Characterization of anti-soybean agglutinin (SBA) IgY antibodies: a new strategy for neutralization of the detrimental biological activity of SBA

Anti-soybean agglutinin (SBA) IgY was produced, and its potential to neutralize the haemagglutinating activity of SBA in vitro was tested. Thirty-five-week-old hens [treatment (n = 5) and control (n = 5)] were immunized with SBA or injected with saline 4 times every 15 days. Eggs were collected after the last immunization, and IgY was extracted using the polyethylene glycol (PEG) method. Serum anti-SBA IgY titres in immunized hens increased after the first immunization and reached a plateau between days 45 and 60. In contrast, specific IgY titres in the control group remained at basal levels throughout the evaluation. Average IgY titres were significantly higher in the treatment group on days 15, 30, 45, and 60. Total IgY content in the egg yolk extract was 38.7 ± 1.6 and 37.7 ± 1.5 mg/ml for the treatment and control groups, respectively. The specific anti-SBA IgY titer detected in the egg yolk extract was significantly higher (p < 0.001) for hens in the treatment group compared to the control group, with OD450nm values of 0.98 ± 0.05 and 0.058 ± 0.02, respectively. The specificity of anti-SBA IgY was confirmed by the Western blotting, and the inhibition of SBA-induced haemagglutination in vitro was compared with D-galactose, a known molecule that binds to SBA and blocks its binding to erythrocytes. The inhibition of SBA-induced haemagglutination by the anti-SBA IgY reached 512 units of haemagglutination inhibition (UHI), compared to 8 or 256 UHI, respectively, when IgY from control chickens or D-galactose was used. Thus, anti-SBA IgY antibodies were efficiently produced in large quantities and effectively inhibited SBA-induced haemagglutination in vitro.


Introduction
Lectins are natural bioactive proteins and glycoproteins, particularly abundant in legumes (1) and seeds (2), with specific affinity to bind simple or complex sugars (3).The most studied lectins are concanavalin A (convA) and soybean agglutinin (SBA), which are the phytohaemagglutinins present in the faba bean (Canavalia ensiformes) and soybean (Glicine max) (4).
SBA has a molecular weight of 120 kDa (5) and consists of four identical tetramers of 30 kDa each (6).Each SBA tetramer consists of an oligosaccharide chain containing N-acetylgalactosamine and mannose in its structure (3).In addition, several polar amino acid residues (Lys, Arg, Asp, Thr, Ser, Asn, and Gln) have been described within its polypeptide chains (7) with two specific binding sites for sugars (8).
Soybean is a major protein ingredient in commercial broiler and layer diets, accounting for ∼30% of the diet at each stage of production (9).Although soybean is a major source of essential amino acids in poultry diets, the presence of antinutritional factors, some of them thermo-resistant and others thermolabile, limits the use of soybean in poultry diets and hinders its inclusion as a raw ingredient (10).SBA is one of the major antinutritional factors present in soybeans (10).As SBA is a thermolabile molecule, it can be reduced but not completely inactivated during the roasting process of soybean (11,12).SBA is also resistant to the action of proteolytic enzymes (12,13).
SBA has a highly specific binding affinity for N-Acetyl-Dgalactosamine, a carbohydrate present in the cell membrane of enterocytes (14).It binds to the small intestinal epithelium, mainly at the brush border, causing morphological changes in these cells, such as microvillar atrophy and hyperplasia (9).Upon binding, SBA acts as a chemical messenger, initiating an inflammatory response and inducing cytotoxic damage to the enterocytes (15).The above is relevant to the feeding of young stock (chicks or jakes) in the poultry industry or piglets since the presence of SBA alters the digestive process, the feeding efficiency, growth, and health status of the animals by impairing cell proliferation and permeability and cell signaling pathways, ultimately inducing apoptosis and autophagy (16).All these lesions affect the barrier function of the small intestinal mucosa, the absorption and transport of nutrients across the intestinal wall, and the reduction of enzymes such as hydrolases; they also destroy endocrine cells and, consequently, the reduction of gut hormones (14,15).
IgY technology, based on the immunization of chickens and the extraction and purification of specific antibodies from their egg yolks, has been used in the development of products for the prevention or treatment of various pathologies or health conditions in animals and humans (17).This technology has been increasingly studied and applied in the last two decades in several contexts of animal production and animal health, proving to be low-cost, animal welfare-friendly, and more efficient compared to the traditional production of antibodies from live animals (18).
Against this background, this study aimed to produce anti-SBA IgY and evaluate its potential to neutralize the biological activity of SBA in vitro.

Ethics statement
All procedures were reviewed and approved by the Bioethics Committee (Certification N • 10-21) of the Catholic University of Temuco, Chile and were performed in accordance with the animal care protocols established by the institution.

Immunization of hens
Lohmann Brown laying hens (n = 10; 35 weeks old) were individually housed in wire cages with a light/dark cycle of 17/7 h and a room temperature ranging from 15 to 20 • C for 3 months.Hens were given ad libitum access to water and a commercial feed and were acclimated to the environment and handling for 2 weeks prior to the start of the experiment.Hens in the treatment group (n = 5) were immunized 4 times every 15 days (days 0, 15, 30, and 45).For the first immunization, 100 µg of soybean lectin (SBA; Sigma-Aldrich, USA) was emulsified with Freund's complete adjuvant and injected intramuscularly into the pectoral muscles at 4-5 different injection sites (0.5 ml/hen), as suggested by several authors (17,19,20).Booster doses consisting of 50 µg SBA emulsified with Freund's incomplete adjuvant were administered by the same route (20).Hens in the control group (n = 5) were injected with 0.5 ml of saline per hen, using the same procedure described for the treatment group.Before the first immunization and 2 weeks after each immunization (days 0, 15, 30, 45, and 60), blood samples were collected from the cubital vein of all hens and transferred to 2 ml Eppendorf tubes.Blood was held at room temperature for 12 h, and serum samples were collected by centrifugation after clotting and frozen at −20 • C until analysis.After the last immunization, eggs were collected from both groups for 15 days and stored separately at room temperature (15-18 • C) until processing.

IgY isolation and purification
IgY was extracted from a pool of randomly selected eggs, separately for the treatment and control groups (n = 10 eggs/group), using the polyethylene glycol (PEG) 6000 precipitation method described by Pauly et al. (18).In our experience, once standardized, this method of IgY extraction is very efficient, and the IgY samples obtained performed well in various immunological assays (18).For each group, the egg yolk pool was diluted at a ratio of 1:2 in PBS (pH 7.2), precipitated with 3.5% (w/v) PEG, and centrifuged at 4 • C for 20 min at 10,000 rpm.The delipidated supernatant was filtered and subjected to two further precipitations with 8.5 and 12% PEG.The final pellet was diluted in 800 µl PBS and transferred into a tubular dialysis membrane (PM 14 kDa, Sigma-Aldrich, USA).The extract was dialysed against 0.017 M NaCl for 12 h at room temperature, diluted in PBS, and subjected to a second dialysis under the same conditions.The total protein content of the final extract was measured by the bicinchoninic acid (BCA) method using a commercial kit (Pierce TM BCA Protein Assay, USA).

Determination of the extract purity and specificity of anti-SBA IgY
The protein profile of the egg yolk extracts from the treatment and control groups was analyzed by SDS-PAGE under reducing conditions and stained with Coomassie blue 25% (blue R250, Sigma-Aldrich, USA).A molecular weight marker (range 11-245 kDa; Page Ruler Plus Prestained Protein Ladder, Thermofisher Scientific) and a commercially purified IgY standard (Sigma-Aldrich, USA) were included.Proteins were then electrotransferred to polyvinylidene difluoride membranes (PVDF, Thermo Fisher Scientific, USA) using a Mini Trans-Blot cell system (Bio-Rad, USA).Membranes were incubated with anti-IgY (heavy chain) conjugated to peroxidase (Jackson ImmunoResearch 303-0035-008, USA) and detected with diaminobenzidine (DAB, Thermofisher Scientific, USA).
Specific detection of the SBA by the obtained IgY was also performed using Western blotting.The SBA (Sigma-Aldrich, USA) was subjected to an SDS-PAGE run and electroblotted onto PVDF membranes.After blocking with 3% milk, the membrane was incubated at 37 • C for 1 h with the IgY extracts from the treatment and control groups at 1:1.000 dilution in PBS.After three washes, the membrane was incubated at 37 • C for 1 h with an anti-chicken IgY-peroxidase antibody at 1:5.000 dilution in PBS.Finally, the membranes were developed using DAB (Thermofisher Scientific, USA).

The quantification of IgY in blood serum and egg yolk
The quantification of IgY in serum and egg yolk from immunized and control hens was performed by indirect ELISA.Microplates (Maxisor; Thermo Scientific, USA) were coated with 1 µg/well of purified SBA in 100 µl of PBS and incubated at 20 • C for 12 h.The microplates were then washed three times with PBS, then blocked with 2% skimmed milk at 37 • C for 1 h, and washed again.Serial dilutions of the sera and the yolk extracts were made with PBS-Tween, and 100 µl of the diluted extracts were added in duplicate to the wells and incubated at 37 • C for 1 h.The wells were then washed with PBS-Tween, and 100 µl of anti-IgY antibody conjugated to peroxidase (Jackson ImmunoResearch 303-0035-008, USA; dilution 1:140.000)was added to each well.After 1 h of incubation, the wells were washed again with PBS-Tween, and 100 µl of the tetramethylbenzidine substrate solution (TMB, Thermo Fisher, USA) was added.After 15 min, 50 µl of 1 M phosphoric acid (Merck, USA) was added to stop the reaction.Absorbance was measured at 450 nm using a microplate reader (Sunostik-SPR-960, Sunostik Medical Technology, China).

In vitro inhibition of SBA haemagglutinating activity by anti-lectin IgY
Haemagglutination SBA-induced haemagglutination reaction was performed according to Spackman (21).Blood samples (type A) were obtained from volunteers within our research group after signing an informed consent form.Blood was collected in 5 ml tubes containing EDTA and centrifuged at 4.000 rpm for 10 min to obtain plasma.The erythrocytes were washed four times in saline and then resuspended in the same solution (2% v/v) until use.Haemagglutination was performed in U-shaped multiwell plates (Nunc TM , Thermo Fisher, USA).In total, 50 µl of PBS was added to all wells.The first well in each row was used as the control.A total of 50 µl of SBA (Sigma-Aldrich, USA; 1 µg/µ) was added to the second well of each row.Serial dilutions were then performed, and 50 µl of erythrocyte solution (2%) was added to all the wells.The microplate was then gently vortexed and incubated at 20 • C for 2 h.All the samples were tested in duplicate.The haemagglutination response observed corresponded to 512 units of haemagglutination/50 µl (UHA/50 µl).One UHA/50 µl was defined as the reciprocal of the maximum dilution, giving positive haemagglutination (21).

Inhibition of haemagglutination
For the inhibition of haemagglutination, 50 µl of PBS and 98 ng of SBA (the amount of SBA present in the maximal positive haemagglutination dilution; 512 UHA/50 µl) were added to all the wells of the microplate.The inhibitory activity of serially diluted D-galactose (Sigma-Aldrich, USA; 350 µg/50µL), IgY from nonimmunized chickens (1.250 µg/50 µL), and anti-SBA IgY (1.250 µg/50 µL) from immunized chickens were measured after the addition of 50 µl of erythrocyte solution (2%) to each well.The microplates were incubated at 20 • C for 2 h.All samples were assayed in duplicate.Inhibition of haemaggutination results were expressed as units of haemagglutination inhibition (UHI), which is defined as the reciprocal of the maximum dilution of the tested solutions that completely inhibited haemagglutination (21).

Statistical analysis
Blood serum IgY titres in immunized and non-immunized hens were compared using a Statistical Analysis System (SAS, version 9.1.2,2004; SAS Institute, Cary, NC, USA) with the mixed procedure for repeated measures.The model included treatment, time, and treatment-by-time interaction using time as the repeated variable.IgY concentrations in blood serum and egg yolk were compared between groups using Student's t-test.
Values are expressed as mean ± SD.The p-values of ≤0.05 were considered significant.

Results
IgY titres in the blood serum of the hens immunized with SBA increased significantly (p < 0.01) after the first immunization and subsequent boosters, reaching a plateau between days 45 and 60.The mean maximum IgY titer was observed 45 days after the first immunization (Figure 1).In contrast, IgY titres in the control group remained at basal levels throughout the evaluation period (Figure 1).
The protein content of the egg yolk extract from immunized and non-immunized hens was 38.7 ± 1.6 and 37.7 ± 1.5 mg/ml, respectively.Since the average volume of the egg yolk extract obtained from each egg was 2.5 ml, the total amount of protein obtained from each egg was 96.8 ± 4.0 and 94.8± 3.0 mg for immunized and non-immunized hens, respectively.
The specific anti-SBA Ig Y titer detected by ELISA in the yolk extracts was significantly higher (p < 0.001) in immunized hens than in the non-immunized control group, with OD 450nm values of 0.98 ± 0.05 and 0.058 ± 0.02, respectively.The electrophoretic pattern of the egg yolk extract from immunized and non-immunized hens consisted of two bands of ∼65 y ∼27 kDa, corresponding to the molecular weight of the heavy and light chains of IgY, as can be contrasted with the commercial IgY (control, Figure 2).
The electrophoretic pattern of the soybean lectin consisted of a band at 30 kDa (Figure 3).Western blot clearly shows the specific detection of the soybean agglutinin (30 kDa band) by the anti-SBA IgY.In contrast, agglutinin was not detected by the IgY obtained from eggs of non-immunized hens (Figure 3).
The inhibition of SBA-induced haemagglutination by the anti-SBA IgY reached 512 UHI, compared with 8 UHI and 256 UHI, The inhibition of haemagglutination is expressed in the form of units (UHI) and percentage (%).* UHI is defined as the reciprocal of the maximum dilution of the tested solutions that completely inhibits haemagglutination.
respectively, when IgY was obtained from eggs of control hens or when D-galactose (positive control) was used (Table 1).The magnitude of inhibition of haemagglutination induced by Dgalactose and non-specific IgY from the control group was 50 and 1.6%, respectively, of that obtained when anti-SBA IgY antibodies were used (Table 1).

Discussion
Our results show that it is possible to produce high concentrations of anti-SBA antibodies using IgY technology, which effectively inhibits the haemagglutination capacity of SBA in in vitro assays.This antinutritional factor of the soybean has been overlooked and only superficially studied with respect to its deleterious effects on poultry and pig feeding efficiency (22).SBA specifically binds with high affinity to cell receptors N-Acetyl-D-galactosamine present in the enterocyte membrane of poultry and mammals, causing deleterious morphological changes due to an induced inflammatory response (22).All of the morphological changes induced by SBA in the small intestinal mucosa result in enzymatic inhibition at the brush border, which alters digestion and absorption of nutrients and ultimately adversely affects the growth and health status of the animals (9,23).In addition, severe negative effects on the local immune system have been reported (16,24).
Our immunization protocol (i.e., four immunisations every 15 days) induced a rapid and sustained immune response in the hens, resulting in an abrupt and significant increase in blood anti-SBA IgY after the first immunization.In this regard, it has been described that some hens respond with mammalian-like antibody kinetics, while other birds may respond with a rapid rise in antibody titer after the first immunization (25), as observed in our assays.Hens in the immunized group maintained high IgY titres until the end of the evaluation period, reflecting a beneficial effect of booster immunisations on increasing IgY antibody titres, as previously described (26).Similar immune responses have been reported in hens following immunization with various bacterial antigens, such as Escherichia coli (27), Campylobacter jejuni (28), Salmonella typhimurium (29), fungi (30), viruses (31), parasites (32), or viper (33), and honeybee venom (34).
The total IgY concentration (i.e., total protein concentration) in the egg yolk extract was higher than previously reported by other authors (32, 34-36) who described protein concentrations ranging from 5.4 to 33 mg/ml.The differences in total protein extracted in these reports compared to the present study may be due to the extraction technique used (37).On the other hand, the similar total IgY concentration in egg yolk from immunized and non-immunized hens is explained by the fact that only 1-10% of the total IgY present is specific (20).
The recovery rate of total protein per egg was also higher than previously reported by Barroso et al. (37) and Carroll and Stollar (38).Similarly, Shin et al. (39) and Sunwoo et al. (40) highlight that the IgY concentration in the egg yolk ranges from 9.4 to 133 mg/ml, depending on the extraction technique used (37).This finding suggests that the use of PEG may maximize the extraction of IgY, although scaling up of this method may be more complex than other methods described for the purification of the immunoglobulin from the egg yolk, as discussed by Pereira et al. (17).
On the other hand, collecting the eggs 10 days after the last immunization could provide the highest concentration of specific IgY in the yolk due to the strong correlation between the immunoglobulin in the yolk and the blood (41, 42).
The specificity of the anti-SBA IgY produced in this study was demonstrated by Western blotting and the haemagglutination inhibition (HI) test, where anti-SBA IgY blocked SBA-induced haemagglutination up to a high dilution.This finding suggests that the specific IgY could efficiently bind to the active sites of SBA, probably by blocking its association with the surface of the erythrocytes.As IgY are polyclonal antibodies, they can recognize and bind to different epitopes of a single antigen (43).
These antibodies may also lead to conformational changes, in addition to the epitope binding mechanism, that could enhance the inactivation of the SBA haemagglutination activity.This finding is supported by the high percentage of inhibition of SBA haemagglutination activity observed with the specific IgY compared not only to IgY from non-immunized hens but also to D-galactose, which was considered as a positive inhibition control in the assays.
To date, the detrimental antinutritional effects of SBA in animal diets have been underestimated.It has been found that low levels of SBA, even as low as 0.5-2 mg/g of soybean meal for chicks and piglets, can cause alterations in enterocyte morphology and growth rate (14).However, few strategies have been proposed to reduce the level of SBA in the final animal diet.Some of these strategies include (a) the development of SBAfree soybean varieties ( 44), (b) the addition of oligosaccharides (12) or N-acetylgalactosamine (45) to the animal diet, or (c) the supplementation of diets with probiotic bacteria expressing Nacetylgalactosamine in their surface (46).However, none of these strategies seem fully effective and thus have not been implemented in the industry.
IgY technology has been extensively studied and applied to various aspects of animal and human health (17).The use of specific IgY antibodies has been repeatedly reported in different animal species as a highly effective prophylactic strategy to confer passive immunity to animals, neutralize bacterial toxins, and inhibit bacterial adhesion to cells or block enzymes from various bacteria {[Escherichia coli; (47)], [Campylobacter jejuni; (48)], and [Salmonella Typhimurium and Salmonella Enteritidis, (49)]}.Specific IgY has also been used against other infectious agents such as fungi (30), parasites (50), and viruses (51), or molecules present in venom/toxins from snakes, scorpions, and bees (34,52,53).All these previous studies strongly support and encourage the use of IgY technology against antinutritional factors present in animal feed, especially those that have shown positive results after oral IgY administration.
Based on our results, we can conclude that anti-SBA IgY antibodies can be efficiently produced at high concentrations and are specific and effective in inhibiting SBA-induced haemagglutination in vitro, doubling the inhibitory capacity of D-galactose.In this context, administering anti-SBA IgY in the diets, for example, as an egg yolk powder additive, may be an effective alternative to reduce the impact of the antinutritional adverse effects of diets formulated with soybean meal.Future trials should evaluate the addition of anti-SBA IgY to the diets of chicks and piglets, as these categories of animals are more susceptible to the detrimental effects of SBA on their intestinal morphology and function.

FIGURE
FIGUREIgY antibody levels in the blood serum of hens immunized with purified soybean lectin (SBA; treatment group; n = ) or saline (control group; n = ).Absolute absorbance values (OD at nm) are expressed as mean ± SD. ↓ indicates the day of immunization or saline administration, and * indicates the day of blood collection for both groups.ODs on day correspond to the first sample taken before the first immunization.

FIGURE
FIGUREElectrophoretic profile (SDS-PAGE % under reducing conditions) of chicken IgY.Lane : molecular weight ladder.Lanes and : purified IgY (control sample) heavy and light chains.Lanes and : egg yolk protein extracts from immunized hens (n = ).Lanes and : egg yolk protein extracts from non-immunized hens (n = ).

FIGURE(
FIGURE(A) Electrophoretic profile (SDS-PAGE % under reducing conditions) of purified soybean lectin (SBA; Sigma-Aldrich, USA; lanes and ).Lane : molecular weight ladder.(B) Immunoblot analysis of soybean lectin incubated with IgY from the treatment (lanes and ) and control (lanes and ) groups, using a rabbit anti-chicken IgY peroxidase antibody.