新旧版国家标准《高致病性禽流感诊断技术》血凝和血凝抑制试验内容比较

为进一步做好高致病性禽流感强制免疫抗体效价评估工作,国家市场监督管理总局和国家标准化管理委员会于2020年12月14日发布实施了新版《高致病性禽流感诊断技术》(GB/T 18936—2020)。新版标准中重新规定了高致病性禽流感血凝和血凝抑制试验的技术要求,与旧版本比较内容有所变动。为尽快理解新版标准要求,熟练掌握操作要领,对新、旧版标准中血凝和血凝抑制试验进行了比较。经对比分析发现,新版标准增加了对血凝抑制试验4 HAU抗原配制的验证,鸭、鹅等水禽血清的预处理内容,并对其结果判定予以补充。新版标准在操作步骤上更为精准详细,在结果判定上更加科学准确,弥补了旧版本存在的诸多不足。本文为广大兽医实验室检测人员更好地理解新、旧标准差异以及新版标准优点,从而提高试验结果准确性提供了参考。     

血凝-血凝抑制试验中配制四单位抗原试验

血凝-血凝抑制(HA-HI)试验是鉴定病毒及检测相应抗体常用的检测方法之一,是兽医实验室人员必须掌握的试验技能,主要应用于家禽H5N1/H7N9型禽流感、鸡新城疫的免疫效果评估。试验中根据血凝(HA)试验的终点判定,正确计算和配制四单位(4HAU)抗原是直接影响试验成败的关键,配制4HAU抗原后需进行抗原回滴验证,确保血凝抑制(HI)试验结果无误。     

血凝与血凝抑制试验4单位病毒抗原配置与标定

为了解决基层兽医实验室检测人员在血凝与血凝抑制（HA和HI）试验中4单位病毒抗原的配置和标定问题的困扰，本试验对GB/T 18936-2020《高致病性禽流感诊断技术》中规定的4单位病毒抗原的配置和标定方法进行了探讨，试验结果表明，通过标定调整的4单位病毒抗原准确性高，从而减少血凝抑制实验的误差。     

血凝抑制试验在实际应用中若干问题的探讨

<正>血凝抑制试验作为一种简便快速的检测方法应用广泛,但实际操作中会受温度、红细胞悬液配制、结果判定等因素影响试验结果的准确性,本文主要对血凝抑制试验操作的关键点进行阐述。1抗原配制HI试验在实际应用中首先应注意4单位(HAU)抗原的     

马动脉炎病毒血凝抑制试验研究及其应用

用马动脉炎病毒（EAV）免疫SPF豚鼠，4周后采血做血凝抑制试验（HI），其抗血清可以抑制血凝抗原对小鼠红细胞的凝集反应，抗原和抗血清在4℃感作24h后可以检测到最高的HI抗体效价，同时结果显示HI抗体与中和抗体产物呈正相关。对561份来自新西兰、吉尔吉斯、沙特阿拉伯及内蒙古、新疆的马血清用HI试验和微量血清中和试验（NT）进行EAV抗体检测，HI和NT阳性符合率为94．7％，血凝抑制抗体与中和抗体效价呈显著的正相关。     

用微量血凝和血凝抑制试验诊断水貂病毒性肠炎

用8HA单位水貂肠炎病毒（MEV）细胞培养活毒标准抗原、8HI单位兔抗MEV血清标准抗体、0．5％戊二醛化猪RBC进行微量血凝（HA）－血凝抑制（HI）试验诊断水貂病毒性肠炎（MVE），具有敏感、准确、简便、省时等特点，适于MVE的确诊、检疫、流行病学调查和免疫水监察，可在接到病料后4小时内报告结果，因本试验只能活毒标准抗原，所以需好消毒关，防止散毒。     

血凝(HA)—血凝抑制(HI)试验操作要点

兽医实验室检测禽流感和鸡新城疫抗体采用血凝(HA)-血凝抑制(HI)试验。该试验有些因素会影响试验结果,是试验的关键环节,如配制1%鸡红细胞时其浓度的高低会影响抗体滴度的高低;4单位抗原的配制需要回滴正确;试验的反应时间和温度也要符合要求。只有掌握好试验的关键点,才能得出准确无误的试验结果。对该试验的操作要点及注意事项进行了介绍。     

猫泛白细胞减少症病毒的血凝及血凝抑制试验检测

猫泛白细胞减少症病毒是由细小病毒引起的传染病,能感染各种猫科动物。通过血凝及血凝抑制试验(HA/HI),检测猫细小病毒的抗原和抗体效价,从而确定一种简易、快速操作简单、敏感、准确的诊断方法,并为免疫程序提供可靠依据。     

浅谈血凝试验（HA）和血凝抑制试验（HI）影响因素及应用

血凝试验（HA）和血凝抑制试验（HI）是目前兽医实验室常用的检测方法,作为血清诊断技术之一,是利用某些病毒具有凝集红细胞的特性,且这种特性能被特异性抗血清抑制的原理,用于已知病毒检查血清中相应抗体状态和已知血清鉴定未知病毒的一种兽医实验室最常用的实验方法。HA和HI试验具有特异性好、操作方便、可同时进行大量样本试验、对试验环境要求不高等优点,     

鸡醛化红细胞在血凝和血凝抑制试验中的应用

应用鸡醛化红细胞 (FoRBC)代替新鲜配制红细胞 (FrRBC) ,进行血凝试验 (HA)和血凝抑制试验 (HI) ,以检测病毒滴度及其对应抗体效价。结果 :0 75 %FoRBC与 1 %FrRBC所测病毒HA和相应抗体HI效价一致。表明 0 75 %FoRBC可以替代 1 %FrRBC ,且具有不易破裂 ,保存期长 ,使用方便 ,无自凝 ,图象清晰、稳定 ,结果易判定等优点     

利用不同源红细胞检测鹅禽流感抗体效价的报告

从某一已免疫禽流感的种鹅场采取30份血样，分别以健康鸡红细胞悬液和健康皖西白鹅红细胞悬液为指示剂，对30份血样进行禽流感血凝和血凝抑制试验。结果：用鸡红细胞悬液作指示剂进行血凝试验，测出抗原4HAU为1：32；用鹅红细胞悬液作指示剂进行血凝试验，测出抗原4HAU为1：8以1：32倍数稀释抗原，分别用鸡红细胞和鹅红细胞悬液作指示剂进行血凝抑制试验，测出30份鹅血清禽流感H5免疫抗体效价的平均值分别为2^5.8和2^3.9；以1：8倍数稀释抗原，用鹅红细胞悬液作指示剂进行血凝抑制试验，测出30份鹅血清禽流感免疫抗体的平均效价为2^5.1。     

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2¹³ per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4℃. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.     

提高鸡传染性支气管炎病毒血凝抗原滴度途径及抗原灭活

将７株传染性支气管炎病毒（ＩＢＶ）标准株（Ｍ４１、Ｈ１２０、Ｈｏｌｔｅ、Ｇｒａｙ、Ｃｏｎｎｅｃｔｉｃｕｔ、Ｉｏｗａ６０９和Ｔ株）和６株分离株（ＮＩＢＶ、ＧＩＢＶ、Ｍ、ＳＨ、Ｊ和Ｈ株）分别接种于鸡胚，收获尿囊液，经浓缩后，用魏氏梭菌培养液处理，制备血凝抗原。其中，Ｈ１２０株血凝滴度最高，Ｔ、Ｍ、Ｊ和Ｈ株无血凝性。应用含有不同滴度ＩＢＶ母源抗体的鸡胚增殖病毒制备抗原，效价与用ＳＰＦ鸡胚增殖病毒制备的抗原效价一致。尿囊液经反复冻融后再制备抗原会使血凝价降低。抗原分别用甲醛、高碘酸钠、硼氢化钾和ＳＤＳ灭活，其中甲醛灭活效果最理想。抗原对氯仿敏感，对乙醚稳定。适宜浓度的Ｎａ＋、Ｍｇ２＋可显著提高抗原的血凝性     

Hemagglutination by calf diarrhea coronavirus

The virus was grown in BEK-1 cells, a stable cell line from bovine embryo kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, room temperature and 37°C. HA was observed at all temperatures with chicken, mouse, rat, and hamster erythrocytes but not with erthyrocytes of human (O), cattle, horses, sheep, guinea pigs, geese, ducks, pigeons and 1-day-old chicks. Chickens showed an individual variation in agglutinability of their erythrocytes, requiring selection of birds to obtain erythrocytes for HA. HA reaction was inhibited by specific antiserum. Some factors involved in HA and HA inhibition (HI) were investigated and standard HA and HI tests were worked out.     

猪细小病毒血凝抑制试验抗原、阳性血清与阴性血清的制备与鉴定

猪细小病毒（Porcine Parvovirus,PPV）血凝抑制试验抗原、阳性血清和阴性血清在猪细小病毒制品的效力检验中不可或缺,对猪细小病毒相关生物制品的质量控制至关重要。试验采用PPV 7909株病毒同步接种PK-15细胞制备血凝抑制试验抗原,用制备的抗原乳化后免疫豚鼠制备阳性血清,同时用未免疫的阴性豚鼠制备阴性血清。对抗原、阳性血清和阴性血清进行鉴定,结果表明,制备的PPV血凝抑制试验抗原HA效价达1:512;阳性血清HI效价达1:1024;阴性血清HI效价＜1:8,且特异性均良好。利用制备的血凝抑制试验抗原、阳性血清与不同PPV疫苗株的灭活抗原、阳性血清进行交叉反应试验,结果表明,制备的抗原与阳性血清具备良好的血清学交叉反应性,可用于PPV制品的统一评价。     

禽流感病毒H5亚型血凝抑制试验抗原国家参考品的研制

按照国家一级标准物质要求和禽流感病毒（H5亚型）血凝抑制试验抗原制造及检验规程规定的抗原制备方法,研制了一批禽流感病毒（H5亚型）血凝抑制抗原国家参考品,用于检测禽流感病毒（H5亚型）制品生物效价和血凝抑制操作过程中的校验。该批抗原经过8家单位对其协作标定后对结果进行统计学分析,确定该批禽流感抗原的血凝效价为1：80。另对所研制的禽流感抗原均一性、稳定性、无菌检验、剩余水分测定、真空度测定等各项指标进行检测,结果均符合禽流感病毒（H5亚型）血凝抑制试验抗原制造及检验规程和国家一级标准物质要求。说明该禽流感病毒（H5亚型）血凝抑制抗原可以作为国家参考品。     

Hemagglutination and hemagglutination inhibition with Chuzan virus.

Chuzan virus agglutinated erythrocytes of several species of animals including bovine. The hemagglutinating (HA) activity against bovine erythrocytes was dependent on NaCl molarity and was expressed best at 0.6 M, but it was independent of pH and temperature. Three strains of Chuzan virus isolated from 2 cows and a pool of culicoides midges had indistinguishable HA antigenicity. All cattle infected with the virus developed high titers of hemagglutination inhibiting (HI) antibody which changed in parallel with neutralizing (NT) antibody titers. Correlation between HI and NT antibodies was very high and the antibodies persisted for one year or more. Therefore it was concluded that the HI test is applicable for survey of Chuzan virus infection among cattle in place of the NT test.     

Hemagglutination and antigenic comparison of rabbit hemorrhagic disease virus

The hemagglutinating activity and serological properties of three strains of rabbit hemorrhagic disease virus, Chinese, Korean and Shizuoka, which was first isolated in Japan, were examined by hemagglutination (HA) and cross hemagglutination inhibition (HI) test with human erythrocytes. Similar results were observed between the Chinese and Korean strains, both of which gave positive HA at 4 degrees C with O, A, B and AB, and at 22 degrees C with B and AB blood groups. In the Shizuoka strain, positive HA was observed at 4 degrees C with O, A, B and AB, at 22 degrees C with A, B And AB, and at 37 degrees C with B blood group. In experimentally infected rabbits, HI antibody in these animals showed a titer of 16,384 or 32,768 at 4 weeks after inoculation. No serological difference was observed in three strains by cross HI test.     

影响血凝试验和血凝抑制试验的因素分析

血凝试验和血凝抑制试验是检验新城疫、禽流感等疫病抗体效价的一种常用方法,该方法具有所需器材简单、操作方便、试验结果判定快速等优点,对保护动物健康和减少养殖损失有重要的临床指导意义。但该试验影响因素很多,常导致试验结果不准确。文章从试验器材、稀释液、抗原和阳性血清、待检血清、红细胞及其他影响因素进行综合分析,旨在避免或减少不利因素的影响,提高试验的准确性。     

Hemagglutination by canine parvovirus: serologic studies and diagnostic applications

Conditions for canine parvoviral hemagglutination (HA) and hemagglutination-inhibition (HI) reactions were defined. The HA phenomena were used to differentiate canine parvovirus (CPV) from feline panleukopenia virus (FPV), mink enteritis virus (MEV), and minute virus of canines. Serologic comparisons of the CPV, FPV, and MEV by HA-HI and serum-neutralization tests indicated that CPV, FPV, and MEV were antigenically similar but were different from minute virus of canines. Diagnostic application of HA tests to fecal samples from acute cases of enteritis was discussed. Combinating HA tests with HI tests on fecal samples provided a rapid and specific diagnostic method for CPV infection. Secular seroprevalence studies indicated the emergence of CPV infeciton in the United States dog population-at-large in 1978.