standardized extracts stinging and biting insects

CLIN REV ALLERGY 75 5:75-88,1987

Standardized Extracts Stinging and Biting Insects

Donald R. Hoffman and David B. K. Golden

H i s t o r y a n d E p i d e m i o l o g y

The state of the art in insect allergy before 1973 consisted of obtaining a history, sometimes performing skin tests with one or more insect whole body extracts (WBEs), and, if convinced of the veracity of the history, treating the patient with WBE. Although some allergists, especially Dr. Mary Loveless, 1 chal- lenged this practice and suggested that venom and not insect bodies con- tained the allergens, they were generally ignored for lack of convincing scientific evidence.

A 4-year-old boy was seen at the allergy clinic of Johns Hopkins University in 1973 who had experienced 2 near fatal reactions to honeybee stings, the second following a course of WBE immunotherapy. His sister had previously died from bee sting anaphylaxis, and his father was a commercial beekeeper. Lichtenstein and colleagues 2 had the father collect bee venom and attempted rush desensitization. At the equivalent of 1/3 sting (15 ~g), systemic symptoms began and they became severe at a dose of 50 ~,g. The patient was placed on a slower injection protocol and eventually could tolerate 100 p,g. At this time serologic studies showed a substantial rise in IgG antibodies against venom and the patient tolerated an intentional sting challenge.

This single case was the beginning of an extensive research program at Johns Hopkins University and other institutions, which led to controlled scientific studies of many of the variables in insect venom allergy and, ulti- mately, to the licensing of bee venom and vespid venom protein extracts for human use. Present standards for dose, regimen, and some of the indications for immunotherapy are based on controlled studies; but many questions are still unanswered, such as the ability to predict severe reactions in untreated individuals, cross-reactivity and multiple reactivity among the venoms, and the duration of immunotherapy. Venom allergy will be an active area of research for many years.

The prevalence of severe reactions to insect stings is unknown. Studies performed mainly by tabulating questionnaires filled out by parents and phy-

From the Department of Pathology, East Carolina University, School of Medicine, Greenville, North Carolina; and Division of Clinical Immunology, Johns Hopkins University, Baltimore, Maryland.

Address correspondence and requests for reprints to Donald R. Hoffman, Ph.D, Department of Pathology, East Carolina University, School of Medicine, Greenville, NC 27858.

�9 1987 Elsevier Science Publishing Co., Inc. 0731-8235/87/$03.50 52 Vanderbflt Ave., New York, NY 10017

76 D.R. Hoffman and D. B. K. Golden

sicians of boys from 10-16 years of age indicated a prevalence of a history of a systemic reaction to a sting in about 0.4-0.8%.3 This figure has been widely quoted over the years with little consideration of factors such as geography and the age and nature of the study group. In the epidemiologic survey reported by Golden et al, 4 4% of the subjects interviewed had histories of systemic reactions and 17% had large local reactions; 12% of those with no histories of adverse reactions had positive venom skin tests. This study sug- gests that as many as 25% of the population has either a history of a reaction to a sting or is skin test positive to venoms, although sensitization after a sting is often transient. In control groups studied by Hoffman et al 5 using measurements of specific IgE antibodies, prevalence of positive reactions ranged from 3% in the midwest to 50% in rural field workers in eastern North Car- olina. Although it is difficult to ascertain the prevalence of nonfatal reactions and risk factors for sting allergy, the rate of documented fatal reactions appears to be relatively stable at about 40 annually in the United States. 6,7 These figures have not changed significantly since compilation began.

In sec t s T h a t C a n C a u s e Al le rg ic R e a c t i o n b y Bi t ing or S t i n g i n g

There are thousands of species of bees, wasps, and ants of the order Hy- menoptera that are capable of stinging man. The great majority of these insects rarely cause difficulty because they are solitary and use their venom to par- alyze prey rather than for defense. Most solitary wasps and bees will only sting if held and squeezed. Most species of ants will not sting man. Those Hymenoptera that are primarily social and live in medium to large colonies are generally protective of their nest and will sting mammals readily. The Hymenoptera that are known to cause venom allergy in various areas of the world are listed in Table 1. Commercial venom or venom sac extracts are only available for honeybees, Vespula species yellowjackets, 2 species of Dolichov- espula hornets, Polistes species paper wasps, and Vespa crabro (only in Europe). A prototype commercial extract has been prepared from Solenopsis invicta, imported fire ant venom.

There are also thousands of insects that can bite man. In general, allergy to insect saliva is relatively uncommon with the exception of allergy to Triatoma (kissing bug). 8 A short list of biting insects known to cause allergic reactions is given in Table 2. Commercial extracts from biting insects are generally WBEs. There is a prototype commercial extract of Triatoma protracta saliva currently undergoing clinical trials. 9

Co l l ec t ion of Mate r i a l a n d P r e p a r a t i o n of Extracts

The raw material for manufacturing Hymenoptera venom protein extracts is obtained by 2 different procedures. Honeybee venom is obtained by placing an electrically charged wire grid over a piece of thin rubber sheet at the entrance to a hive. When the bees touch the wires and receive a shock, they sting through the rubber sheet.l~ The venom is allowed to air dry and then

Standardized Extracts 77

Table 1. Hymenoptera That Cause Venom Allergy in Man

Super family Family Subfamily Genus Species

Apoidea Apidae (bees)

Halictidae (sweat bees)

Vespoidea Vespidae (social wasps)

Formicoidea Formicidae (ants)

Apis (honeybees) mellifera (domestic honeybees), 5 spp found in SE Asia

Bombus 39 spp in N America, many others (bumblebees) worldwide

Polistinae Polistes (paper wasps)

Vespinae

Ropalidia (tropical)

Provespa Vespa

(old world hornets)

Vespula (yellow jackets)

Dolichovespula (aerial hornets)

Myrmicinae Pogonomyrex (harvester ants)

Solenopsis

Several hundred spp, important in N America include exclamans, fuscatus, metricus, annularis, apachus

3 spp in SE Asia Only crabro in N America

important in Old World

19 spp 13 found in N America, important spp include maculifrons, germanica, squamosa, vulgaris, pensylvanica, flavopiIosa, vidua, consobrina

14 spp, 5 in N America, important include arenaria, maculata

22 spp in N America

15 native spp in N America, 2 introduced important spp are invicta and richteri

Table 2. Insects That Cause Allergic Reactions From Bites in Man

Order Family Genus

Hemiptera Reduviidae Triatoma (kissing bug, conenose bug) (bugs) Cimicidae (bedbugs)

Diptera Culicidae (mosquitoes) (flies)

Siphonaptera (fleas)

Simuliidae (blackflies) Tabanidae Tabanus (horseflies)

Chrysops (deerflies) Therevidae (stiletto flies) Phlebotominae (sandflies and Phlebotomus

gnats) Culicoides


is collected. Venom collected in this manner is relatively pure and uncontam- inated by pollens and fecal matter. 11

Although it is possible to collect vespid venoms in a similar manner, the product is usually contaminated with fecal material. 12-13 There are also tech- nical problems with collecting at vespid nests as well as the danger of thousands of agitated yellowjackets. Commercial extracts are prepared from insects that are trapped, fresh frozen, and shipped to the manufacturer. After species identification and quality control for parasites, dirt, and other contaminants, the insect venom apparati are removed. The venom sacs are homogenized in a beta-alanine buffer, pH 4.5. The extracts are then centrifuged and filtered. Most of the sac proteins are insoluble at the acidic pH, but the venom proteins are highly soluble under these conditions. Experimental batches of vespid venoms have been prepared by individual hand milking, individual shocking, and by squeezing of dissected venom sacs. 13-15 However, all of these procedures are too labor intensive for commercial application.

Venom protein extracts are quality controlled by several procedures. Each lot of extract is checked by nondenaturing electrophoresis with Coomassie blue staining and by staining the electrophoretic gel for phosphatases and esterases. The results are compared with reference patterns. The "protein" content is measured by both Lowry et a116 and ninhydrin procedures. Activ- ities of the enzymes phospholipase and hyaluronidase are also measured. 17 The hyaluronidase activity defines acceptable lots. Some preparations such as yellowjacket and paper wasp are mixtures of species prepared in standard

Figure 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of imported fire ant (Solenopsis invicta venom). S = venom obtained by individual milking of live insects, and E = "venom" obtained by mass electrical stimulation.


ratios. At present, all of the vespid venom protein raw material sold is derived from a single supplier. The final extracts are diluted, sterilized, bottled, and freeze-dried. Samples of each lot are tested for sterility, pyrogenicity, and toxicity by the final manufacturer. Lots also are tested for enzyme activities for standardization purposes.

Fire anffextracts currently available commercially are WBEs manufactured by grinding up worker ants. Unlike bee and wasp WBEs, fire ant body extracts contain significant allergen activity. 18 More enriched preparations are defi- nitely better for diagnosis, and there are presently 2 types of prototype venom extracts being evaluated. One is laboriously prepared by individually milking the ants, and the other is prepared by electrical stimulation. These 2 types of extracts are compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 1). The venom extract obtained by milking contains 3 strong protein bands, 2 of which correspond to the strong bands in the electrically stimulated extract. The extract obtained by electrical stimulation contains a number of protein bands not found in the naturally stung venom.

Triatoma extract is prepared from salivary glands removed from cultured insects. The dissected glands are extracted in phosphate buffered saline (PBS), sterilized by filtration and the extract standardized by radioallergosorbent test (RAST) or enzyme-linked immunosorbent assay (ELISA) inhibition. 9 This extract is not commercially available. Other biting insect extracts are WBEs with no meaningful standardization.

Venom Allergens Insect venoms are among the most well characterized of all allergen extracts. The known venom allergens are listed in Table 3 along with their molecular weights from SDS-PAGE, their approximate content in venom, and an esti- mate of their allergenic importance. Bee and wasp venoms both contain at least 3 major proteins. 13"14"19-24 All of the venoms contain hyaluronidases of similar enzyme specificity and molecular weights from 41,000-47,000 daltons. These hyaluronidases are closely related among the vespids with the exception of Polistes. The Vespula, Dolichovespula, and Vespa hyaluronidases also are cross-reactive with bee hyaluronidases from both honeybees and bumblebees. This cross-reactivity can be seen by immunoblot studies with rabbit antisera in Figure 2, and by RAST inhibition studies with human allergic sera in Figure 3. It appears that the hyaluronidases are the major source of cross-reactivity between bees and vespids.14

All of the venoms also contain phospholipases; the bee phospholipases are of molecular weight about 15,800 daltons and are of A2 specificity, cleaving only the middle fatty acid. The vespid phospholipases are of molecular weight 33,000-37,000 daltons and are of A1B specificity cleaving both fatty acids from phosphatidyl choline. The bee enzymes are antigenically unrelated to the vespid enzymes. The vespid phospholipases exhibit a variable degree of cross- reactivity. 25~27 Studies with antisera raised in mice, rabbits, and human IgG and IgE have shown inconsistent patterns of cross-reactivity characterized by a nonreciprocity of antigen and antisera reactivities.


Table 3. Known Venom Allergens

Allergen Molecular weight % of venom Importance

Honeybee (Apis mellifera) Phospholipase A2 15,800 6-12 Strong Hyaluronidase 47,000 1 Strong Acid phosphatase 98,000 (dimer) <1 Moderate Allergen C 102,000 <1 Moderate Melittin 2,848 40-60 Weak Other proteins 20,000-50,000 1 ?

Yellowjackets and hornets (Vespula, Dolichovespula, and Vespa) Antigen 5 22,000 15--40 Strong Phospholipase AIB 33,000-37,00(Y 10-25 Strong Hyaluronidase 41,000-46, 00(Y 2-5 Strong Vmacl b 97,000 1 Moderate Vmac3 b 3%000 ? Weak

Paper wasps (Polistes) Phospholipase AIB 34,000 23 Strong Antigen 5 24,500 18 Moderate Hyaluronidase 42,000 1.5 Weak Pool 3 10,000-38,000 2 ? 76,000 + 78,000 76,000-78,000 1 ?

Imported fire ant (Solenopsis invicta) Phospholipase ? ? ? Hyaluronidase ? ? ? Unidentified ? ? ? protein

'Two forms of differing molecular weight and/or charge are found in many venoms. bExamples are from Vespula maculifrons venom; similar materials are found in the other venoms.

Honeybee venom contains a large amount of the surface active peptide melittin, which is present mainly as dimers in the native venom. Melittin is responsible for much of the pain of bee stings. 28 It is a weak allergen of clinical importance in only some individuals. 2~ There is no analogous material in any of the other venoms. Bee venoms also contain acid phosphatase, which in honeybee venom is mainly a dimer of 98,000 molecular weight. It has moderate allergenic activity in most allergic individuals. An additional pro- rein, named allergen C, of 102,000 molecular weight is also an allergen for many people. Honeybee venom does contain a number of other proteins as can be seen in the map illustrated in Figure 4, but these are present in relatively small amounts and have not been characterized. Bumblebee venoms contain a number of additional proteins including an unusual trypsin-like enzyme, but they have not been characterized in detail.

Each of the vespid venoms contains a major protein of molecular weight 21,000-25,000 daltons called antigen 5, which is probably a presynaptic neurotoxin used against the insect's prey. 3~ The antigen 5's exhibit significant cross-reactivity, which is incomplete and not always reciprocal between antigen and antibody. Polistes antigen 5 is a weaker allergen than Vespula antigen 5's, but cross-reacts with Vespula antigen 5. Antigen 5 from Vespa appears to be the least related. Vespid wasp venoms also contain small amounts of some


Figure 2. Immunoblot using rabbit antibodies against honeybee, paper wasp; and Vespula raaculifrons yellowjacket hyaluronidases with various insect venoms. Abbre- viations: Std = standard for allignment, Vm = Vespula maculifrons, Vs = Vespula squamosa, Vv = Vespula vidua, Dm = Dolichovespula maculata, Vc = Vespa crabro, Pe = Polistes exclamans, Am = Apis mellifera, and Bp = Bombus pennsylvanicus. The antibody against Polistes hyaluronidase only reacts with Polistes exclamans venom, while the antibodies against bee and yellowjacket hyaluronidases react with all of the species except Polistes. The extra bands and lower molecular weight of the Dm hyaluronidase are caused by partial decomposition of the proteins.

additional proteins that appear to have some allergenic activity. These components have not been studied in detail. The SDS-PAGE pattern of Vespula squamosa venom and its fractions is shown in Figure 5.

The venom of the imported fire ant, Solenopsis invicta, is quite different from the bee and wasp venoms in that it contains about 95% water insoluble piperidine alkaloid. 31 Because of the small size of the ants and the small amount of aqueous phase, studies have been limited. As is shown in Figure 1, there appear to be at least 3 protein components, as has been reported. 32 One of them appears to have phospholipase A activity and another hyaluronidase activity. They have not yet been isolated. A few patients who experienced allergic reactions from fire ant stings reported that their reactions are the result of their first fire ant stings and many of the victims show IgE antibodies against other venoms. In one limited study cross-reactivity has been demonstrated between fire ant and yellowjacket venoms. 33


8 0

Z 0 b- n

rn m

"l- Z n

6 0

4 0

2 0

I I I I i

- 0 . 5 0 0 . 5 1 1 . 5

Log(microgram inhibitor)

Figure 3. Radioallergosorbent inhibition of binding of serum 10841 to VespuIa maculifrons hyaluronidase by honeybee hyaluronidase illustrating the cross-reactivity between the enzymes from different superfamilies.

Figure 4. Protein map of honeybee venom showing the numerous proteins present. The right-hand margin shows SDS-PAGE of honeybee venom for reference. The known proteins are labeled: C = allergen C, Hy = hyaluronidase, AP = acid phosphatase, Mel = melitfin, and PLA = phospholipase A. The map is stained with a polychro- matic silver stain.


Figure 5. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Vespula squa- raosa venom (V) and its fractions. 1 = Vsql (trace high molecular weight), H = hyaluronidase, 3 = Vsq3, P = phospholipase A~B, Pd = partially digested phospholipase, 5 = antigen 5. The gel is stained with a monochromatic ammoniacal silver stain.

Clinical Aspects

Honeybee

Honeybee venom was the first of the Hymenoptera venoms to be clinically evaluated because of its availability and ease of collection, and the necessity to treat beekeepers more effectively than with WBE. The initial efforts in the early 1970s began with empiric observations of appropriate venom concen- trations for diagnostic skin testing. This also served as a guideline for the dose at which to start immunotherapy. Venom skin tests were found to be highly sensitive and specific for identifying sting-allergic patients, whereas WBEs could not distinguish allergic subjects from nonallergic controls. 34,35 The degree of venom skin test sensitivity is not a reliable indicator of the severity of the allergic sting reactions. The venom RAST is less sensitive (10-20% fewer positives compared with skin tests). 36 With standard methods, venom skin tests only rarely cause systemic adverse reactions (as do all allergens).

The effective (protective) dose to be attained was reasoned to be the equivalent of 2 honeybee stings; each sting was estimated to deliver 50 p,g of venom protein (the average for venom droplets deposited by honeybees stinging through plastic wrap). Using an immunizing schedule of rapidly increasing doses, this treatment was found to ablate the systemic allergic sting reactivity


of subjects who had previously experienced severe reactions despite injections of "whole body extract". 2 The efficacy of honeybee venom immunotherapy has now been established in many clinical trials.

However, the reported 98% rate of complete prevention of systemic sting reactions may not apply to therapy with any single venom. Systemic symptoms occur after live sting challenge in 15% of patients treated with 100 ~g maintenance doses of honeybee venom, although most of these reactions were less severe than pretreatment sting reactions. These results were not improved by immunotherapy with phospholipase A (the major honeybee venom allergen). 37 Patients who are incompletely protected by therapy at the 100-~g dose have a poor immune response to treatment (low serum levels of venom-specific IgG-"blocking" antibodies)38; the clinical and immunologic responses can both be corrected by double-dose (200 ,,g) maintenance venom immunotherapy. Many immunized beekeepers have been able to successfully maintain their immunity with frequent and regular, year-round live bee stings instead of venom injections.

The safety of honeybee venom immunotherapy has been shown in several ways. Adverse reactions during initial treatment commonly include large local swelling but systemic reactions are felt to be no more frequent than the 5--15% rates observed with inhalant allergen immunotherapy. Most of the relevant observations have been made with yellowjacket (and mixed vespids) venom immunotherapy. Few patients have been reported to have problems with systemic reactions during maintenance treatment, and only an occasional patient must give up therapy because of repeated systemic reactions and an inability to tolerate adequate therapeutic doses.

The long-term safety of honeybee venom immunotherapy is assumed, based on a lack of biochemical, hematologic, or clinical abnormalities in beekeepers who sustain thousands of bee stings in a lifetime. 39 Adverse reactions (other than immediate systemic) have been rare during prolonged venom immunotherapy up to 10 years. Vague rheumatologic symptoms occur spo- radically without laboratory abnormalities and without a clear clinical rela- tionship to venom treatment.

Wasp

Polistes venom sensitivity is usually associated with vespid (yeUowjacket) venom allergy, but may occur as a distinct entity. Although the diagnostic accuracy and therapeutic utility of Polistes wasp venom have been established, there have been few long-term studies of its safety (distinct from other venoms). Suspected cross-sensitivity with yellowjacket venom can be tested by RAST inhibition techniques. As with other venoms, clinical protection is complete in about 85% of patients receiving only Polistes venom injections (without vespid venoms).

Yellowjacket Venom

Yellowjacket venom sensitivity is the most common venom allergy in many areas, and its clinical use has been most extensively studied. Prick/puncture skin tests are highly specific but the intradermal test is more sensitive and


has been better characterized. Intradermal skin tests at venom concentration up to 1.0 ~g/mL only rarely cause false-positive reactions (<5%); most affected patients are sensitive to ~<0.1 ~g/mL. Skin tests with 3 purified allergens (antigen 5, phospholipase A, hyaluronidase) have shown that in vivo sensitivity is strongest and most frequent to antigen 5. *o

Yellowjacket venom immunotherapy has been extensively studied (usually with concomitant hornet venom therapy) in over 500 patients 42 and has been monitored in more than three thousand others. 43 Complete protection from a systemic reaction to challenge sting is achieved in 85% of patients treated with yellowjacket venom at the recommended 100-~g maintenance dose (98% with 200 ~g yellowjacket venom or with 300 ~g mixed vespid venom). This level of protection is sustained during 10 years of maintenance therapy, but is lost if therapy is prematurely stopped. ~ Patients who did have a systemic sting reaction while on venom immunotherapy had much less severe reactions than before treatment. 43

Adverse reactions are not common. Systemic symptoms occur in 5-15% of patients during initial induction therapy, but most are mild and do not really disrupt the course of treatment. 4s The frequency of systemic reactions (1.5 per 100 injections) is the same with slow or "rush" induction regimens. Main- tenance doses are much less likely to cause systemic symptoms. Large local reactions at the injection site are more common, especially during initial treatment (20 reactions per 100 injections). Full-dose therapy is associated with fewer large local reactions (6 per 100 injections). No long-term side effects have been observed.

Hornet Venoms Only scanty data are available on specific hornet venom allergy diagnosis or treatment, since more than 95% of those with "hornet" sensitivity are allergic to yellowjacket venom. The "hornets" referred to are the New World hornets (Dolichovespula), which are phylogenetically and immunochemically closely related to the yellowjackets. Isolated hornet allergy is so uncommon that no data have been available on any number of such individuals. However, hornet stings certainly do occur and may cause a sensitivity to unique hornet allergens even in yellowjacket allergic patients. Radioallergosorbent test inhibition techniques have shown that about 5% of patients with multiple vespid venom sensitivities have IgE specific for hornet allergens. .6 Skin tests and RASTs both indicate that yellow hornet (D arenaria) venom shows the least reactivity in vespid venom sensitive patients. White-faced hornet (D maculata) venom is more reactive but still generally less than yellowjacket venom. These observations also are reflected in the fact that hornet venom sensitivity declines and disappears more frequently and more rapidly than yellowjacket venom sensitivity. 44

Over 90% of patients with multiple vespid venom sensitivities can be clinically protected by immunotherapy with yellowjacket venom alone. However, mixed vespid venoms give a higher level of protection because of the higher dose of yellowjacket venom allergens (due to the yellowjacket cross-reactive antigens in the hornet venoms). ~ Therapy with Vespa venoms is not available in North America due to the sparsity of the insects. Few data have been


reported on the clinical use of Vespa venoms in Europe, but it would be expected to be quite similar in all respects to other Hymenoptera venoms.

Fire Ant Extracts

Controlled studies are just starting with fire ant venom-enriched preparations. There are no controlled studies of the efficacy of WBE immunotherapy with intentional sting challenges reported. There is significant evidence from comparative diagnostic studies that the commercially available WBE in some cases contains significant venom allergen, ls'47 In comaparative RAST studies WBE appears to have from 10-25% of the activity of venom; however, it is clear that venom or venom-enriched preparations are superior. There has been a single preliminary report of immunologic changes during immunotherapy with WBE. 4s Specific IgE antibodies declined and specific IgG antibodies in- creased during therapy. The venom appears to be safe for human adminis- tration, since it is not uncommon for people to receive thousands of stings with only local pustule formation.

Biting Insect Extracts

The only biting insect extract that has been evaluated in controlled studies is Triatoma protracta saliva. 9 Insects were grown in the laboratory to avoid possible contamination with Trypanosoma cruzi and blood borne diseases such as hepatitis. Patients with a history of anaphylaxis from Triatoma bites who were skin test and IgE radioimmunoassay (RIA) positive were treated until maintenance at 1000 U monthly. All patients tolerated a bite challenge and showed significant rises in IgG-blocking antibody against the Triatoma salivary gland extract. It is very important to recognize that triatoma allergy shows a very strong species specificity. Triatoma rubida is almost totally noncross-reactive with T protracta.

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43. Peppe B, Lockey R, Madden J, et ah AAA1 committee on insects: Hymenoptera venom study, treatment results, 9/30/82. J Allergy Clin Immunol 71:120, 1983

44. Golden DBK, Johnson K, Addison BI, et al: Clinical and immunologic observations in patients who discontinue immunotherapy. J Allergy Clin Immunol, 77:435--442, 1986

45. Golden DBK, Valentine MD, Kagey-Sobotka A, et al: Regimens of hymenoptera venom immunotherapy. Ann Intern Med 92:620-624, 1980

46. Golden DBK, Valentine MD, Kagey-Sobotka A, et al: Cross-reactivity of vespid venoms. J Allergy Clin Immunol 67:57, 1981

47. Strom GB, Boswell RN, Jacobs RL: In vivo and in vitro comparison of fire ant venom and fire ant whole body extract. J Allergy Clin Immunol 72:46-53, 1983

48. Johansson SGO, Nordvall S, Ledford D, et ah Specific IgE and IgG responses to immunotherapy with imported fire ant whole body extract in IFA hypersensitivity. J Allergy Clin Immunol 75:208, 1985

standardized extracts stinging and biting insects

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