atopic individuals with sensitization to tree and grass pollens

Sera of atopic individuals with predominant sensitization to either tree pollen (TAs) or tree and grass pollens (TGAs) as well as of nonatopic subjects (NAs) were analyzed for IgE, IgG, and IgG4 antibodies specific for grass pollens allergens. Of 600 atopic individuals with serum IgE antibodies specific for birch pollen allergens, 54% also had serum IgE antibodies specific for grass pollen. The mean titers of IgG antibodies specific for grass pollen proteins were about 10 times higher in the sera of TGAs than those in the TAs and NAs. SDS-PAGE immunoblotting analysis of grass pollen proteins using sera of TGAs, TAs, and NAs with respect to the binding of these proteins with IgE and IgG antibodies in these sera exhibited a similar pattern of variation. Quantitation by enzyme immunoassay of the antibody binding to a recombination grass pollen allergen, rKBG8.3, further demonstrated the elevated IgG antibody levels in TGAs are mainly due to a broader range of specificities, and not to high specific binding to the individual protein. Statistically significant correlation was found between IgE and IgG4 antibodies specific for the Kentucky bluegrass (KBG) extract, but not for the isolated recombinant allergen. These results indicate that the grass pollens elicit a complex array of antibody specificities in both atopics and nonatopics, and that the profile of antibodies specific to the pollen extract and pure allergens differs, suggesting that single grass allergens may be inadequate for replacing grass pollen extracts for immunotherapy.

A two-site monoclonal antibody ELISA for the quantification of group V allergens in grass extracts.

A two-site solid-phase enzymimmunoassay was used for the quantification of group V allergen in grass pollen extracts in mass units. The assay is based on the monoclonal antibodies (MoAbs), 1D11 and 3B2 which recognize different epitopes on the standard Phl p V. The MoAb-ELISA is very sensitive (15-100 ng/ml Phl p V) and highly specific for group V allergens. Six pollen extracts of different grasses demonstrated parallel binding curves. The group V content ranged between 73 and 673 micrograms/ml in the extracts. The International Standard of Phleum pratense (IS 82/520) contains 400 micrograms/ml Phl p V. A good correlation was observed between the group V content and RAST inhibition, with the exception of Poa pratensis.

Evaluation of allergenic potency by REAST inhibition. A new tool for the standardization of allergenic extracts

The potency of allergenic extracts can be determined in vitro by RAST inhibition, and this has become the preferred method for the standardization of allergens. A disadvantage of this technique is the impossibility of obtaining data about allergens bound to the solid phase, i.e., the counterpart of the inhibiting extract. The REAST (reverse the reaction of captured IgE with biotinylated allergen and the development of a colour reaction by subsequent addition of streptavidin-peroxidase and chromogenic substrate. The addition of an allergen extract in a dose-response fashion competes with the biotinylated allergen and inhibits the test. In the present study REAST inhibition has been evaluated with Dermatophagoides pternyssinus, Parietaria judaica and mixed grass pollen extracts. The correlation of REAST inhibition with RAST inhibition and both intra-assay and inter-assay reproducibility have been evaluated. REAST inhibition is a potentially valuable new tool for the standardization of allergenic extracts.

INFLUENCE OF GRASS POLLEN IMMUNOTHERAPY ON CELLULAR INFILTRATION AND CYTOKINE EXPRESSION DURING ALLERGEN-INDUCED LATE-PHASE CUTANEOUS RESPONSES

Purpose of the Study. The mechanism by which immunotherapy (IT) provides its beneficial effects are poorly understood. This study examines the effects of grass pollen IT on cellular infiltration and cytokine mRNA expression during allergen induced late phase cutaneous responses. Methods. Forty subjects were enrolled in this double blind, placebo controlled study to receive twice weekly injections of either a standardized grass pollen extract or placebo. Once maintenance was reached, injections were administered monthly for 9 months. Skin biopsies following intradermal infection with Timothy extract were performed prior to and following IT. Monoclonal antibody staining was used to detect surface markers on lymphocytes. In situ hybridization was used to identify cells expressing cytokine mRNA. Findings. Both patient groups were matched for age, disease severity, and duration. Thirty-seven patients (17 placebo, 20 active) completed the study. The treated group had a significant decrease in symptoms, medication use, and size of the immediate and late phase reaction (LPR) after intradermal allergen injection upon completion of the IT. Skin biopsy cell counts from actively treated subjects obtained after subcutaneous allergen challenge showed a significant decrease in T helper and eosinophil cell numbers compared to the placebo group. Increases in the expression of CD25 and HLA-DR (markers of T cell activation) were seen in the treatment group. No differences in amount of HA and IL5 mRNA-expressing cells (TH2) were noted, but there was an increase in IFN-γ and IL2 mRNA expressing cells (ie, TH1) in the actively treated group. A significant correlation was noted between the size of the LPR and the numbers of CD4+ cells in the placebo group.

A double‐blind, placebo‐controlled trial by the sublingual route of immunotherapy with a standardized grass pollen extract

Fifty-eight patients with well-documented history of seasonal rhinoconjunctivitis caused by grass pollens were allocated randomly on a double-blind basis to receive either sublingual therapy with a solution of purified, standardized allergen preparation (Stallergènes) or a matched placebo for 17 weeks. The assessment of the effect of oral immunotherapy, done with drops of five-grass allergen extract, was on the clinical symptoms and on the medication score of the authorized rescue treatments. The actively treated patients had significantly (P < 0.05 to P < 0.01) fewer symptoms of rhinitis (sneezing and rhinorrhea) and of conjunctivitis (redness and tears) during the pollen season than the placebo group. Consumption of nasal solution of sodium cromoglycate and of betamethasone and dexchlorpheniramine was significantly less in the desensitized group (P < 0.01). Side-effects were negligible. This study concludes that perlingual immunotherapy with grass pollen extract in grass-pollen-sensitive seasonal hay fever and conjunctivitis patients is effective, easy to perform, inexpensive, and safe.

DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

Booster immunotherapy (BIT).

This study tries to answer two questions: 1) how long does the therapeutic effect of successful immunotherapy (IT) last after termination of the treatment? and 2) what is the best treatment for recidivist patients? To answer the first question, we asked 108 patients with rye/grass pollen allergy who had previously undergone IT for 3-4 years and had responded well to treatment to complete a questionnaire on the course of their recovery after termination of the therapy. Evaluation of the answers revealed a recidivist rate of approximately 30% in the first 3 years. According to our results, the risk of a relapse after this period seems to be low. To answer the second question, we included 40 patients suffering from type I allergy to grass/rye pollen in a clinical study. Each of them had previously undergone specific IT with a grass pollen extract mixture and had terminated this therapy after 3-4 years practically free of symptoms during the grass pollen season. As the symptoms returned and increased from year to year after the end of IT, new therapeutic steps had to be considered. We investigated the efficacy of a short preseasonal injection treatment called "booster immunotherapy" (BIT). BIT was performed with two different injection-regimens, a low-dose schedule comprising six injections and a high-dose schedule with 11 injections, in both cases administered as a build-up regimen. In the next pollen season, 28/40 (70%) patients reported strong improvement or even complete remission of the allergy symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of grass group I allergens in timothy grass pollen

Using Phl p V-depleted timothy grass pollen extract ( Phleum pratense) as immunogen, we obyained a monoclonal antibody, QG 4, which recognized proteins of 33, 35, and 37 kd as determined by Western blotting. The antibody cross-reacted with pollen proteins of other grass species in the molecular weight range of 30 to 37 kd. By means of two-dimensional polyacrylamide gel electrophoresis blot of timothy grass pollen extract, we demonstrated at least seven protein spots: two of 37 kd with isoelectric points of 6.4 and 6.6; four of 35 kd with isoelectric points of 6.5, 6.8, 7.1, and 7.3; and one of 33 kd with an isoelectric point of 8.5. These protein spots were also detected by patients' pooled serum. Microsequencing of the 20 N-terminal amino acid residues revealed structures with sequence identities up to 90% to the well-established allergen, Lol p 1 of ryegrass ( Lolium perenne). Therefore we assume that the monoclonal antibody QG 4 recognized the corresponding allergen Phl p I in timothy grass pollen.

Monoclonal antibody immunoassay for quantitative analysis of group V allergens in grass pollen extracts.

A two-site monoclonal antibody (MoAb) ELISA has been developed for the quantification of the Phleum pratense major allergen, Phl p V. The assay is based on two MoAbs which recognize different non-overlapping epitopes on the Phl p V molecule; one antibody (1D11) was immobilized on the solid phase and the other (3B2) was biotinylated. An affinity-purified Phl p V preparation (purity of 95%) was used as standard. The assay has a sensitivity of 10 ng/ml of allergen and is suitable for the detection of group V allergen in aqueous grass extracts. The specificity of the assay was investigated with 14 grass pollen and five non-grass pollen extracts. Different levels of group V allergen were detected in extracts of grasses, but not in non-grasses. The assay gives a good correspondence with allergenic activity of extracts as determined by ELISA inhibition using serum pool of allergic patients. The results indicate that the two-site MoAb ELISA could be very useful in the standardization of allergenic extracts from grass pollen.

Extended evaluation of diagnostic skin testing practices in Orange County, California.

In an effort to evaluate conformity with published guidelines for diagnostic allergy skin testing in their locality, a committee of members of the Orange County (California) Society of Allergy and Clinical Immunology surveyed their membership for the number, identity, and rates of sensitization of aeroallergens in a selected patient population. Test data were analyzed on patients who were judged appropriate to undergo immunotherapy. Complete test results of 271 patients were obtained from one third of local allergy specialists. A total of 115 different aeroallergen extracts were identified. By category, tree pollen allergens (32) were followed in declining order by weed pollens (28), molds (22), environmentals (18), and grass pollen extracts (15), for the total of 115. The average number of aeroallergens used by a practice in a comprehensive evaluation was 77 (range, 47-114). When only unequivocally positive reactions were considered (3+ equivalent or greater), Bermuda grass pollen extracts elicited the highest rate, 85%, and the environmental, rat dander, was the lowest at 1.8%. At least 20% of the patients reacted strongly on skin tests to 97% of the extracts. The disparity between the recommendations of a maximum of 30 aeroallergens for a comprehensive evaluation and actual practice procedures merits efforts at resolution.