Treatment with carbonate buffer decreases antigenicity of nervous necrosis virus (NNV)

Gye, Hyun Jung,Nishizawa, Toyohiko Elsevier 2019 Aquaculture Vol.500 No.-

<P><B>Abstract</B></P> <P>Antigens are commonly immobilized after suspending them in an alkaline buffer such as carbonate buffer (pH 9.6) for enzyme-linked immunosorbent assay (ELISA). The objective of this study was to determine the effect of 100 mM carbonate buffer (pH 9.6) on antigenicity of nervous necrosis virus (NNV) known to be pathogenic to fish. ELISA values of NNV antigens immobilized using carbonate buffer (pH 9.6) were ≤ 0.23, significantly lower than those by dry immobilization (ELISA values of approximately 1.0). NNV antigenicity was drastically decreased by treatment with carbonate buffer (pH 9.6), but not by treatment with Tris-HCl buffers at pH 8.0 or pH 9.6. Thus, the decrease of NNV antigenicity was due to carbonate/bicarbonate, not pH. NNV infectivity was also decreased after treatment with carbonate buffer (pH 9.6). The anti-NNV serum could recognize conformational structures of NNV surface protrusions related to NNV infection. Therefore, NNV surface protrusions might have been denatured by treatment with carbonate buffer (pH 9.6). Results of this study also demonstrate that coating NNV antigens by buffer immobilization might be far from practical. However, the sensitivity of ELISA detecting NNV antigens can be greatly improved by dry immobilization of antigens.</P> <P><B>Highlights</B></P> <P> <UL> <LI> NNV antigens were easily denatured by treatment with carbonate buffer (pH 9.6). </LI> <LI> Infectivity of NNV was also decreased by treatment with carbonate buffer (pH 9.6). </LI> <LI> The denaturation of NNV was due to carbonate/bicarbonate, not pH. </LI> <LI> Sensitivity of ELISA detecting NNV antigens can be improved by dry immobilization. </LI> </UL> </P>

겔여과크로마토그라피에 의한 질트리코모나스의 항원분획 분석

임미혜,심재찬,민득영,안명희,류재숙,최영길 한양대학교 의과대학 1993 한양의대 학술지 Vol.13 No.1

This study was aimed to analyze the antigenicity of T. vaginalis using a high performance gel filtration chormatography(HPLC), enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectotransfer blot (EITB). Soluble antigen was prepared using a bead beater. Antiserum was collected from rabbit immunized previously. The results obtained are as follows: the crude antigen of T.vaginalis was separated into 7 peak by HPLC and the fraction 3 revealed the highest reactivity in DLISA. The immunogenic bands of fraction 3 were visualized at 38, 45, 47, 55, 64, 78, 94, 115, 126 and 180 KDa and the bands at 38KDa and 180KDa showed stronger reactivity than that of equivalents of the crude antigen in EITB. In conclusion the fraction 3 of the crude antigen of T. vaginalis obtained by chromatographic separation may have a high antigenicity and proteins ay 38KDa and 180KDa of the fraction 3 may play an important role in antigen-antibody reaction in T. vaginalis infection.

효소 가수분해에 따른 대두 2S 단백질의 항원성 및 알레르기성 변화

성동은,임승용 한국산업식품공학회 2026 산업 식품공학 Vol.30 No.-

This study investigated the effects of high pressure (HP) treatment combined with enzymatic hydrolysis on the antigenicity and allergenicity of soybean 2S proteins. HP treatment alone (100–600 MPa) did not induce noticeable changes in the protein profiles or antigenicity. Enzymatic hydrolysis resulted in enzyme-dependent changes in protein degradation and antigenicity. Among the enzymes tested, alkaline protease showed the most substantial degradation of soybean 2S proteins, resulting in the residual antigenicity of < 10%. IgE-binding analysis using sera from patients with soybean allergy further demonstrated that alkaline protease treatment markedly decreased allergenicity to a level comparable to that of the negative control, whereas papain treatment only resulted in limited allergenicity reduction due to partial protein degradation. Alkaline protease treatment conditions were optimized to reduce the enzyme concentration or hydrolysis time while maintaining the low antigenicity level of soybean 2S proteins; however, the ideal conditions were not achieved yet, necessitating further investigation. These findings suggest that alkaline protease- mediated hydrolysis shows promise for hypoallergenic soybean protein production.

韓牛에 있어서 anaplama marginale의 抗原性에 관한 硏究

Byeong Kiri BAEK(白秉杰),Chan Moon CHIN(陳墡,文),Byeong Su KIM(金秉洙),Jin Ho KIM(金眞鎬),Chon Hu CHO(趙鍾厚),Joo Mook RHEE(李周默),Byung Tak JUNG(丁炳鐸) 한국예방수의학회 1989 예방수의학회지 Vol.13 No.3

A splenectomized 5 month old Korean calf was inoculated with cryopreserved Anaplasma marginale infected blood. At peak parasitemia(40.1%) blood was collected, infected erythrocytes washed, lysed and A. marginale initial bodies isolated by differential centrifugation. Antigenic profile of isolated A. marginale organism was analized by SDS-PAGE and Western blotting technique. Coomassian blue stained SDS-PAGE gels revealed at least ten specific protein bands of approximately 28Kd-30Kd, 38Kd, 42Kd, 58Kd, 66Kd, 72Kd and 198Kd molecular weight. Only few protein bands recognized with polypeptides in the noninfected bovine erythrocyte antigen used as control. A. marginale protein recognized by specific anti-A. marginale antibodies demonstrated 8 polypeptide at 18KD, 28-31Kd, 38Kd, 55-58Kd, 97Kd, and 198Kd molecular weight range. Negative reactivities were observed with normal bovine serum with either A. marginale or erythrocytic antigens, respectively. Predominant protein were found at 18Kd, 38Kd and 97Kd molecular weight. On going studies will analyze the relative importance of those antigens.

Vibrio vulnificus 박테리오 파아지의 특성

오양효,윤소겸,공은진,김민정,김영부,박영민 대한감염학회 1995 감염 Vol.27 No.5

목 적 : 이미 보고되어 있는 파아지들의 경우, 일반적인 형태, 숙주역과 몇 가지의 성질만 연구되어 있으므로 보다 상세한 특성에 대한 연구가 필요한 상태이다. 본 연구에서는 해양으로부터 분리한 V. vulnificus를 숙주균으로 하여 투명한 용균반을 형성하는 파아지를 분리해 낸 다음, 분리된 V. vulnificus 파아지의 특징을 연구하고자하였다. 방 법 : V. vulnificus 를 숙주균으로 하여 해산물로부터, 숙주균에 특이적인 파아지를 분리하고, 농축 정제과정을 거쳐 파이지입자를 순수분리하였다. 파아지의 형태를 전자현미경으로 관찰하였으며, 분리된 파아지의 숙주특이성을 조사하고, 파아지 입자의 물리적 특성을 검토하기 위해, 온도 안정성시험, pH 안정성 시험을 실시하였다. 파아지의 흡착율, 파아지의 흡착율에 미치는 무기염 이온의 영향, 온도의 영향과 pH의 영향을 관찰하였다. 파이지의 일단증식곡선을 구하였으며 토끼 면역과 교차 중화 시험, 파아지 핵산의 특성, 구조단백질의 특성을 조사하였다. 결 과 : 전자현미경관찰결과 본 연구에서 분리한 파아지는 두부와 미부를 가지는 형태로 미부에 꼬리판과 꼬리섬유를 가지고 있었다. 파아지의 물리적성질시험 결과 파아지는 50℃이하의 온도에서 안정하였으며 중성pH영역에서 안정하고 산성과 알칼리 영역에서는 활성을 잃었으며 자외선에는 35초 조사에 50% 정도 불활성화 되었다. 파아지는 약 65%의 흡착율을 나타내었으며 흡착에 ??, ??을 요구하였다. 파아지입자가 안정한 온도와 pH영역에서는 흡착에 영향을 받지 않았으며 일단증식시험 결과 108 PHU/ml의 파아지 입자를 생산하였다. 정제한 파아지를 항원으로 하여 토끼에 면역한 항혈청에 파아지가 중화되었으며 파아지 핵산은 이중나선 DNA였다. 파아지의 구조단백질은 SDS-PAGE polyacrylamide gel 전기영동 결과 2개의 밴드를 나타내었다. 그리고 Western blotting 결과 파아지의 전기영동 결과와 동일한 위치의 밴드에서 항원성을 나타내었다. 결 론 : 인체병원균으로 중요성이 인식되고 있는 V. vulnificus는 해산물의 위생과 국민건강에 직접적인 영향을 미칠 수 있는 세균으로 이에 대한 활발한 연구가 요구되는 실정이다. 파아지의 분리율은 숙주균의 분리율에 의존하므로 숙주균의 해수내 분포에 영향을 미친다고 추측되며, 파아지는 숙주균에 감염되어 숙주균을 용균시키므로 숙주균의 개체수를 조절하여 생태학적 측면에서 중요한 기능을 담당하는 것으로 여겨진다. Background : Vibrio vulnificus causes severe wound infection and life-threatening septicemia. In this study we present the characteristics of V. vulnificus bacteriophage and the basis of pathogenicity study of V. vulnificus related on bacteriophage. Methods : We isolated bacteriophage for V.vulnificus from marine products and examined its physiological and biochemical characteristics, and the ultrastructure by electron microscopy (TEM,SEM). And also, we investigated host ranges, temperature stability, pH stability, inactivation by UV irradiation, DNA isolation and analysis and structural protein analysis of the bacteriophage. Results : The adsorption rate of bacteriophage was above 80% and had a little influence at 10-45℃ and pH5-10. Isolated bacteriophage was used to produce antiserum against rabbits and its antigenicity was investigated by cross-neutralization. Western blot analysis showed the capsid protein of bacteriophage had antigenicity. The bacteriophage had double-strand DNA. In restriction analysis, the phage DNA was digested HinfI. The structural protein of phage showed two bands of 34.7kDa and 18.4kDa on SDS-PAGE. Conclusion : The specificity of bacteriophage may be due to the specific binding site that be possessed by host strain surface. Therefore, it seems that virulent bacteriophage controls the number of host strain and then takes a role on natural marine environment.

Production of low antigenic cheese whey protein hydrolysates using mixed proteolytic enzymes

Shin, Hyun Soo,Kim, Sang Bum,Kang, Soo Cheol,Khan, Muhammad Ajmal,Kim, Hyeon Shup,Shin, Hyun Jung,Chang, Chi Hoon John Wiley Sons, Ltd. 2007 Journal of the Science of Food and Agriculture Vol.87 No.11

<P>This study examined the effect of different proteolytic enzymes on the production of cheese whey protein (CWP) hydrolysates with low antigenicity. Four enzyme combinations (1:1) trypsin + papain W-40 (TP), trypsin + neutrase 1.5 (TN), papain W-40 + protease S (PP) and papain W-40 + neutrase 1.5 (PN) were added at the rate of 1% of the CWP and it was incubated for 15, 30, 60, 90, 120 and 180 min at 50 °C. CWP hydrolysis and its non-protein nitrogen concentrations were higher with TP and TN compared with PP and PN at all incubation times. The SDS-PAGE revealed complete removal of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) from hydrolysates produced by trypsin-containing enzyme mixtures. Reverse-phase HPLC analysis ascertained the CWP hydrolysis and SDS-PAGE results. The lowest antigenicity in CWP hydrolysates was observed with the use of trypsin-containing enzyme mixtures compared with other enzyme combinations. Present results suggested that TP and TN combinations were the most effective for CWP hydrolysis for the removal of β-LG from CWP. Further research is warranted to identify the peptides in CWP hydrolysates produced with these enzyme combinations that may help enhance the utilisation of whey protein in human food. Copyright © 2007 Society of Chemical Industry</P>

토끼 항 ${\beta}-Lactoglobulin$ 항혈청에 대한 유청단백질 가수분해물의 항원성

이수원,하월규,전석락,김정완,손동화,이재영,Lee, Soo-Won,Ha, Woel-Kyu,Juhn, Suk-Lak,Kim, Jung-Wan,Shon, Dong-Hwa,Lee, Jae-Young 한국식품과학회 1994 한국식품과학회지 Vol.26 No.5

In order to investigate the lowering effects of in vitro enzymatic hydrolysis by the treatment of chymotrypsin, trypsin, pancreatin, or protease from Aspergillus oryzae on the antigenicity of whey protein(WPI) against rabbit anti ${\beta}-LG$ antiserum, competitive inhibition ELISA(cELISA) and passive cutaneous anaphylaxis(PCA) test using guinea pig were performed. The results of cELISA showed that the monovalent antigenicity of the whey protein hydrolysates(WPH) to the antiserum was decreased to $10^{-1.7}{\sim}10^{-4.1}$ and less by the hydrolysis. Especially, the antigenicity of OUP(hydrolysate by protease from Asp. oryzae with preteatment of pepsin) was found almost to be removed. By the heterologous PCA the polyvalent antigenicity of the WPH was decreased to $1/2{\sim}1/128$ and less. Especially, the polyvalent antigenicity of OUN(hydrolysate by protease from Asp. oryzae without preteatments) was found almost to be removed, although OUN did not have so high degree of hydrolysis(DH) or so low monovalent antigenicity (reduced to $10^{-3.2}$). Therefore, this result was assumed to come from effective destruction of antigenic determinants on ${\beta}-LG$ in WPI, not to produce polyvalent antigenic peptides that are closely associated with induction of allergy. This finding suggested that WPH prepared by the treatment of microorganic protease from Asp. oryzae would be a material for hypoallergenic infant formula due to the removal of the polyvalent antigenicity of ${\beta}-LG$, the major milk allergen in WPI.

Streptomyces 속 균주들의 생장에 따른 외피 항원성 변화의 혈청학적 분석

김재헌,조성기,Kim, Jae-Heon,Jo, Sung-Kee 한국미생물학회 2007 미생물학회지 Vol.43 No.4

The changes in antigenicity during cultivation of streptomycetes were determined by immunodiffusion assay and indirect ELISA. New precipitin lines in immunodiffusion assay began to appear in the growth period of the soluble pigment production and became thickened thereafter. The increase in the antigenicity was also confirmed by ELISA. The antigenic development was relatively weak for S. lavendulae and S. viridochromogenes while that was strong for S. lavendulae and S. viridochromogenes. The results indicated that Streptomyces strains, even though not proved for some strains, changed the compositions of cell envelope during submerged growth and this could be estimated quantitatively by serological method.

Peptic and Tryptic Hydrolysis of Native and Heated Whey Protein to Reduce Its Antigenicity

Kim, S.B.,Ki, K.S.,Khan, M.A.,Lee, W.S.,Lee, H.J.,Ahn, B.S.,Kim, H.S. American Dairy Science Association 2007 Journal of dairy science Vol.90 No.9

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10min at 100<SUP>o</SUP>) WPC (2% protein solution) were incubated at 50<SUP>o</SUP> for 30, 60, 90, and 120min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.

우유단백 항원의 열처리 및 가수분해에 따른 단백의 분포와 항원성 변화

이희선 ( Hee Seon Lee ),김미나 ( Mi Na Kim ),홍정연 ( Jung Yeon Hong ),허원일 ( Won Il Heo ),김경원 ( Kyung Won Kim ),손명현 ( Myung Hyun Sohn ),김규언 ( Kyu Earn Kim ),이경은 ( Kyung Eun Lee ),박중원 ( Jung Won Park ) 대한소아알레르기호흡기학회 2014 Allergy Asthma & Respiratory Disease Vol.2 No.4

Purpose: Cow`s milk protein is one of the most common and strongest food allergen. We investigated the effects of heat treatment on the distribution and antigenicities of major allergens from cow`s milk. We also compared the protein distribution and antigenicities among cow`s milk formula and its substitutes. Methods: We heated α-casen, β-lactoglobulin (BLG), α-lactalbumin (ALA), and crude extract of cow`s milk in 100°C boiling water for 1 hour. We prepared crude extracts from cow`s milk formula, partially hydrolyzed milk formula (pHF) and extensively hydrolyzed milk formula (eHF). The protein compositions of all the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenicities were determined by IgE immunoblotting with pooled serum collected from 11 patients with milk allergy. Results: After heating, no significant alteration was found in casein, and the aggregates of ALA and BLG were detected with molecular weights of about 30 and 45 kDa, respectively. The antigenicities of newly detected aggregates were increased. The new aggregates of BLG with increased antigenicities were also found in heated milk total protein. Major milk allergens were not found in pHF, and residual components with a molecular weight below 10 KDa did not show IgE-binding activity. We failed to observe the residual components and antigenicities of eHF. Conclusion: Changes in protein distribution and antigenicity of milk total protein induced by heat treatment may not be significantly different from those of each major allergen. The residual components of pHF could have little IgE-binding capacity, and there may be few or no antigenic components in eHF.