Determination of Specific IgG to Identify Possible Food Intolerance in Athletes Using ELISA

Abstract

Nutrition is considered one of the foundations of athletic performance, and post-workout nutritional recommendations are fundamental to the effectiveness of the recovery and adaptive processes. Therefore, at present, new directions in dietetics are being formed, focused on the creation of personalized diets. To identify the probable risk of somatic and allergic reactions upon contact with food antigens, we used the method of enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of IgG antibodies in the blood plasma of athletes against protein–peptide antigens accommodated in food. The study enrolled 40 athletes of boating and fighting sport disciplines. We found that the majority of the studied participants were characterized by an elevated IgG level against one or two food allergens (barley, almond, strawberry, etc.). Comparative analysis of the semiquantitative levels of IgG antibodies in athletes engaged in boating and fighting did not reveal significant differences between these groups. As a result, foods that are likely to cause the most pronounced immune response amongst the studied participants can be identified, which may indicate the presence of food intolerances. An athlete’s diet is influenced by both external and internal factors that can reduce or worsen the symptoms of a food intolerance/allergy associated with exercise. The range of foods is wide, and the effectiveness of a diet depends on the time, the place, and environmental factors. Therefore, during the recovery period (the post-competition period), athletes are advised to follow the instructions of doctors and nutritionists. An effective, comprehensive recovery strategy during the recovery period may enhance the adaptive response to fatigue, improving muscle function and increasing exercise tolerance. The data obtained may be useful for guiding the development of a new personalized approach and dietary recommendations covering the composition of athletes’ diet and the prevalence of food intolerance.

Dataset: ELISA data were deposited at the Mendeley Database, v2, Malsagova Kristina (2021), “Determination of IgG specific for the diagnosis of food intolerance in athletes”, https://data.mendeley.com/datasets/gmmkwh26r9/2, accessed on 11 November 2021.

Dataset License: CC-BY 4.0

1. Summary

Any atypical reactions after the consumption of food, such as food hypersensitivity, including food intolerances and food allergies or food aversions, are defined as adverse food reactions [1]. Food intolerance (FI) affects up to 20% of the world’s population, but the true prevalence remains unknown due to a data deficiency [2] and the lack of a standardized assessment for FI [3]. Routine clinical tests for FI can be conditionally divided into two types: cellular (the study of changes in blood cells) and humoral (the study of the level of antibodies specific to food antigens (AGs)) [4].

Cellular tests (CT) include the Cito test [5] and the Prime test [4] and were the first to be introduced into clinical practice for FI determination [6] The antigen leukocyte antibody test (ALCAT) method automatically calculates the linear dimensions or size distribution of leukocytes as the end markers of type 2 reactions between blood cells and food AGs [7,8]. The mediator release test (MRT) is based on flow cytometry using laser radiation scattering [9]. The main disadvantages of such methods include a high degree of subjectivity due to the visual assessment and manual curation of results, the poor reliability and reproducibility, the selectivity of affinity interactions, and the duration of the analysis. Currently, the enzyme-linked immunosorbent assay (ELISA) is the main method for the determination of the antigen–antibody reaction and is widely recognized in allergology and immunology due to its high selectivity, reproducibility, and specificity [10,11]. In ELISA, the diagnostic marker is a measure of antigen-specific immunoglobulins of class G (IgG) or subclass G4 (IgG4) [12]. In addition, approaches such as the immunological solid-phase allergen chip (ImmunoCAP-ISAC) and Western blot are based on the ELISA approach [3].

There are key pathophysiological differences between food allergies and food intolerances. A food allergy occurs as a result of an immunological reaction to a certain kind of food and ubiquitously affects the body system. In contrast, food intolerance is a non-immunological reaction initiated by a food component in a normally tolerated dose. However, the subsequent response occurs with the implication of cellular and humoral reactions of the immune system [13]. While a food allergy is usually mediated by IgE antibodies, a food intolerance is mediated by IgG antibodies [14,15,16]. Such differences between the two pathological conditions require divergent diagnostic and therapeutic strategies [14,15,16].

Patients with food intolerances who present with allergic symptoms are usually screened for specific IgE antibodies, which they might not bear. In this case, the time to produce IgG can vary from several days to several months [17]. Consequently, this group of patients may remain undiagnosed and suffer from frolicking symptoms, which are, in turn, associated with significant financial costs for both patients and healthcare resources. [17,18,19].

It is believed that IgG-mediated FI is caused by increased intestinal permeability, which leads to the release of food chemical compounds into the circulation and induces the production of specific IgG [15]. Increased generation of IgGs and low generation of anti-inflammatory cytokines (IL-10 and TGFβ1) are associated with irritable bowel syndrome [20].

The combination of dyes, preservatives, and hardeners in biologically active additives (dietary supplements) and food supplements entails an overload of the immune system and the consequent development of numerous chronic diseases. With a single instance of consumption of such products, the formation of a disproportionate number of immune complexes does not lead to a noticeable disturbance in the body’s functioning. However, the continual presence of such products in the diet leads to a significant increase in immune complexes, an extremely high loading on the immune system, and the emergence of many chronic diseases.

Current investigations show that the influence of the immune system on digestive processes is the basic factor affecting human health in general [21,22]. Ignoring this knowledge can lead to the development of such pathological conditions as obesity, chronic fatigue, type 2 diabetes mellitus, cardiovascular diseases, and allergies [14]. Moreover, drug therapy and traditional nutritional approaches are ineffective in this case. It has been shown that, with the development of a food intolerance, the use of an elimination diet, which involves the exclusion of foods causing undesirable reactions, leads to a significant improvement in symptoms [23].

Sports nutrition is a newly emerging multidisciplinary approach that combines elements of physiology, biochemistry, valeology, pharmacology, and sports pedagogy and aims to perform the following tasks:

(1)

improvement of the general and special performance of athletes;

(2)

acceleration of recovery and prevention of overstraining after exertion;

(3)

acceleration of adaptation to climate and normalization of biological rhythms when moving over long distances;

(4)

stabilization of the immunity of athletes;

(5)

regulation of body weight; and

(6)

maintenance of the optimal hydration balance and metabolism of basic minerals.

However, the use of food supplements with complex ingredients can lead to an immunological conflict, a deterioration in the state of the body, and, as a consequence, to a decrease in athletes’ abilities.

This study intended to determine food intolerances in a group of athletes (n = 40) by the method of solid-phase non-competitive indirect enzyme-linked immunosorbent assay (sandwich type). Due to the bivalent interaction of antibodies with antigene molecules, very strong cyclic complexes [24,25] can be formed when using the sandwich method. Being energetically more favorable, cyclic complexes are organized even at a low concentration of the antigen [24], which improves the analytical sensitivity meaningfully [24,25]. The sandwich-type immunoassay tests permit the achievement of a reliable analytical sensitivity and specificity due to the recognition of two different epitopes and the accommodation of strong cyclic complexes.

The assay was performed using a commercial ELISA kit for the semi-quantitative determination of allergen-specific IgG antibodies.

The study included blood plasma samples obtained from athletes enrolled by the A.I. Burnasyan State Research Center at the Federal Medical Biophysical Center of the Federal Medical and Biological Agency of Russia (https://fmbafmbc.ru/en/, accessed on 11 November 2021). All subjects provided informed consent before participating in the study.

2. Data Description

2.1. Study Participants

The study involved 40 athletes from various sports disciplines, including 22 athletes engaged in boating (6 females aged 21 ± 0.75 years old, 16 males aged 22 ± 0.8 years old) and 18 athletes engaged in fighting (males aged 27 ± 4.46 years old) (

Table 1. Main characteristics and attributes of the plasma sample donors.

Sample	Sport Discipline	Age	Sex	Weight, kg	Height, cm	Sample	Sport Discipline	Age	Sex	Weight, kg	Height, cm
1	Boating	22	male	90	198	30	Fighting	35	male	75	182
2	Boating	22	male	99	198	31	Fighting	29	male	67	167
3	Boating	21	male	85	186	32	Fighting	23	male	75	176
4	Boating	21	male	90	90	36	Fighting	23	male	97	181
5	Boating	21	male	92	190	37	Fighting	24	male	86	182
7	Boating	22	male	88	193	38	Fighting	26	male	97	191
8	Boating	22	female	73	178	39	Fighting	36	male	95	193
9	Boating	21	male	93	192	42	Fighting	25	male	69	173
10	Boating	21	female	82	182	46	Fighting	23	male	82	180
11	Boating	23	male	97	194	47	Fighting	36	male	100	183
12	Boating	21	female	85	186	50	Fighting	31	male	66	171
13	Boating	23	male	88	188	67	Fighting	29	male	81	185
14	Boating	20	female	80	180	68	Fighting	29	male	90	173
15	Boating	22	male	71	180	69	Fighting	27	male	135	189
16	Boating	23	male	90	184	70	Fighting	27	male	95	165
17	Boating	22	male	98	196	71	Fighting	22	male	115	180
21	Boating	21	female	80	176	74	Fighting	25	male	65	175
22	Boating	21	male	97	200	75	Fighting	28	male	92	179
25	Boating	22	male	92	194
26	Boating	20	female	59	158
28	Boating	21	male	87	189
29	Boating	23	male	93	198

).

2.2. Results of IgG-Specific Determination

The study population comprised six females and 16 males aged 21.6 ± 0.9 years old engaged in boating and 18 athletes engaged in fighting (males aged 27.6 ± 4.4 years old) (

Table 2. Demography.

	Boating		Fighting
	Male (n = 16)	Female (n = 6)	Male (n = 18)
Age	21.9 ± 0.8	20.8 ± 0.7	27.6 ± 4.4
Weight, kg	90 ± 6.6	76.5 ± 9.4	87.8 ± 18.1
Height, cm	179.3 ± 36.4	176.6 ± 9.7	173.6 ± 27.9

).

The result of the determination of IgG specific to 30 food antigens (Figure 1) demonstrates a narrow range of optical density (OD) scattering (from 0.07 to 0.15) and the presence of outliers. However, data heterogeneity is typically tracked within a wider signal range and achieves 0.2–0.4 OD (Figure 1).

In particular, the most pronounced signals, and, accordingly, immune responses were detected against beef protein, coconut, pomegranate, corn, wheat peanut protein, and whey proteins.

It should be noticed that the immune response does not depend on the type of sport discipline (Figure 1). The analysis of the ELISA signal did not demonstrate a gender-specific distribution of the signal (Wilcoxon test at p < 0.05) with the lowest p = 0.07. The most definite difference in the immune response between the boating and fighting groups was observed regarding the following food products: rice, peanut protein, sunflower, and strawberry. The variety of distinct food products is, probably, caused by both personal diet preferences and individual nutritional programs coordinated by the coaching staff to improve athletes’ abilities and recovery during the training period.

An intolerance to an almost complete set of allergens was revealed in subjects 25, 26, 67, and 68 (Figure 2). However, the disclosure of outliers might have been caused by a certain pathological condition (e.g., an inflammatory or chronic disease) at the sampling time, while a definite response to only one particular food compound (such as honey, spinach, or peanut protein) is more typical and was observed within the study (Figure 2).

3. Methods

Following overnight fasting, the blood samples were collected in 3.8% sodium citrate (IMPROVACUTER, Guangzhou Improve Medical Instruments Co., Ltd., Guangzhou, China) vacutainers from the cubital vein and centrifuged at 3000 rpm for 6 min at room temperature. Each plasma sample (500 μL) was split into two aliquots and transferred into polypropylene test tubes stored at −80 °C.

This study was approved by the independent Local Research Ethics Committee of the A.I. Burnasyan State Research Center at the Federal Medical Biophysical Center of the Federal Medical Biological Agency of Russia (Protocol no. 40 issued on 18 November 2020). All data presented in this study were obtained during the out-of-competition period.

In order to identify the risk of somatic–allergic reactions upon contact with antigens, we employed a set of ELISA reagents designed for research and educational purposes (LLC, Immunovet, Russia) that realize a semiquantitative determination of IgG specific to protein-to-peptide antigens in food. The assay is based on a non-competitive indirect sandwich-type enzyme immunoassay. One single kit is designed for the analysis of 30 allergens (the list of allergens is available at 10.17632/gmmkwh26r9.1(accessed on 11 November 2021) in three test samples and includes five calibration samples and one control sample.

This work was supported by the World-class Scientific Center “Digital Biodesign and Personalized Healthcare” within the framework of the national project “Science”. The Center brings together the efforts of several universities and research institutes as members of the scientific Consortium for the Digitalization of Healthcare and the Development of Approaches to Human Health Management over the 2020–2025 period.

The significance of the between-group comparison was determined using the Wilcoxon test at a p-value cut-off of less than 0.05. The inter-quartile (IQR) criterion means that all observations above q0.75 + 1.5 IQR or below q0.25 − 1.5 IQR (where q0.25 and q0.75 correspond to first and third quartile, respectively, and the IQR is the difference between the third and first quartile) are considered to be potential outliers. In other words, all observations outside of the interval are considered to be potential outliers. With the percentile method, all observations outside the interval between the 2.5 and 97.5 percentiles were considered to be potential outliers. Hampel’s filter consists of considering as outliers the values outside the interval (I) formed by the median, plus or minus 3 median absolute deviations (MAD): I = [median − 3 MAD; median + 3 MAD], where MAD is the median absolute deviation and is defined as the median of the absolute deviations from the data’s median ~X = median(X): MAD = median(|Xi − ~X|). Grubbs’s test allows us to detect whether the highest or lowest value in a dataset is an outlier. Dixon’s test was used to test whether a single low or high value was an outlier. Rosner’s test was used to detect several outliers at once.

4. Discussion

The present study aimed to assess the nutritional performance of age-aligned athletes belonging to boating or fighting sports disciplines. We performed an intergroup analysis of the 30 food allergen panel recommended for athletes. The designed test system targets 24 allergens of plant origin (soy protein, honey, grapes, citrus fruits, strawberries, etc.) and six allergens of animal origin (animal protein, yeast protein, etc.). We found no significant intergroup (between sport disciplines) differences (at a cut-off of p < 0.05) in FI and allergy reactions, and no significant difference if the immune response regarding gender and age matters.

We determined that studied participants predominantly only responded to one or two food allergens (Figure 2). Additional questioning of the studied participants for self-diagnosis of a possible food intolerance to certain foods revealed a kind of discomfort and unwanted symptoms observed after (participant #1 (male), #8 (female), and #14 (female); all athletes were involved in the boating discipline): eating more than five tangerines at one meal, which resulted in a rash on the face (participant #14) and eating fried meat, which was accompanied by nausea (participant #1 and #8).

A proper balance between regular training intensity, rest, recovery, and fit-for-purpose nutritional support is key to maximizing and optimizing athletes’ performance.

Dysfunction of the intestinal barrier increases the risk of uncontrolled immune responses to food and microbial antigens, as well as to environmental toxins [26]. Such maladies stimulate the production of IgGs specific to food antigens. If the “dietary” antibody level keeps rising, this may eventually result in IgG-mediated food intolerances to that particular food [27].

A recent longitudinal study showed that elimination diets based on an IgG-mediated food intolerance led to significant improvements in gastrointestinal discomfort symptoms and exercise performance [28].

Most athletes did not notice a clear deterioration in their condition after eating certain foods. The reaction to tangerines (participant #14) may have been caused by a manifestation of an allergic reaction. Feeling unwell after eating fried meat (participant #1 and #8) has been associated with the type of food preparation, intolerance to the foods used in its preparation (oil, spices, etc.), and/or a violation of the digestive process.

In dietetics, new directions are actively being developed that are focused on personalized dietary programs [29]. Such directions include (1) genetic studies that aim to determine the predisposition to a particular type of food and the risk of foodborne illness [30]; (2) studies on the human microbiota diversity, which characterizes the digestion performance and the state of the intestinal barrier [31]; and (3) studies on the individual immune response to food antigens that cause changes in food tolerance and the reactivity of the adaptive immune response.

There is a high level of food intolerance among athletes. Some studies have linked the FI in professional athletes with excessive physical activity [29]. To assess the impact of a food intolerance on the athletic performance and health of elite athletes, a three-month experimental longitudinal study was conducted [29]. Based on the obtained result, an individual elimination diet was drawn up. Blood tests showed a reduction in post-diet food intolerances in each athlete, indicating that the elimination diet significantly improved the athlete’s well-being, resulting in a more rapid decrease in heart rate after cardiopulmonary testing.

Symptoms of food intolerance can vary and include gastrointestinal upset (bloating, loose stools, abdominal pain) and/or extraintestinal symptoms (fatigue, headaches, and cognitive problems) that appear several hours or days after eating. Some of these symptoms coincide with those of irritable bowel syndrome and exercise-induced functional gastrointestinal disorders [32]. Given the ambiguous nature of food intolerance, its diagnosis, generally, is carried out by athletes on their own, followed by the cancellation of certain foods or a group of foods [33].

5. Conclusions

Currently, a wide range of testing methods can be used to determine the prevalence of intolerance to certain foods and/or their components. In the present study, we examined the possible food intolerance amongst 40 athletes belonging to boating or fighting sport disciplines. The majority of study participants had an increased immune response to one or two food allergens, while some subjects may have had an intolerance to several food products. We did not observe a correlation between the immune response and anthropometric characteristics or the kind of sport discipline under consideration (boating or fighting).