The necessity of developing antibodies for autoimmune diseases has recently increased due to the outbreak of new virus such as avian influenza (AI) and foot-and-mouth disease. In the development of antibody treatment, immunological and pathological importance of N-glycosylation of immunoglobulin glycoprotein (IgG), composed of two heavy chains and two light chains, has been recognized. Because all N-glycosylation phenotype of mice is not identical to human’s, antibody production from mice has been difficult. It has been devoted to develop antibodies that are humanized with containing N-glycosylation of a mouse IgG2b hinge region, more resistant to the attack of protease and providing a high level of complement fixation. Major saccharides found in human glycoprotein are xylose (pentose); fucose, galactose, glucose, mannose, N-acetylgalactosamine and N-acetylglucosamie (hexose); and N-acetylneuramic acid (nonose) (Kim et al. 1996).

GAGs are polysaccharides linked between glyco, amino and glycan by glycosidic linkage. It is a type of carbohydrate molecules making up the blood vessel inner walls and is known to influence directly and indirectly on various functions such as metabolism of low density lipoprotein (LDL) cholesterol, blood coagulation and the formation of cellular matrix. Such GAGs have structurally various and complex form through partial changes in biosynthetic pathway. This research of GAGs derived from insects showed, unlike other GAGs, the effects of significance on the subjects (mice) with chronic inflammation reproducibly in the 3^rd repeat animal testing (Ahn et al. 2013). The B. terrestris extract was also seen to have anti-inflammatory effects and the effect to significantly control proinflammatory cytokines (Ahn et al.2012). Therefore, to verify more validity of GAGs derived from insects, the possibility was determined by attempting to develop antibodies.

Fresh insects (1. male silkworm pupae 2. clony shell of B.ignitus 3. worker of B. terrestris 4. queen of B. ignitus 5. Isaria sinclairii 6. Catharsius molossus 7. queen of B. terrestris 8. Tabanus bivittatus 9. Gryllus bimaculatus) were dried at room temperature after leaving it for two d in acetone, an organic solvent, to remove its lipid component. 50 g (dry weight) of each sample was put into 500 mL of 0.05 M carbonate buffer (pH 9.0) respectively and then 8 mL of alkaline solution, a type of heat-resistant protease, was added. Crude GAGs of each insect were gained by breaking down the protein by incubating 2.5% weight of alkaline solution (sigma) derived from Bacillus lichenformis for 48 h at 60℃ as proteinases; depositing the protein by adding a refrigerated 50% trichloroacetic acid to be the total concentration of 5% and leaving it at 4℃ for three hours; removing proteolysis by centrifugation (≥8,000 rpm); coprecipitating the saccharide in the residue with the doubled amount of ethanol (100%) and potassium acetate; depositing the precipitate in the doubled amount of cetylpyridinium (5% weight); depositing again the precipitate in the doubled amount of ethanol; and freeze-drying after dialyzing with the dialysis membrane method with distilled water (molecular weight: ≥ 3,500).

After dissolving crude GAGs in water and removing substances insoluble in water by centrifugation, 5% cetylpyridinium chloride was added to 1/4 volume and the mixture was deposited. The precipitate was purified by dissolving in 2.5 M sodium chloride by centrifugation and depositing with alcohol. Polysaccharides were purified by freeze drying after dialyzing by dissolving the purified sample in a small amount of water. Through the repetition of the process, each insect GAG fraction was prepared by collecting sugar acid fraction to determine the purity by electrophoresis; purifying more with a salt gradient (0, 0.1, 0.5, 1, 2.5M NaCl in phosphate buffer) via ion exchange chromatography (Sephadex DEAE A-25); and collecting the fraction containing uronic acids.

The titer assay was conducted by preparing polyclonal antibodies for research on mass production techniques of the insect GAG antibodies. Polyclonal antibodies were prepared by mixing 10 mg/kg of the insect GAGs with the same amount of complete Freund’s adjuvant (sigma CO, USA) in the 1^st immunization; injecting the homogenate emulsified for a day to BALB/c mice intraperitoneally; at 12-day intervals as the 2^nd and the 3^rd, mixing incomplete Freund’s adjuvant (sigma, CO., USA) with the same amount of the purified GAGs; injecting intraperitoneally after emulsifying for a day; and collecting serum after 2 wk. After taking the blood sample, antibodies in this research were prepared by preparing sodium citrate 3.8%; coating it in the syringe for 1/9 of the blood to enter; collecting blood; hardening fibrin by centrifugation; and diluting the serum with 1% FCS-tween to 1/10.

In the titer assay, the GAGs as an antigen were dissolved in 0.1 M coating buffer (1.593g Na₂CO₃, 2.93g Na HCO₃) in a 96 well ELISA plate and coated overnight with 100, 200 and 500 ng and then the plate was washed two times with PBS-0.05% tween. The unattached impurities were incubated with PBS-0.05% tween buffer containing 1% fetal calf serum for an hour and dried to prevent antibodies from combining at nonspecific binding sites. Sandwich ELISA was performed after diluting the serum, containing the antibodies gained from immune induction by emulsifying the GAGs in the 1^st to the 3^rd with the complement and injecting intraperitoneally, 100-fold, 250-fold, 500-fold and 1000-fold and incubating at room temperature. Serum was put in a half of the ELISA plate as dilution ratio and bovine serum albumin (BSA), with serum-free, i.e. serum-immunoglobulinfree, from commercial sigma was put in the other half of the plate as the equal dilution ratio (100, 250, 500, 1000:1) as serum. The method to determine the antibody production was applied by comparing the both parts of the plate for coloring reaction at 414 nm. As measuring coloring reaction at 414 nm with antispecies (mouse) IgG alkaline phosphatase as 2^nd antibodies and WesternBreeze Chromogenic from Invitrogen as the reagent, the WesternBreeze manual of Invitrogen (Carlsbad, USA) was applied to the ELISA plate (Choi et al. 1997).

As seen in the Table 1, the insect GAGs of the nine species (1. male silkworm pupae 2. clony shell of B.ignitus 3. worker of B. terrestris 4. queen of B. ignitus 5. Isaria sinclairii 6. Catharsius molossus 7. queen of B. terrestris 8. Tabanus bivittatus 9. Gryllus bimaculatus) were obtained by preparing new insect GAGs from the residue of the insect extracts.

[Table 1.] Yield of purified insect glycosaminoglycan from each extract residue

Yield of purified insect glycosaminoglycan from each extract residue

The insect shells left after the preparation of alcoholic extract from the nine species were dried to be utilized and 1.1 g(yield= approximately 0.1%) was obtained by drying each insect shell; removing protein by proteolysis with proteinases; and dialyzing with ethanol and distilled water after removing impurities by precipitation. In particular, the yield was gained with 4.9% from queen of B. ignites; 2.2% from queen of B. terrestris; 1.5% from male silkworm pupae; 1.1% from Catharsius molossus; and 0.52% from Tabanus bivittatus. The sample used for the injection to the mice for the antibody was prepared by collecting sugar acid fraction to determine the purity by electrophoresis; purifying more with salt gradient (0, 0.1, 0.5, 1, 2.5M NaCl in phosphate buffer) via ion exchange chromatography (picture 1); and collecting fraction containing uronic acids. The content of the prepared GAGs was measured. The Table 1 below indicated the content of crude GAGs derived from each insect of male silkworm pupae, clony shell of B.ignitus, worker of B. terrestris, queen of B. ignitus, Isaria sinclairii, Catharsius molossus, queen of B. terrestris, Tabanus bivittatus and Gryllus bimaculatus. It was shown that a respectable amount for medical use could be obtained mainly in the mucosal cells of thick insect shells.

In the titer assay result of polyclonal antibodies, antibody production could be derived from a variety of insects. 1. PSG (male silkworm pupae GAG, Fig. 1 A): Significance was p<0.05 against serum 500:1 with 100 ng and 200 ng of coating antigen; serum 250: 1 with 100 ng; serum 100:1 with 200ng and 500 ng; and contrast BSA. 2. IGPCG (clony shell of B.ignitus GAG, Fig. 1 B): The significance of the coloring reaction was p<0.05 against contrast BSA when reacting serum 500:1 with 100 ng and 200 ng of coating antigen and serum 250:1 with 10 ng and 100 ng. 3. BTWG (worker of B. terrestris GAG, Fig. 1 C), 4. IQG (queen of B. ignitus GAG, Fig. 1 D), 5. ISG (Isaria sinclairii GAG, Fig. 1 E), 6. CaG (Catharsius molossus GAG, Fig. 1 F), 7. BTQG (queen of B. terrestris GAG, Fig 1 G), 8. Tabanus bivittatus GAG (Similar to the above) 9. Gryllus bimaculatus GAG (Similar to the above): Significance was p<0.05 against serum, containing antibodies produced by injecting the 3^rd immune complement mixture, 250:1 with 100 ng of coating antigen; serum 500:1 with 100 ng and 200 ng; and contrast BSA.

[Fig. 1.] Profile of purification of some glycosaminoglycan by ion exchange chromatography

[Fig. 2.] Immunoassay of produced polyclonal antibody of each glycosaminoglycan

In conclusion, if producing methods of antibodies derived from insect GAGs of the various species in this research further improve, it is considered to be possible to produce antibodies that are more stable form than the method producing antibodies derived from the GAGs of pigs with the possibility of contamination by infection. This is because it is injected into mammals or animal cells as safer replacement than heparin, GAG derived from pigs and the like with the possibility of having infectious diseases such as foot-andmouth disease.