Int. Archs Allergy appl. Immun. 48 : 812-823 (1975)

Human Antibodies to Bovine /-Globulin Occurrence in Immunological Disorders and Influence on Allergy Radio-imniunoassays 1

T. F oucard, H. Bennich, S. G. O. J ohansson and U. L undkvist The Blood Centre, University Hospital, the Biomedical Centre, University of Uppsala, and the Research Department, Pharmacia Diagnostics, Uppsala Abstract. Antibodies to bovine /-globulin (anti-BGG antibodies) were detectable by a radio-immunoassay in 70%> of healthy blood donors but, generally, the titres were low. Significantly increased concentrations of anti-BGG antibodies were found in patients lacking IgA but not in patients with allergic disorders. The anti-BGG an­ tibodies were shown to give rise to falsely high IgE values in the radio-immuno­ sorbent test for IgE determination (RIST) when a sheep anti-IgE antiserum was used. Furthermore, falsely positive results can sometimes be caused by such anti­ bodies in the determination of cow-dander- or cow’s-milk-specific IgE by the radio-allergosorbent test (RAST). When a rabbit anti-IgE antiserum was used instead of the sheep anti-IgE, normal IgE levels and negative RAST results were obtained. The difference is explained by the higher degree of cross-reactivity between the an­ ti-BGG antibodies and sheep /-globulin than between anti-BGG antibodies and rab­ bit /-globulin.

The presence of antibodies to animal proteins in some human sera is well known [6, 7, 14, 16] but their biological significance is still obscure. In immunological tests using animal antisera for the quantitative determi­ nation of human proteins at low concentrations, such antibodies may in­ terfere with the specific reaction and give rise to false results [2], In this study, the influence is demonstrated of human antibodies to bovine /-globulin (BGG) on the results of IgE determinations by the ra­ dio-immunosorbent test (RIST) and on the quantitation of IgE antibodies specific to cow dander and to cow’s milk by the radio-allergosorbent test Received: November 25, 1974.

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1 This work was supported by the Swedish Medical Research Council, grants No. 16X-105 and 13X-3556.

F oucard/B ennich /J ohansson/L undkvist

813

(RAST). In addition, the occurrence of such antibodies in the sera from healthy blood donors and from patients with various immunological dis­ orders was studied by radio-immunoassay. Material and Methods Sera were obtained from healthy blood donors and from patients attending var­ ious clinics at the University Hospital, Uppsala, Sweden. Antigens. Cow dander allergen extracts at the highest concentrations available (1/10 w/v or 10,000 PNU), were obtained from Vitrum Laboratories, Stockholm, Sweden, Dome Laboratories (Meda Ltd., Gothenburg, Sweden), Beecham Laborato­ ries, England, and the Allergological Laboratory, Copenhagen, Denmark. Commer­ cially available skimmed cow’s milk was also used. Bovine serum albumin (BSA) was purchased from Sigma Chemical Company, USA. Crystallized BSA and rabbit y-globulin were purchased from Mann Research Laboratories, USA and y-globulins from cat, cow, dog, guinea pig, horse, pig and sheep from Pentax Corporation, USA. Goat serum was obtained from the Agricultural College of Sweden, Uppsala, and y-globulins were isolated by salt precipitation at 25 °C, using 18 g anhydrous sodium sulphate per 100 ml serum. 20 fig of BGG, human IgE and immunosorbent purified rabbit and sheep anti-IgE were each labelled with 2 mCi of 125I by the chloramine-T method as de­ scribed by W ide et at. [19]. Antisera. Sheep and rabbits were immunized with Fc fragments from E myeloma ND in Freund’s complete adjuvant as described by J ohansson and Bennich [8] and specific antibodies were isolated by immunoabsorption on Sepharose 4B-IgE beads followed by elution at acid pH [2]. Gel filtration. The cow dander allergen extract was fractionated on Sephadex G-100 in 0.05 m Tris buffer, pH 7.6. The protein concentration of the effluent frac­ tions was determined by spectrophotometry using 1%, 1 cm 14.0. h280 nm Buffers. All radio-immunoassays were performed in a 0.05 M phosphate buffer, pH 7.4, containing 0.15 m NaCl, 0.3°/o human serum albumin, 0.1°/o Tween 20 and 0.1°/o NaN3. Saline containing 0.1°/o Tween 20 was used for washing. RIST [9] was used for estimation of the serum IgE concentrations. The y-globulin fraction of specific anti-Fc? from rabbit or sheep was coupled to CNBr-activated Sephadex particles. Approximately 25 «g of these particles were incubated with 50 /(I test serum diluted 1/10 and ,25I-label!ed IgE with an activity of 10,00020,000 cpm as previously described [9]. The concentration of IgE in the sample was calculated from its capacity to inhibit the binding of labelled IgE to particle-bound antibodies as compared to a reference serum with known IgE concentration.

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Immunological Techniques

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F oucard/B knnich/J ohansson/L undkvist

RAST [10, 19J was used to determine the presence of allergen-specific IgE anti­ bodies. 1 ml of the most concentrated allergen solution (1/10 w/v or 10,000 PNU or cow’s milk undiluted) was coupled to 100 mg CNBr-activated cellulose particles to make an allergen polymer complex (APC). Of this APC, 0.5 mg in 0.5 ml buffer was incubated with 50 «1 reaginic serum overnight at room temperature. After five washings, immunosorbent-purified, ,25I-labelled anti-Fce of either rabbit (rabbit RAST) or sheep (sheep RAST) origin, corresponding to approximately 10 ng AbN with an activity of 80,000-100,000 cpm, was added, and after a further overnight incubation period followed by five washings, the remaining radioactivity on the APC's was measured in a gamma scintillation counter. Assay of allergen. Inhibition experiments were performed by mixing 50 «1 of re­ aginic serum with 25 «1 of fractions from a gel filtration separation of cow dander allergen, y-globulin of various animal origins or human E myeloma protein ND, re­ spectively for 45 min at room temperature, after which the mixture was tested by RAST. Cow dander allergen was measured in every second fraction from the gel filtra­ tion by direct RAST [5] in the following way: 200 id eluate was coupled to 2 CNBractivated filter paper discs. The discs were used as APC in rabbit RAST against 2 cow dander reaginic sera. The allergen concentration was expressed as the amount of radioactivity (cpm) bound to the disc. Assay of BGG. The concentration of BGG in cow allergen extracts or their frac­ tions was estimated by their capacity to inhibit the sheep RAST reaction of serum T. F. to BGG as compared with known concentrations of BGG. Assay of antibodies to BGG. Anti-BGG antibodies were quantified by a ra­ dio-immunoassay which measured the capacity of the antibodies (in samples or standards) to bind radio-labelled BGG to insolubilized (particle-bound) BGG [11, 20]. 1 ml (2 mg/ml) BGG was coupled to 200 mg CNBr-activated cellulose particles and 0.1 mg in 0.5 ml buffer was incubated with 50 /d serum 3 h at room tempera­ ture. ,25I-labelled BGG (100,000 cpm) was added and the tubes were rotated at room temperature. After six washings, the remaining radioactivity on the particles was measured in a gamma scintillation counter. The results were expressed as a per­ centage of a reference serum (serum T. F.). Radioactivity uptake of more than 0.5“/o of that of the reference serum (corresponding to about twice the background activ­ ity) was regarded as positive. Assay of cross-reacting antibodies. 1 mg of y-globulin from cat, dog, goat, guinea pig, horse, pig and rabbit, respectively, was coupled to 100 mg cellulose par­ ticles. Of these complexes, 0.1 mg in 0.5 ml buffer was incubated with 5 0 //I human serum for 3 h at room temperature after which *25I-labelled BGG was added as de­ scribed above.

Results

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This study was initiated by the finding lhat one of us (T. F.) was strongly RAST-positive to a cow dander extract and to skimmed cow’s milk without being skin-test-positive to the dander extract or having clini-

Human Antibodies to BGG

Number of serum sample

815

control

Fig. I. The results of RAST analyses of 10 serum samples and a negative buffer control against a cow dander APC using a sheep anti-IgE (open bars) and a rabbit antiIgE (hatched bars), respectively. Note the positive reactions of sera 3 and 4, the only cow-reaginic sera in this experiment, when rabbit anti-IgE was used.

cal hypersensitivity to cow or cow’s milk. When the serum was tested by RAST using a rabbit anti-IgE instead of the sheep anti-IgE primarily used, a negative result was obtained. As seen from figure 1, several other sera from individuals with no apparent cow dander allergy also gave a positive result in RAST with the sheep anti-IgE (sheep RAST), but not with the rabbit anti-IgE (rabbit RAST). When IgE was assayed with the RIST technique using a sheep anti-IgE, higher values were often obtained than when using a rabbit anti-IgE, and in some sera the difference was pronounced (fig. 2). The antibody activity to the cow dander extract detected by sheepRAST did not decrease after heating serum T. F. to 56 °C for 2 h, indi­ cating that the antibodies giving the positive sheep-RAST reaction did not belong to the IgE class. The RAST reaction obtained with this serum could be completely inhibited, not only by most of the cow dander ex­ tracts, but also with highly diluted whole cow’s milk and isolated BGG or sheep y-globulin at low concentrations. y-Globulin from other animals at corresponding concentrations or human myeloma IgE inhibited the reac-

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Experimental Studies

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F oucard/B ennich /J ohansson/I. undkvist

Number of serum sample Fig. 2. The results of IgE determinations of 8 serum samples by RIST using a sheep anti-IgE (open bars) and a rabbit anti-IgE (hatched bars), respectively. The sera tested are not the same as in figure 1. Table I. Inhibition of the sheep RAST reaction of serum T. F. to whole cow’s milk allergen by y-globulin from different animal species and by human myeloma IgE (ND) Inhibiting substance

Uptake of radioactivity at different concentrations of the inhibiting substance, cpm 0.1 mg/ml 0.01 mg/ml 1 mg/ml

Bovine y-globulin Sheep y-globulin Rabbit y-globulin Horse y-globulin Pig y-globulin Myeloma IgE

217 427 34,531 17,386 32.700 26,692

233 798 35,730 23,589 33,000 33,550

19,756 3,558 35,066 29,000 33,961 23,700

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tion only slightly or not at ail (table I). A crystalline BSA preparation of 1 mg/ml gave a partial inhibition corresponding to that of a BGG solution of 0.1 /

Human antibodies to bovine alpha-globulin.

Int. Archs Allergy appl. Immun. 48 : 812-823 (1975) Human Antibodies to Bovine /-Globulin Occurrence in Immunological Disorders and Influence on Alle...
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