Antigens in penicillin allergy. III. Antigen and antibody levels in mice treated with pure and contaminated penicillins.

Using a radioimmunoassay, it was shown that commercially available ampicillin preparations often contain penicilloylated high molecular weight impurities. These possess immunological activities and stimulate penicilloyl-specific antibody formation in mice treated according to a therapeutic schedule. Using purified and experimentally contaminated preparations it was also found that exposure of the animals to Escherichia coli and Bordetella pertussis bacteria could increase the antibody formation to small amounts of impurities. In addition, penicilloylated antigen could be recorded in serum from treated animals. The antigen formed by penicilloylation in vivo, however, was very weak and did not induce much antibody formation when injected together with Freund's adjuvant in mice or rabbits.     

Solid-phase radioimmunoassay for detection of antibodies to myelin basic protein.

A solid-phase double-antibody radioimmunoassay was developed for the detection of anti-myelin basic protein (BP) in sera. Antigen was adsorbed to glass test tubes, reacted with rat anti-BP sera, followed by 125I-labeled rabbit anti-rat IgG. This assay was capable of detection of specific antibody at low nanogram per ml levels, was technically simple, and the results correlated well with established procedures.     

A radioimmunoassay for the detection of adrenal autoantibodies.

A solid phase radioimmunoassay for adrenal antibodies is described. In the assay plastic tubes coated with adrenal microsomes (100 micrograms/ml) were incubated with human sera diluted from 1:50 to 1:5000 and the retained antibodies detected by subsequent incubation with 125I-labelled protein A. The method was precise over the range of serum dilution of 1:250 to 1:5000. In the group of 30 patients with Addison's disease 19 had positive results in adrenal antibody radioimmunoassay (RIA). Comparative studies of RIA and immunofluorescence (IFL) revealed that there was partial correlation of adrenal antibody results in patients with high titre antibodies whereas RIA usually was more sensitive than IFL in patients with low titre antibodies. Computerized tomography (CT) displayed bilateral adrenal atrophy in most patients who had adrenal antibodies. On the other hand, patients with low RIA results and negative IFL antibodies had predominantly adrenal calcifications on scans.     

Tests for penicillin allergy in man: I. Carrier effect on response to penicilloyl conjugates

The value of using benzylpenicilloyl (BPO) conjugates rather than benzylpenicillin (B.Pen.) itself in skin tests and in in vitro diagnostic tests for penicillin allergy in man is assessed. The effect of various carriers on the outcome of these tests has also been investigated in order to find the most appropriate. Skin tests with B.Pen. and BPO conjugates (with polylysine, PL, and human serum albumin, HSA) in penicillin allergic patients were positive in 36 per cent and up to 50 per cent respectively. The two carriers used were equally effective. Negative results were obtained in the non-allergic control subjects. For in vitro studies two tests were selected on the basis of their well established value, the lymphocyte transformation test (LTT) and histamine release from sensitized leucocytes (HRL). In the HRL test BPO conjugates with PL, HSA, bovine serum albumin (BSA) and bovine gamma globulin (BGG) were also compared with B.Pen. The BPO conjugates were all more effective than B.Pen. and the proportion of patients giving positive results with these conjugates was much higher than with B.Pen. (up to 86 per cent compared with 29 per cent). The rank order of effectiveness of the various carriers as judged from maximal histamine release by various penicilloyl conjugates was PL<BSA<HSA<BGG, BGG being the most effective. In the LTT, where BPO:PL, BPO:HSA, BPO:BGG as well as BPO human gamma globulin have been used, the BPO conjugates were also more effective than B.Pen. but the difference was relatively less marked than in HRL test (positive results being obtained with conjugates in up to 92 per cent of patients as compared with 57 per cent with B.Pen.). The rank order of effectiveness of the carriers in the LTT, as judged by comparing the maximum response obtained with each BPO-protein conjugate with the maximum response obtained with BPO:PL, was PL<HSA<HGG<BGG, BGG being the most effective. However, BPO:HGG was effective in lower concentrations. The HRL and LTT were negative in nine out of ten non-allergic subjects, and in control experiments with the carrier molecules alone.     

A survey of antibiotic prescribing and knowledge of penicillin allergy.

BACKGROUND: Cephalosporins can cause allergic reactions in patients with penicillin (PCN) allergy. Physicians' prescribing habits for patients with PCN allergy can vary. OBJECTIVES: 1) Survey community and academic physicians, students, residents, and allergists on their tendencies to prescribe cephalosporins and/or perform PCN skin testing in patients with different histories of PCN allergy. 2) Evaluate PCN allergy knowledge in these groups. METHODS: A questionnaire consisting of four case scenarios and five true/false questions on PCN allergy was distributed at various conferences and by mailings. RESULTS:Three hundred seventy-eight completed surveys were analyzed. Given a patient with a history of rash with PCN, an equal number of allergists and nonallergists (36%) prescribed cephalosporins, although there was a difference between pediatricians (56%) and internists (22%). Given a history of PCN anaphylaxis, no allergists but 11% of nonallergists prescribed a cephalosporin. Skin testing was infrequently requested by nonallergists. The correct response rate for the true/false questions was 89% for allergists, community (63%) and academic (67%) physicians, pediatricians (61%), internists (67%), residents (68%), and students (68%). Pediatric residents had the highest (74%) and community pediatricians the lowest (59%) correct response rate. CONCLUSIONS: There is marked variation in prescribing cephalosporins and in requesting PCN skin testing in patients with varied histories of PCN allergy. The survey results indicate a need for increased PCN allergy education.     

A solid-phase radioimmunoassay for detection of human antibodies. I. Measurement of IgG antibody to bee venom antigens.

A solid-phase radioimmunoassay (SPRA) has been developed to measure IgG antibodies to bee venom (BV) and phospholipiase A2 (PLA) in human sera. The principle of the test is similar to that of the radioallergosorbent test (RAST) measuring IgE antibody. Cyanogen-bromide-activated paper discs coupled with BV or PLA followed by supplementary coupling with human serum albumin were incubated with standard or test sera, washed, and incubated with 125I-labeled anti IgG. The serum levels of the IgG antibody have been temporarily expressed in arbitrary units. the reaction between the antigen and antibody was specific and the results were reproducible. Sera from 19 beekeepers, 42 beesting-sensitive patients and 20 blood donors (controls) were assayed by the SPRA. IgG antibodies to BV and PLA could not be detected (less than 4 U/ml) in all control sera, in 25 of the 42 patients and in one beekeeper. The IgG antibodies in 17 patients ranged between 5 to 58 U/ml (mean 7.6 U/ml), and in the 18 beekeepers ranged between 8 to 160 U/ml (mean 59 U/ml).     

Antigens in penicillin allergy. II. The influence of the number of penicilloyl residues on the antigenicity of macromolecules as determined by radioimmunoassay (RIA), passive cutaneous anaphylaxis (PCA) and antibody induction.

The present communication reveals a relationship between the epitope density of penicilloylated protein antigens and their antigenic activities in a radioimmunoassay (RIA), in passive cutaneous anaphylaxis (PCA) and in inducing antibody formation in mice. In the RIA and PCA a critical number of 2-4 penicilloyl residues per protein molecule was noted. At this level small changes in the number of substituents considerably influenced the antigenic activities. The molecular weight and the nature of the carrier proteins, myoglobin, bovine serum albumin (BSA) and dimeric BSA also affected the threshold concentration for efficient antigenic activity. The results with the RIA and PCA were significantly correlated to each other. Using penicilloylated BSA as immunizing antigen in mice it was found that an epitope number higher than 11 penicilloyl residues per protein molecule induced significant antibody formation after a single injection. Antigens with a lower degree of penicilloyl substitution were less immunogenic. An antigen carrying 0.6 penicilloyl residues per BSA molecule did not induce penicilloyl-specific antibodies even after three injections. The capacity of heavily penicilloylated proteins to induce and elicit penicillin allergy as revealed by the present results stresses the importance of limiting their presence in penicillin preparations.     

Female sex as a risk factor for penicillin allergy.

BACKGROUND: Identification of risk factors is an integral part of a physician's evaluation of a patient. OBJECTIVE: To determine whether female sex is an independent risk factor for penicillin allergy. METHODS: Rates of positive penicillin skin test (PST) results, according to sex, were determined in patients with a history of penicillin allergy undergoing penicillin allergy evaluation with major and minor determinants of penicillin between June 1, 2002, and June 30, 2004. Univariate and multivariate logistic regression analyses were used to calculate unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for sex differences in the rates of positive PST results. RESULTS: Of the 1,921 patients, 1,759 underwent PST and 157 did not; 5 medical records were not available for review. The mean patient age was 60 years. Sixty-four patients (4%) had a positive PST reaction; of these, 53 (83%) were females and 11 (17%) were males (OR, 3.6; 95% CI, 1.9-7.2; P < .001). In a multivariate logistic regression analysis adjusted for age, history of multiple drug allergies, and elapsed time from the initial penicillin adverse drug reaction to PST, female sex again had a significant risk of a positive PST reaction (OR, 3.2; 95% CI, 1.6-6.7; P = .001). CONCLUSION: A greater risk of penicillin allergy exists in association with female sex in patients with a history of penicillin allergy.     

A radioimmunoassay for detection of circulating antibodies reacting with the hepatic asialoglycoprotein receptor protein

To determine whether circulating antibodues against the liver-specific membrane lipoprotein complex (LSP), which occur in a number of liver disorders, are directed at the hepatic asialoglycoprotein receptor protein (hepatic lectin) — recently shown to be a constituent of LSP — a radioimmunoassay for anti-hepatic lectin antibodies was developed based on the use of protein A in situ on staphylococcal cells to precipitate ¹²⁵I-labelled rabbit hepatic lectin/anti-lectin immune complexes. The assay was used to test sera from 30 patients (15 anti-LSP-positive and 15 anti-LSP-negative) with autoimmune chronic active hepatitis (CAH), hepatitis B virus-positive CAH or primary biliary cirrhosis. Anti-lectin antibodies (at titres of 1 : 200 to 1 : 1600) were found in all 15 anti-LSP-positive sera and were not detected in the 15 anti-LSP-negative patients. Hepatic lectin is a highly purifiable, liver-specific, hepatocellular surface component and the anti-lectin assay (which proved sensitive, reliable and easy to perform) will permit further exploration of the role of autoimmune mechanisms in the pathogenesis of various liver diseases.     

Four-layer radioimmunoassay for detection of adenovirus in stool.

A four-layer antispecies radioimmunoassay (RIA) was developed for the detection of adenovirus in stool specimens. Polystyrene beads were used as the solid phase, anti-adenovirus guinea pig immunoglobulin (1 microgram per bead) was used as the primary antibody, anti-adenovirus rabbit immunoglobulin (16 micrograms/ml) was used as the secondary antibody, and 125I-labeled sheep anti-rabbit immunoglobulin was used as the indicator antibody. A highly purified, crystallized adenovirus type 2 hexon antigen was used as the immunizing antigen for the production of hyperimmune sera. The sensitivity of the test was 1 ng of hexon protein per ml. Each of the 13 stool specimens positive for adenovirus by electron microscopy was positive for adenovirus by the RIA. Of 200 nonconcentrated stool specimens negative by electron microscopy, 14 additional specimens were positive by the RIA, increasing the detection rate from 6% by electron microscopy to 13% by the RIA. A confirmatory test was done on the RIA-positive, electron microscopy-negative specimens, and the test indicated a true specific result with each specimen. A confirmatory test was also done on each specimen with a low positive counts per minute value. The specificity of the RIA was further demonstrated by the fact that a positive result was found with only 3 of 295 specimens positive by the rotavirus RIA. In two of these three specimens, adenovirus and rotavirus were also detected simultaneously by electron microscopy, and the third specimen was from a patient with serological evidence for a dual infection. The adenovirus and rotavirus RIAs are now in a routine diagnostic laboratory, and in the 307 stool specimens tested during the first 5 months, the positive rate was 32% for rotavirus and 9.5% for adenovirus.     

Solid-phase radioimmunoassay for the detection of rotavirus.

A solid-phase radioimmunoassay method has been developed for the detection of rotavirus in the form of a purified antigen and in stool. The parameters of the radioimmunoassay were examined and optimized to give high sensitivity and same-day results. Compared with electron microscopy, the assay is up to 10 times as sensitive for detection of the virus in stool and up to 128 times as sensitive for detection of a purified virus antigen. In a field study on stool specimens it was at least as efficient as electron microscopy.     

Detection of penicillin allergy of the immediate type by radioimmunoassay of reagins (IgE) to penicilloyl conjugates

Reaginic antibodies (IgE) to penicilloyl were detected by an in vitro method, a radioimmunological technique (RAST), in the sera of nine patients out of eleven with a recent history of penicillin hypersensitivity of the immediate type. Provocation tests in two patients with negative RAST were negative as well as in twelve patients having had a possible penicillin hypersensitivity reaction 7 months to 14 years ago. Results from skin tests and RAST agreed. Skin tests and RAST reactions were negative for thirty-two patients with a history of penicillin reactions of the delayed type. The results of this study indicate that the RAST is a valuable alternative to the more dangerous intracutaneous test with penicilloyl-polylysine for-detection of penicillin hypersensitivity of the immediate type.