The anti-penicillin antibodies levels in sensitive and insensitive people to intradermal skin test

Abstract

BACKGROUND:

The hypersensitivity reaction to penicillin is a public health problem. Immunological responses to penicillin and other beta-lactam antibiotics can be classified into immediate and non-immediate responses. The immediate hypersensitivity is mediated by IgE; however, the non-immediate sensitivity is facilitated by other isotypes of antibody or T lymphocytes.

OBJECTIVE:

This research detected the non-IgE antibody value against penicillin in allergic and normal people.

METHODS:

Thirty-eight samples from patients with positive or negative intradermal skin testing results of penicillin allergy were included in this study. The total antibody and IgM levels against penicillin G were defined by in-house ELISA test.

RESULTS:

The results showed a significant ( $P<$ 0.05) elevation in total immunoglobulin and non-IgM anti-penicillin antibody of sensitive groups; however, the anti-penicillin IgM was significantly greater in non-sensitive peoples.

CONCLUSIONS:

Although the sensitized people to penicillin cannot be certainly detected with the total antibody, specific IgG and IgM value against penicillin, these values are good indicators for prediction of immediate and late response of the immune system to penicillin.

Keywords

Penicillin sensitivity antibody ELISA human

1. Introduction

Beta-lactams antibiotics are mentioned as one of the most frequent causes of immediate and non-immediate hypersensitivity reactions to drugs [1, 2]. The $\beta$ -lactam antibiotics covalently bind lysine residues on protein [3]; the antibody responses against hapten are generated whenever hapten cross-link with a carrier protein to make it immunogenic. It is thought that T cells recognize the carrier determinants of an antigen, whereas B cells are thought to recognize the haptens [4]. This drug-protein binding initiates the immune responses [5]. Immediate allergy to penicillin injection, which is determined as one of the public health problems, is the most commonly reported hypersensitivity reaction [6]. The incidence of immediate immune response to penicillin varies from 1% to10%; while skin testing (PST) could not detect many of these patients [7].

The onset of the hypersensitivity symptoms following penicillin and other beta-lactam antibiotics administration may accrue as immediate or non-immediate. The immediate signs are mediated by IgE and usually occur in length of one hour after injection while non-immediate symptoms are non-IgE-mediated and generally happen more than 60 minutes to several days following administration. Determinations of these immediate and non-immediate reactions are generally difficult mainly when the immediate allergy tests are negative in condition of a normal clinical history [8].

The in-vivo or in-vitro methods based on the IgE mediated reactions are generally utilized for detection of the allergic people; the primary diagnosis is based on a clinical history including time and type of the reactions [9]. The approximately accurate detection of the type I and IV hypersensitivity reactions could be provided by skin testing to beta-lactams. Although recognized as safe, systemic reactions can occur in 0.7% to 11% of those with positive test results after the skin test [10]. On the other hand, the lack of commercially available tests for determining the anti-penicillin antibody, the time required to obtain results and interpretation, and low confidence of practicing physicians have contributed to the limited use of penicillin skin testing. Finding the correlation of the circulatory of anti-penicillin antibody levels and the skin test result, as well as prediction of the non-immediate hypersensitivity reactions incidence and estimation of the effectiveness of the beta-lactam treatment could be accessed by determining the other antibodies rather than IgE antibody. This study aimed at designing an ELISA for measuring the level of total antibody and IgM against penicillin in patients with the positive and negative results of intradermal skin test.

2. Materials and methods

2.1 Sample providing

The serum samples were taken from patients on whom physicians administrated penicillin in different hospitals in Khozestan, Iran. The 10,000 U/mL of potassium G penicillin was prepared in addition to isotonic sodium chloride to a vial containing 1 million units. The diluted reagent at value of approximately 0.1 ml was injected to the intraderm of volar surface of the patients’ forearms. The penicillin skin test was recorded as negative if no alteration was observed on the tested site; the result was considered positive when there was a papule elevation, erythema and pruritus, with a diameter at least 2 mm. Then, the total of 18 and 20 serum samples were prepared from patients with positive and negative skin test results respectively.

2.2 The coupling procedure

Molecules of penicillin and bovine serum albumin were cross-linked together using glutaraldehyde [11]. Penicillin G potassium (Sigma, P7794) 1 ml (10 mg/ml) in a solution containing 0.5 M Ethylene diamine tetra acetic acid (EDTA) was incubated with 1.0 ml of 5 mM GA for 3 hours with stirring. 100 $\mu$ l of bovine serum albumin (5 mg/ml) in phosphate buffered saline (PBS) was added to this mixture, and the entire mixture was incubated for 24 hours with slow stirring at room temperature. The coupling mixture was dialyzed for 72 hours against several changes of PBS at 4 ${}^{\circ}$ C.

2.3 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized according Laemmli’s method [12] using 11% polyacrylamide gel. The samples were mixed in a 62.5 mM Tris-HCl sample buffer (pH 6.8) containing 2.5% SDS. An aliquot (25 $\mu$ l) of the sample solution was then loaded onto each well, and the gel was run at a constant voltage (100 V) for 90 min, using SDS running buffer. After electrophoresis, the gel was stained with Coomassie brilliant blue G-250.

2.4 Antibacterial test

The presence of the penicillin in produced conjugates was tested by using the antibiogram method [13]. The Bacillus anthraces bacteria were prepared from the archive of microbiology department of veterinary medicine at Shahid Chamran university of Ahvaz, Iran; and were cultured in blood agar plate media. The 10 $\mu$ l of prepared conjugates of penicillin was loaded on the plate. The penicillin G 1 mg/mL and phosphate buffered saline were loaded onto the other plates as positive and negative control plates respectively. The presence of the penicillin in produced conjugates was interoperated by formation of a clear ring or zone of inhibition.

2.5 Immunization

The conjugated penicillin to BSA was used to immunize two rabbits; the methods of immunization were the same as those described previously [14]. The immunized blood was directly collected and allowed to clot. Serum was pipetted out, centrifuged at 1500 RPM for 10 minutes, isolated in a sterilized vial and finally stored in 4 ${}^{\circ}$ C. After the confirmation of good titers (checked by passive hemagglutination), sufficient amount of sera were prepared.

2.6 Passive hemagglutination

Sheep Red Blood Cells were collected in the tubes containing 45 mmol/L of sodium citrate and centrifuged (for 6 min at 300 $\times$ g); SRBCs were washed with saline and were suspended to a final concentration of 5% in saline. The SRBCs were incubated with tannic acid (0.05 g/ml) for 30 minutes at 37 ${}^{\circ}$ C and then washed three times with saline. The sensitized SRBCs were treated with penicillin or bovine serum albumin (as negative control) and incubated overnight at 4 ${}^{\circ}$ C. The sensitized SRBCs were then washed three times with saline containing 1% bovine serum albumin, and suspended in the same buffer. Hemagglutination was applied by two-fold serial dilutions of all serum samples in “U” botton 96-well plates. The sensitized SRBCs were added to a 1:1 vol/vol rate to serum and plates were incubated for 90 minutes at 37 ${}^{\circ}$ C. The sensitized SRBCs with BSA or SSRBCs $+$ dilution buffer were used as negative controls [15].

2.7 ELISA for antibody titer against penicillin

Flat-bottomed ELISA plates were coated overnight with conjugated penicillin (1 $\mu$ g/well) in 1 M sodium bicarbonate pH 9.6. The plates were then washed three times with phosphate-buffered saline (PBS)/0.05% Tween 20 (PBST) and blocked for 2 hours at 37 ${}^{\circ}$ C with 2% of Tween 20. A part of samples were treated with 0.1 M beta-2mercaptoethanol (2ME); the samples got dispensed in duplicate wells. The plate was incubated for 1 hour at the room temperature. After being washed for three times with PBST, the purified specific rabbit’s IgG against penicillin (2 $\mu$ g/well) were added to each well. After incubation and washing, the horseradish peroxidase-conjugated goat anti-rabbit IgG (Immuno Chemistry Technologies company, USA, HRP Affi Pure Goat anti-Rabbit IgG Fc, Catalog Number: 6293) which was diluted as 1/10000 was added to each well; the plate was incubated for the next 1 hour in the room temperature. The unbound antibody was removed by washing with PBST. Finally, the substrate solution of TMB was added to the wells. The reaction was stopped with 3 M sulfuric acid before being read by the absorbance at 450 nm. The standard curve was created by using serial dilution of specific IgG against the tested samples. The specific IgG was prepared by extraction of the total IgG. The IgG fraction was purified by performing Ion Exchange chromatography on DEAE-C column (Sigma, Product Number: D3764) according to Hay and Westwood [16]. The specific IgG was purified by affinity chromatography according to Khoobdel et al. [17]; modifying it, the column was prepared by conjugating 20 mg of penicillin with 5 ml of activated CH Sepharose 4B (Sigma-Aldrich, Product Number: 4B200). The individual specific antibody against penicillin (A) was calculated as in below:

A ( $\mu$ g/ml) $=$ 2 – value of the banded specific IgG of the rabbits

2.8 Statically analysis

In this study, statistical significance of the differences between the study species or the target isotypes level was assessed using the one-way analysis of variance (ANAVA). In all statistical analyses, P value of less than 0.05 was considered significant.

3. Results

In this study, the penicillin was coupled to bovine serum albumin (BSA) by a short cross-linker reagent (glutaraldehyde, GA). The optimum condition along with thorough dialysis to remove unbound penicillin resulted in good coupling efficiency of penicillin to bovine serum albumin as shown by SDS-PAGE (Fig. 1), antibacterial (Fig. 2) and passive hemagglutination test (Fig. 3). The immunized rabbits gave an equivalent titer of 1/640 by passive hemagglutination test since positive result at 1/320 titer was detected after treatment of serum with $\beta$ -2 mercaptoethanol. The obtained hapten-protein conjugates had hapten molecular weight on carrier protein coupling ratio (weight of penicillin molecules per carrier) 20:1.

Figure 1.

The SDS-PAGE analysis shows that penicillin conjugates to BSA. 1. BSA, 2 and 3. conjugated penicillin-BSA. Arrow shows bovine serum albumin () band and the conjugated penicillin ().

The presence of anti-penicillin antibody was detected by ELISA in sero-positive and sero-negative cases. The results showed a significant elevation of total immunoglobulin ( $P<$ 0.032) and non-IgM ( $P<$ 0.001) against penicillin in the allergic people; however the anti-penicillin IgM was significantly ( $P<$ 0.009) elevated in non-sensitive people. Also in contrast to insensitive people, the value of non-IgM anti-penicillin and IgM anti-penicillin was significantly different ( $P<$ 0.001) in sensitized people (Table 1).

Figure 2.

Passive hemagglutination test detects anti-penicillin antibody in serum of immunized rabbits. Columns 1 and 2 show the anti-penicillin titer of the immunized rabbits; column 3 shows the non-sensitized red blood cells as negative control samples.

Figure 3.

The anti-biogram assay analysis shows that penicillin conjugates to BSA. a. Phosphate buffered saline as the negative control; b. Penicillin as the positive control; c and d. conjugated penicillin to bovine serum albumin.

Table 1

The level ( $\mu$ g/ml) of the total antibody and IgM against penicillin is shown in tested people by intradermal skin test of penicillin. The uppercase and lowercase letters show the significant difference between the people with positive and negative reaction

Skin test	Main Ig		Main non-IgM		Main IgM
	value $\pm$ sd		value $\pm$ sd		value $\pm$ sd
Positive	1.8 $\pm$	0.39 ${}^{\text{A}}$	1.29 $\pm$	0.32 ${}^{\text{B}}$	0.32 $\pm$	0.08 ${}^{\text{Cb}}$
Negative	1.35 $\pm$	0.42 ${}^{\text{a}}$	0.57 $\pm$	0.18 ${}^{\text{b}}$	0.59 $\pm$	0.26 ${}^{\text{cb}}$

Ig: Immunoglobulin, sd: standard deviation.

4. Discussion

The immunogenicity of the conjugated antigen is dependent on the hapten density on the carrier protein. The optimum ratio of BSA-penicillin conjugation was obtained on 1/20 proportion. Bovine serum albumin has 26 available surface lysines that could bind up to 26 hapten molecules. Because the various conjugation presses have different effects on conformational change of the carrier protein, diverse ratios were reported previously [4]. Therefore, higher ratio might have resulted in a low affinity IgM response greater than that of IgG. Several drug classes, including the $\beta$ -lactam antibiotics, bind no covalently at specific sites on albumin [18]. In this study, EDTA and GA were utilized as short cross-linker reagents to aid penicillin linking to BSA. This method produced a stable cross-linking of penicillin with BSA along with acid amide and carbons bridge formation with satisfactory IgG responses subsequent to immunization.

Skin test had more sensitivity for penicillin hypersensitivity mediated by IgE than in vitro test; nevertheless approving a negative skin test needed more additional approach. The in-vitro test could facilitate detection of truly at risk individual for clinically significant IgE-mediated allergy [9]. In the present study, the value of anti-penicillin IgG and anti-penicillin IgM was significantly different in sensitized people compared to insensitive individuals. These finding could be helpful in approving the skin test results.

The presence of the IgG and IgM antibodies against penicillin might have caused hypersensitivity reactions. The manifest outcome of the penicillin injection could range from any types of the hypersensitivity reactions. The reported effects of the penicillin administration are included as vasculitis, purpura and petechiae often involving the lower extremities, fever, urticaria, arthralgias, lymph adenopathy, elevated erythrocyte sedimentation rate, and low complement levels [19]. The present research confirmed the occurrence of types two and three of hypersensitivity symptoms following penicillin injection because of the elevated levels of the anti-penicillin IgG in sensitive individuals and presence of the anti-penicillin IgM in normal people.

In addition to the drugs, most of food and drink products are polluted with penicillin [20]. Generally, IgM antibody titers are detected in normal people. These anti-penicillin antibodies may cause pharmacologic inhibition. Klaus et al. reported a significant difference in anti-penicillin titers of IgG compared to IgM across all age groups, with the IgG level being always higher than IgM [21]. According to the obtained results in this study, the normal people showed the same IgG and IgM titers. However, difference in the time and location of the study might have been an effective factor in individuals’ reaction to penicillin which is important in immune responses to various drugs.

In spite of the protective role of the IgG in allergic diseases, because of high dose of drugs administration, the IgG and IgM circulatory could have developed an immune complex and caused the third class of the hypersensitivity reactions. In addition, it may have caused induction of the cell cytotoxicity reaction [22] against antigen attachment to the cell surface membrane. The current study suggested that the presence of the high value of specific IgG against penicillin might predict the outcome of injection in vulnerable people. It’s valuable to note that, the IgG was previously reported as the anaphylaxis promoting agents [23, 24]. For example, researchers investigated the isotypes and specificities of anti-penicillin IgG and IgE [25]. The effects of IgG antibodies were also reported in the immediate allergic reactions to different determinants of benzyl penicillin, amoxicillin, and ampicillin [26]. However, the protective role of specific IgG in the development of anaphylaxis could not be confirmed. Similar to the current study, researchers found higher IgG levels of specific to various allergen components in allergic subjects [27]. Moreover, a higher prevalence of anti-penicillin IgM and IgG were detected in patients with negative skin tests but typical symptoms, especially the individuals who claimed to have an adverse reaction to penicillin [28].

The humeral immune responses to penicillin were investigated by Lee et al.; they reported a positive correlation between total dose of injection and prevalence of IgG titers [29]. Also a good correlation was reported between skin test and specific anti-penicillin IgE antibodies in children considered to have adverse reactions to penicillin [30]. The significant difference in IgG level of sensitive and insensitive people showed the possible role of the IgG in occurrence of the immediate hypersensitivity. In this vein, an allergy prevention trial also reported an increased risk of egg allergy in relation to elevated anti-ovalbumin IgA and IgG serum levels [31]. Also, most of the investigated samples by Chandra et al. had anti-penicillin IgG, IgM, IgA, and IgE while infants had a high proportion of IgM more than their mothers [30]. Siman et al. suggesting that measurement of allergen-specific IgG antibodies is an important laboratorial parameter [32], then the IgG attachment to allergen-IgE complexes can result in complement activation which may affect allergen processing and presentation, hence influence the allergic response [33].

Finally, the presence of the cross-reacting antibodies should be taken into consideration. All beta-lactams share a four-member ring that may cross-react with each other [34]. It is in spite of the Amali et al. results reporting that fine specificity of piperacillin-specific IgG antibodies can circulate in patients with hypersensitivity, without binding to other $\beta$ -lactam protein conjugates [35].

However, the sensitive people to penicillin could not be certainly detected with the total antibodies, specific IgG and IgM levels against penicillin; these values are good indicators for prediction of immediate and late response of the immune system to penicillin. Further research should be carried out in this area to clarify the immune complex formation or the other late hypersensitivity induction by using penicillin. Finally it should be mentioned that some of the antibiotic resistance may be due to the immune responses to the drugs, not the microbial resistance against the antibiotics.

Footnotes

Acknowledgments

This study was financially supported by the Shahid Chamran University.

Conflict of interest

The authors declare that have no conflict of interest.

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