CN104090101A - Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof - Google Patents

Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof Download PDF

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CN104090101A
CN104090101A CN201410346920.9A CN201410346920A CN104090101A CN 104090101 A CN104090101 A CN 104090101A CN 201410346920 A CN201410346920 A CN 201410346920A CN 104090101 A CN104090101 A CN 104090101A
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hiv
magnetic particles
antigen
antibody
gp41
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CN201410346920.9A
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姚继承
李晓燕
饶志明
闻雯
刘海光
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威海威高生物科技有限公司
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea

Abstract

The invention belongs to the technical field of immunologic diagnosis and particularly relates to a human immunodeficiency virus (HIV) antibody detection kit using a microparticle chemiluminiscence method and a preparation method of the kit. The kit is composed of magnetic microparticles for detecting an HIV antibody, a tracing conjugate for detecting the HIV antibody, a negative control, a I type positive control, a II type positive control and an analyzing buffering solution. The invention further discloses the preparation method of the detection kit, which adopts a particle chemiluminiscence immunoassay technology; compared with ELISA (Enzyme-Linked Immunosorbent Assay), the technology has higher sensitivity and specificity and is suitable for the clinical auxiliary diagnosis of HIV.

Description

人类免疫缺陷病毒抗体检测试剂盒及其制备方法 Human immunodeficiency virus antibody test kit and its preparation method

技术领域 FIELD

[0001] 本发明涉及一种免疫诊断技术,具体地说是一种磁微粒作为载体的人类免疫缺陷病毒抗体检测试剂盒及其制备方法。 [0001] The present invention relates to an immunological diagnostic technique, particularly a virus detection kit and method for preparing an antibody of magnetic particles as carriers of human immunodeficiency.

背景技术 Background technique

[0002] 人类免疫缺陷病毒(Human Immunodeficiency Virus, HIV)是一种感染人类免疫系统细胞的慢病毒,属反转录病毒的一种。 [0002] Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is an infection of the human immune system cells slow the virus, is a retrovirus of. 普遍认为,人类免疫缺陷病毒的感染导致艾滋病(AIDS, Acquired Immune Deficiency Syndrome,后天免疫缺乏综合症,或译作"爱滋病"), 艾滋病是后天性细胞免疫功能出现缺陷而导致严重随机感染及/或继发肿瘤并致命的一种疾病。 Generally believed that infection with human immunodeficiency virus that causes AIDS (AIDS, Acquired Immune Deficiency Syndrome, acquired immune deficiency syndrome, or translated as "AIDS"), AIDS is acquired cellular immune function defects caused severe opportunistic infections and / or following the tumors and a disease fatal. 艾滋病自1981年在美国被识别并发展为全球大流行,至2003年底,已累计导致两千余万人死亡。 Since 1981, AIDS was identified and developed into a global pandemic in the United States, until the end of 2003, has led to a total of more than two million deaths. 人类免疫缺陷病毒通常也俗称为"艾滋病病毒"或"艾滋病毒"。 Human immunodeficiency virus is often known as the "AIDS virus" or "HIV."

[0003] HIV病毒呈球形或卵圆形颗粒,直径100-140nm,是带有包膜的RNA逆转录病毒,在分类上属于逆转录病毒科中的慢病毒亚科。 [0003] HIV virus oval or spherical particle diameter of 100 to 140 nm, is coated with an RNA retrovirus, lentivirus subfamily belonging to the retrovirus family in the classification. HIV病毒单拷贝基因组RNA长约9. 2-9. 7Kb。 Single copy of HIV RNA genome is about 9. 2-9. 7Kb. 目前发现有HIV-1和HIV-2两型,这两种类型在生物学特性上很相似,它们的基因组有40%-50%的同源性,其中两种病毒的核心蛋白具有高度的交叉反应。 There has now been found HIV-1 and HIV-2 type two, both types are similar in biological properties, their genome is 40% -50% homology, viral core protein of two highly cross reaction. HIV有三个主要的结构基因:env、gag、pol,他们分别编码产生不同功能的蛋白质即抗原,抗原会引起机体免疫应答从而产生相应的抗体。 HIV has three major structural genes: env, gag, pol, respectively, they have different functions, i.e. protein antigens, the immune response will cause an antigen to produce antibodies encoded thereby.

[0004] HIV侵入机体后,一般在6周左右产生免疫应答,产生抗体。 [0004] HIV invade the body, the immune response is generally about 6 weeks to produce antibodies. 血清中首先出现HIV 前体p55抗体和核心蛋白p24 ;然后出现外膜蛋白gpl20抗体,跨膜糖蛋白gp41抗体,其他的如P66和p32的抗体也陆续出现。 Serum p55 antibodies and p24 HIV core protein precursors appears first; then appeared outer membrane protein gpl20 antibody, transmembrane glycoprotein gp41 antibodies, other antibodies such as P66 and p32 are starting to appear. 经过几个月至几年后,当病毒再次出现在血液中,血清中的p24、p55抗体水平下降,而其他抗体持续存在或水平上升。 After a few months to a few years later, when the virus reappears in the blood, serum p24, p55 antibody levels decline, while others persist or antibody levels to rise. 通过检测这些不同抗体来诊断是否感染HIV。 By detecting these different antibodies to diagnose whether or not infected with HIV. 在我国流行的主要是HIV-1型。 Endemic in our country are mainly HIV-1 type.

[0005] 爱滋病毒是透过交换体液来传播的,特别是精液和血液。 [0005] HIV is transmitted through the exchange of body fluids to spread, particularly in the semen and blood. 最常见的传染途径是:进行阴道或肛门性交,共用沾污了的针筒,受病毒感染的母亲传播给婴儿。 The most common route of infection is: vaginal or anal intercourse, sharing of contaminated syringes, spread by infected mothers to infants. 另外,亦有越来越多个案显示,感染了病毒的母亲可经喂母乳而把病毒传给婴儿。 In addition, there are also more and more cases show that infected mothers can breast-feed while by the virus to their babies.

[0006] 如果不进行治疗,根据HIV不同亚型,感染艾滋病毒后的的净存活时间平均为9至11年,而诊断为AIDS之后,如果在资源受限导致无法治疗的情况下,根据不同的研究表明, 平均存活时间在6至19个月之间。 [0006] After Without treatment, according to the different subtypes of HIV, net survival time after infection with HIV an average of 9-11 years, diagnosed with AIDS, if resources are limited in the case of lead can not be treated, depending on the study showed that the average survival time is between 6-19 months. 而在医疗资源充足的地区,用高效抗逆转录药物(HAART) 的作为有效治疗手段治疗HIV感染者和AIDS患者,可以让死亡率减少80%,并能将新诊断出的HIV感染者的寿命延长最少30年。 And adequate medical resources in areas with highly active anti-retroviral drugs (HAART) therapy as an effective treatment for HIV-infected people and AIDS patients, allowing 80% reduction in mortality, HIV infection and can newly diagnosed life extend a minimum of 30 years.

[0007] 至今还没有研制成功可以预防艾滋的疫苗,防治艾滋只有早期进行诊断,才能在早期进行治疗并防治病情的进一步恶化,否则将会传染给他人和使自身病情加重、影响身体健康和传给后代。 [0007] has yet to be successfully developed a vaccine to prevent HIV, and prevention and treatment of HIV only early diagnosis, early treatment and to prevention of further deterioration of the condition, otherwise it will infect others and make their condition worse, affecting their health and pass to their offspring. 因此有必要开发出一种性能较好的诊断方法,能有效地在早期就将该疾病诊断出来,及时治疗,减少因艾滋带来的健康和财产的损失。 It is therefore necessary to develop a better performance diagnostic methods, it can effectively diagnose the disease at an early out, timely treatment, to reduce losses caused by orphaned by health and property.

[0008] 人类免疫缺陷病毒抗体的传统检测方法包括酶联免疫法、胶体金法及化学发光检测法,这些方法虽然具有很多优点,但在检测的灵敏度、特异性、稳定性等方面还有待进一步提高。 Traditional methods [0008] Human immunodeficiency virus antibodies by ELISA include colloidal gold and chemiluminescence detection methods, although these methods have many advantages, but in the detection sensitivity, specificity, stability and the like to be further improve. 全自动微粒子化学发光免疫分析是在酶免疫分析基础上结合了高灵敏度的化学发光测定技术和磁性微粒分离技术,与其他方法相比,这种方法有许多独特的优点,首先它用顺磁性微粒作为固相载体,由于颗粒体积小,表面积大,扩大了反应面积,大大提高了检测灵敏度;其次由于使用全自动仪器及配套试剂,使人为因素减至最低,提高了方法的稳定性和结果的重复性,同时也使得批内差异与批间差异都较小。 Automatic luminescent immunoassay chemistry is the combination of fine highly sensitive chemiluminescent enzyme immunoassay based on the measurement techniques and the magnetic particle separation technology, compared with other methods, this method has many unique advantages, first it with paramagnetic microparticles as solid support, since small particle size, large surface area, the reaction area is enlarged, greatly improving the detection sensitivity; secondly the use of automated equipment and reagents, makes factor is minimized, and improve the stability results of the method of repeatability, but also makes intra-assay and inter-assay differences are small differences. 与放射免疫法相比,微粒子化学发光法除具有高灵敏度、高精确度、高可靠性等优点外,还具有如下优点:a.无放射性污染,稳定性好;b.特异性高;c.试剂可随用随取,测定方便迅速,可作为急诊检测项目。 Compared with radioimmunoassay, chemiluminescent addition to having the advantages of high sensitivity, high accuracy, high reliability, but also has the following advantages: a non-radioactive contamination, good stability; high specific B; C agents.. It can be used with the check, to facilitate the rapid measurement can be used as emergency test items. 根据大量的实验结果以及临床应用资料,从实用性、稳定性、准确性及其发展前景来看,该方法逐渐成为取代放射性免疫分析和酶免疫分析的首选。 According to the results of a large number of experimental data and clinical applications, from practicality, stability, accuracy and development perspective, which is becoming replaced by radioimmunoassay and enzyme immunoassay of choice.

发明内容 SUMMARY

[0009] 本发明所要解决的技术问题是克服上述现有技术的不足,提供一种具有高灵敏度和特异性,适合于临床人类免疫缺陷病毒的辅助诊断的微粒子化学发光法HIV抗体检测试剂盒及其制备方法。 [0009] The present invention solves the technical problem is to overcome the above deficiencies of the prior art, to provide a high sensitivity and specificity, suitable for clinical diagnosis of the human immunodeficiency virus HIV antibody chemiluminescent detection kit and methods for their preparation.

[0010] 本发明解决上述技术问题采用的技术方案是:一种人类免疫缺陷病毒抗体检测试剂盒,其包括测HIV抗体磁微粒、测HIV抗体示踪结合物、阴性对照、I型阳性对照、II型阳性对照、分析缓冲液,所述测HIV抗体磁微粒为标记有HIV抗原(gp41、gp36)的磁性微粒子; 测HIV抗体示踪结合物为标记有HIV抗原(gp41、gp36)的异鲁米诺;I型阳性对照为含有HIV-1抗体的人血清/血浆;II型阳性对照为含有HIV-2抗体的兔血清/血浆;分析缓冲液为含牛血清白蛋白的磷酸盐缓冲液。 [0010] Solution to the technical problem of the present invention is employed: one kind of human immunodeficiency virus antibody test kit comprising magnetic particles measuring HIV antibody, HIV antibody test tracer conjugate, a negative control, positive control Type I, type II positive controls, assay buffer, said magnetic particles to measure HIV antibody labeled with HIV antigen (gp41, gp36) magnetic particles; measured HIV antibody conjugate is labeled with a tracer HIV antigen (gp41, gp36) iso-Lu minocycline; type I human positive control containing HIV-1 antibodies in serum / plasma; type II positive control containing HIV-2 antibodies in rabbit serum / plasma; assay buffer is a phosphate buffer containing bovine serum albumin.

[0011] 本发明还提供了上述人类免疫缺陷病毒抗体检测试剂盒的制备方法,其特征是: 具体步骤如下: (1)制备测HIV抗体磁微粒将带羧基磁微粒与EDC按质量比1 : 2,磁微粒与gp41 的比例为每毫克磁微粒标记2(^g的gp41,磁微粒与gp36的比例为每毫克磁微粒标记25Pg 的gp36,在22~26°C条件下进行混匀标记,标记时间1小时;标记后采用甘氨酸封闭多余的位点,使之浓度达到25mM,反应0. 5小时,洗涤三次,加入磁微粒保存液(含1%牛血清白蛋白的0. 01M PBS缓冲系统),使之终浓度达到每20 μ L测HIV抗体磁微粒中分别含有6(^g的磁微粒标记gp41抗原和5〇μ〖的磁微粒标记gp36抗原,2~8°C保存。 [0011] The present invention also provides the above human immunodeficiency preparing virus antibody test kit defect, characterized in that: the following steps: (1) Preparation of measuring antibodies to HIV magnetic particles will take carboxyl magnetic particles with EDC mass ratio of 1: 2, and the ratio of the magnetic particles per mg of gp41 labeled magnetic particles 2 (^ g of gp41, gp36 ratio of the magnetic particles and the magnetic particles per milligram of gp36 25Pg labeled, marked by kneading at 22 ~ 26 ° C conditions, numerals 1 hour; closed after labeling using the extra glycine site, so that a concentration of 25 mM, the reaction 0.5 hours, washed three times, added magnetic particles preservation solution (containing 1% bovine serum albumin in 0. 01M PBS buffer system ), so that a final concentration of 6 (^ g magnetic particles and a labeled gp41 antigen 5〇μ 〖magnetic particles labeled gp36 antigen, 2 ~ 8 ° C storage measured every 20 μ L of magnetic particles contained in the HIV antibody, respectively.

[0012] (2)制备测HIV抗体示踪结合物HIV抗原(gp41、gp36)与异鲁米诺的标记,反应体系为:戊二醛使用浓度为1. 〇%-2. 0%,其最佳使用浓度为1. 25%,gp41与异鲁米诺的质量比为1 : 5,在22〜26°C反应1.5小时,用pH7. 2-7. 4的0. 01MPBS进行透析,透析后加入等体积甘油-20°C存放;戊二醛使用浓度为1. 0%-2. 0%,其最佳使用浓度为1. 25%,,gp36与异鲁米诺的质量比为1 : 5,在22〜26°C反应1.5小时,用pH7. 2-7. 4的0. 01M PBS进行透析,透析后加入等体积甘油_20°C存放;将异鲁米诺标记HIV抗原用示踪结合物稀释液(含20%小牛血清的0. 01MPBS缓冲系统)分别按稀释比例为异鲁米诺标记gp41抗原1 : 4000 稀释,异鲁米诺标记gp36抗原1 : 3000稀释,最终组成测HIV抗体示踪结合物。 [0012] (2) Preparation of tracer antibody conjugate measured HIV HIV antigen (gp41, gp36) labeled with isoluminol, the reaction system is: 1. square glutaraldehyde concentration% -20%, which optimal concentration of 1. 25%, gp41 isoluminol mass ratio is 1: 5, the reaction 22~26 ° C 1.5 hours of 2-74 with pH 7 0. 01MPBS dialysis, dialysis. after addition of an equal volume of glycerol storage -20 ° C; glutaraldehyde concentration of 1.0% -20% of its optimal concentration of 1.25% by mass ratio of gp36 ,, it is 1 and isoluminol. : 5, the reaction 22~26 ° C 1.5 hours, dialyzed against 0. 01M PBS pH7 2-7 4 and dialysis after adding an equal volume of glycerol storage _20 ° C; the HIV antigen labeled with isoluminol. tracer conjugate dilutions (0. 01MPBS buffer system containing 20% ​​calf serum) were diluted by a ratio of a heterologous antigen gp41 isoluminol: 4000 dilution, isoluminol gp36 antigen diluted 1: 3000, final HIV antibody test composition tracer conjugate.

[0013] (3)制备阴、阳性对照选用5份以上HIV抗体检测为阴性的人血清或血浆混合,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为阴性对照;选用5份以上HIV-1 抗体检测为阳性的人血清或血浆混合,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为I型阳性对照;选用HIV-2抗体检测为阳性的兔血清或血浆稀释后,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为II型阳性对照。 [0013] (3) Preparation of negative and positive selection control HIV antibody test is more than 5 parts by negative human serum or plasma mixture by 60 ° C, after 1 hour treatment, sterile filtration, storage at 2~8 ° C, as a negative control; selected HIV-1 antibody test is more than 5 parts by positive human serum or plasma mixture by 60 ° C, after 1 hour treatment, sterile filtered and stored at 2~8 ° C, as a positive control type I; selection HIV-2 antibody is diluted positive rabbit serum or plasma, by 60 ° C, after 1 hour treatment, sterile filtered and stored at 2~8 ° C, type II as a positive control.

[0014] (4)制备分析缓冲液磷酸二氢钠0.39g/L、磷酸氢二钠2.68g/L、氯化钠8. 50g/L、牛血清白蛋白10g/L、硫柳萊1.0g/L,按上述配方配制稀释液。 [0014] (4) Preparation of assay buffer Sodium dihydrogen phosphate 0.39g / L, disodium hydrogen phosphate 2.68g / L, NaCl 8. 50g / L, bovine serum albumin 10g / L, 1.0g thimerosal Levin / L, prepared by dilution of the above formulation.

[0015] 本发明的原理是利用微粒子化学发光免疫分析技术,采用双抗原夹心法,在磁微粒固相标记上HIV抗原(gp41、gp36),加入待测样本,反应形成磁微粒标记抗原-抗体结合物,温育后加入异鲁米诺标记的HIV抗原,反应后形成磁微粒标记抗原-抗体-异鲁米诺标记抗原复合物,建立免疫反应与化学发光的联系。 [0015] The principles of the present invention is a light emitting immunoassay utilizing microparticles chemistry using double antigen sandwich method, the magnetic particles in the solid phase labeled HIV antigen (gp41, gp36), was added a test sample, to form the magnetic particles labeled antigen - antibody conjugate was added after incubation isoluminol HIV antigen, the magnetic particles are formed after the reaction labeled antigen - antibody - antigen complex isoluminol established immune response chemiluminescent contact. 加入激发液催化标记抗原的异鲁米诺发出425nm的光,样本中HIV抗体的含量与发光值(RLU)呈正相关,根据S/C0值对样本检测结果进行判断。 Was added a catalytic excitation of labeled antigen isoluminol emitted light is 425nm, sample content and HIV antibody light emission value (RLU) were positively correlated, the sample is judged according to the detection results of S / C0 value.

[0016] 本发明试剂盒的优点是采用了微粒子化学发光免疫分析技术,比ELISA具有更高的灵敏度和更好的特异性。 Advantages [0016] The kit of the present invention is the use of a chemiluminescent immunoassay, ELISA than with higher sensitivity and better specificity.

具体实施方式 Detailed ways

[0017] 本发明试剂盒采用微粒子化学发光免疫分析技术,检测血清或血浆中是否存在HIV抗体。 The kit of the invention [0017] The use of the present chemiluminescent immunoassay, whether the presence of HIV antibodies in serum or plasma. 下面具体描述HIV抗体检测试剂盒及其制备方法。 HIV antibody test kit and its preparation method described in detail below.

[0018] 一种人类免疫缺陷病毒抗体检测试剂盒,其包括测HIV抗体磁微粒、测HIV抗体示踪结合物、阴性对照、I型阳性对照、II型阳性对照、分析缓冲液。 [0018] A human immunodeficiency virus antibody test kit comprising magnetic particles measuring HIV antibody, HIV antibody test tracer conjugate, a negative control, the positive control type I, type II positive controls, assay buffer. 所述测HIV抗体磁微粒为标记有HIV抗原(gp41、gp36)的磁性微粒子;测HIV抗体示踪结合物为标记有HIV抗原(gp41、gp36)的异鲁米诺;I型阳性对照为含有HIV-1抗体的人血清/血浆;II型阳性对照为含有HIV-2抗体的兔血清/血浆;分析缓冲液为含牛血清白蛋白的磷酸盐缓冲液。 The HIV antibody test is labeled with magnetic particles HIV antigen (gp41, gp36) magnetic particles; measured HIV antibody conjugate is labeled with a tracer HIV antigen (gp41, gp36) of isoluminol; positive control containing Type I HIV-1 antibody is human serum / plasma; type II positive control containing HIV-2 antibodies in rabbit serum / plasma; assay buffer is a phosphate buffer containing bovine serum albumin.

[0019] 本发明上述HIV抗体检测试剂盒的制备方法,其具体步骤如下: (1)制备测HIV抗体磁微粒将带羧基磁微粒与EDC按质量比1 : 2,磁微粒与gp41 的比例为每毫克磁微粒标记2(^g的gp41,磁微粒与gp36的比例为每毫克磁微粒标记25Pg 的gp36,在22~26°C条件下进行混匀标记,标记时间1小时;标记后采用甘氨酸封闭多余的位点,使之浓度达到25mM,反应0. 5小时,洗涤三次,加入磁微粒保存液(含1%牛血清白蛋白的0. 01M PBS缓冲系统),使之终浓度达到每20 μ L测HIV抗体磁微粒中分别含有6(^g的磁微粒标记gp41抗原和5〇μ〖的磁微粒标记gp36抗原,2~8°C保存。 [0019] The present invention is method for preparing the HIV antibody detection kit, the specific steps are as follows: (1) Preparation of measuring antibodies to HIV magnetic particles will take carboxyl magnetic particles with EDC mass ratio of 1: 2, the ratio of the magnetic particles and the gp41 is per mg of magnetic particles labeled 2 (^ g of the gp41, gp36 ratio of the magnetic particles and the magnetic particles per milligram of marks 25Pg gp36, is carried out at 22 ~ 26 ° C mixing condition flag, flag 1 hour; after labeling using glycine extra closure site, so that a concentration of 25 mM, the reaction 0.5 hours, washed three times, added magnetic particles preservation solution (0. 01M PBS buffer system containing 1% bovine serum albumin), so that a final concentration of 20 per HIV antibody test μ L of magnetic particles contained in each of 6 (^ g magnetic particles and a labeled gp41 antigen 5〇μ 〖magnetic particles labeled gp36 antigen, 2 ~ 8 ° C storage.

[0020] (2)制备测HIV抗体示踪结合物HIV抗原(gp41、gp36)与异鲁米诺的标记,反应体系为:戊二醛使用浓度为1. 〇%-2. 0%,其最佳使用浓度为1. 25%,gp41与异鲁米诺的质量比为1 : 5,在22〜26°C反应1.5小时,用pH7. 2-7. 4的0. 01MPBS进行透析,透析后加入等体积甘油-20°C存放;戊二醛使用浓度为1. 0%-2. 0%,其最佳使用浓度为1. 25%,,gp36与异鲁米诺的质量比为1 : 5,在22〜26°C反应1.5小时,用pH7. 2-7. 4的0. 01M PBS进行透析,透析后加入等体积甘油_20°C存放;将异鲁米诺标记HIV抗原用示踪结合物稀释液(含20%小牛血清的0. 01MPBS缓冲系统)分别按稀释比例为异鲁米诺标记gp41抗原1 : 4000 稀释,异鲁米诺标记gp36抗原1 : 3000稀释,最终组成测HIV抗体示踪结合物。 [0020] (2) Preparation of tracer antibody conjugate measured HIV HIV antigen (gp41, gp36) labeled with isoluminol, the reaction system is: 1. square glutaraldehyde concentration% -20%, which optimal concentration of 1. 25%, gp41 isoluminol mass ratio is 1: 5, the reaction 22~26 ° C 1.5 hours of 2-74 with pH 7 0. 01MPBS dialysis, dialysis. after addition of an equal volume of glycerol storage -20 ° C; glutaraldehyde concentration of 1.0% -20% of its optimal concentration of 1.25% by mass ratio of gp36 ,, it is 1 and isoluminol. : 5, the reaction 22~26 ° C 1.5 hours, dialyzed against 0. 01M PBS pH7 2-7 4 and dialysis after adding an equal volume of glycerol storage _20 ° C; the HIV antigen labeled with isoluminol. tracer conjugate dilutions (0. 01MPBS buffer system containing 20% ​​calf serum) were diluted by a ratio of a heterologous antigen gp41 isoluminol: 4000 dilution, isoluminol gp36 antigen diluted 1: 3000, final HIV antibody test composition tracer conjugate.

[0021] (3)制备阴、阳性对照选用5份以上HIV抗体检测为阴性的人血清或血浆混合,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为阴性对照;选用5份以上HIV-1 抗体检测为阳性的人血清或血浆混合,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为I型阳性对照;选用HIV-2抗体检测为阳性的兔血清或血浆稀释后,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为II型阳性对照。 [0021] (3) Preparation of negative and positive selection control HIV antibody test is more than 5 parts by negative human serum or plasma mixture by 60 ° C, after 1 hour treatment, sterile filtration, storage at 2~8 ° C, as a negative control; selected HIV-1 antibody test is more than 5 parts by positive human serum or plasma mixture by 60 ° C, after 1 hour treatment, sterile filtered and stored at 2~8 ° C, as a positive control type I; selection HIV-2 antibody is diluted positive rabbit serum or plasma, by 60 ° C, after 1 hour treatment, sterile filtered and stored at 2~8 ° C, type II as a positive control.

[0022] (4)制备分析缓冲液磷酸二氢钠0.39g/L、磷酸氢二钠2.68g/L、氯化钠8. 50g/L、牛血清白蛋白10g/L、硫柳萊1.0g/L,按上述配方配制稀释液。 [0022] (4) Preparation of assay buffer Sodium dihydrogen phosphate 0.39g / L, disodium hydrogen phosphate 2.68g / L, NaCl 8. 50g / L, bovine serum albumin 10g / L, 1.0g thimerosal Levin / L, prepared by dilution of the above formulation.

[0023] 本发明上述各种原材料的选择要求如下: 1、标记用HIV抗原的选择首先就抗原的外观、浓度、纯度和效价进行研究,抗原应为澄清透明的液体,不含异物,无摇不散的沉淀;用紫外吸收法检测其蛋白含量应不低于lmg/mL ;SDS-PAGE检测纯度应主带清晰,无明显杂带;效价应不低于标示效价且不低于1 : 10000,研究结果表明HIV抗原完全可用于本发明试剂盒的制备。 Select the required [0023] The present invention is the above-described various materials as follows: 1, selection markers Study HIV antigens to antigen first appearance, concentration, purity, and potency, the antigen should be transparent as a clear liquid, free from foreign bodies, no shake ghost precipitate; detected by UV absorption protein content should not be less than lmg / mL; SDS-PAGE the purity of the main belt should clear with no obvious heteroaryl; potency should not be less marked titer not less than 1: 10000, results show complete HIV antigen preparation kit of the invention may be used.

[0024] 2、磁微粒的选择通过对磁微粒的外观,标记蛋白的比率,磁响应性,磁微粒吸附一致性等方面进行分析,经过多次分析研究,将磁微粒混匀,在灯光下观察,易分散,无聚集,无异物;将蛋白采用不同的方法进行标记,标记率应大于90% ;将磁微粒置370-380特斯拉的磁铁上,观察磁微粒的聚集速度,分散均匀的磁微粒在10秒钟内完全聚集;磁微粒吸附一致性CV < 10%。 [0024] 2, the magnetic particles were selected by the appearance aspect of the magnetic particles, the ratio of marker protein, magnetically responsive, the magnetic particles adsorbed consistency analysis, after several analysis, the magnetic particles mixed in the light observed, easily dispersed, no aggregates, no foreign matter; protein labeled using different methods, mark rate should be greater than 90%; the upper magnet facing 370-380 Tesla magnetic particles, the magnetic particles was observed aggregation speed, a uniform dispersion magnetic particles fully gathered within 10 seconds; the magnetic particles adsorbed consistency CV <10%. 研究结果表明直径为0. 90-1. 10 μ m的磁微粒,含有羧基基团,标记率最高,可用于本发明诊断试剂盒的制备。 The results show that the diameter 0. 90-1. 10 μ m magnetic particles, having a carboxyl group, marking the highest rate, the preparation of diagnostic kits of the present invention may be used.

[0025] 3、异鲁米诺的选择将异鲁米诺用DMS0(二甲基亚砜)进行溶解,用纯化水进行稀释至1. 2 X 1(Γ5Μ的量,加入10 μ L异鲁米诺液体,各加入200 μ L激发液,测定其发光值, 发光值应> 160000,经过研究,符合要求的异鲁米诺可作为发光的原料。 [0025] 3, isoluminol isoluminol selection will be dissolved in DMSO (dimethyl sulfoxide) were diluted to an amount of 1. 2 X 1 (Γ5Μ with purified water, was added 10 μ L iso Lu minoxidil liquid, each excitation was added 200 μ L, measured values ​​of light emission, light emission value should be> 160,000, through research, to meet the requirements of isoluminol as a light emitting material.

[0026] 本发明HIV抗体检测试剂盒检测样品中HIV抗体的检测方法是:首先取出浓缩洗液,用纯化水按照倍数进行稀释。 [0026] The present invention HIV antibody test kit for the detection method for detecting HIV antibodies in a sample are: first removed washings was concentrated, diluted with purified water by a factor. 而后取出激发Α液和Β液,放置全自动化学发光测定仪合适的位置。 It was then removed and the excitation Α Β liquid, placing the appropriate position automated chemiluminescence analyzer. 激发A液为含有4%NaOH的缓冲液,激发B液为含有0. 12%H202的缓冲液。 A solution to the excitation buffer containing 4% NaOH, the excitation buffer solution B containing 0. 12% H202 in. 接着将试剂盒从冰箱中取出后,放于仪器试剂区至少混匀30分钟后方可使用,并严格按照设定的程序进行加样和温育。 After the kit then removed from the freezer, placed in the instrument reagent mixing region at least 30 minutes before use, and strictly loaded and incubated in accordance with the procedures set.

[0027] 具体加样方法如下:50 μ L血清/血浆样本、100 μ L分析缓冲液以及20 μ L测HIV 抗体磁微粒在37°C的条件下反应20分钟,加入150 μ L测HIV抗体示踪结合物,37°C反应20分钟,再用稀释后的洗液洗涤3次,最终分别加入200 μ L激发A液和激发B液,测定发光值,根据S/C0值对样本检测结果进行判断。 [0027] DETAILED loading method is as follows: 50 μ L of serum / plasma samples, 100 μ L of assay buffer and 20 μ L measured HIV antibody magnetic particles was reacted at 37 ° C for 20 minutes, was added 150 μ L measured HIV antibody tracer conjugate, 37 ° C for 20 minutes, then washed with diluted wash three times after the final 200 μ L were added to the excitation and the excitation B a solution was measured luminescence value, according to the S / C0 value of the sample results judge.

[0028] 本发明质量控制要求是:试剂盒中的阴、阳性对照用于试验过程控制。 [0028] Quality control requirements of the present invention is: a female kit positive control for controlling the test process. 阴性对照检测结果(S/C0)不得高于0. 8,阳性对照检测结果(S/C0)不得低于10。 A detection result of the negative control (S / C0) not more than 0.8, the detection result of the positive control (S / C0) not less than 10.

[0029] 本发明试剂盒检测HIV抗体国家参考品,检测的结果显示:该试剂盒的阴、阳性参考品符合率、最低检出限、精密度、稳定性等各项质量指标均符合国家要求。 [0029] The kit for detecting national reference, the result of the detection of HIV antibodies according to the present invention shows that: the quality indicators of the kit negative and positive reference materials coincidence detection limit, precision and stability are in line with national requirements . 阴性符合率: 20份阴性参考品符合率20/20 ;阳性符合率:20份阳性参考品符合率20/20,且P12 > P11 ; 最低检出限检测:SI、S2均为阴性,S3、S4、S5和S6均为阳性;精密度:10孔检测变异系数(CV)彡15% ;稳定性:试剂各组分于37°C放置6天,检定结果达到标准。 Negative coincidence rate: 20 negative samples accordance rate 20/20; positive coincidence rate: 20 parts by positive reference product conforms 20/20 ratio, and P12> P11; detection limit of detection: SI, S2 were negative, S3, S4, S5 and S6 are both positive; precision: 10 well assay coefficient of variation (CV) Pie 15%; stability: the components of the reagent placed in 37 ° C 6 days, the test results meet the standards.

[0030] 本发明试剂盒由河南中医学院第二附属医院、河南中医学院第三附属医院、中国人民解放军第一五三中心医院、中国人民解放军艾滋病检测确认实验室、北京市疾病预防控制中心五家临床检测机构进行临床考核,结果: (1)河南中医学院第二附属医院考核307例阴性样本和3例阳性样本,本发明试剂盒阴性样本307例全部检出,阳性样本3例全部检出。 [0030] The kit of the present invention consists of Henan Second Affiliated Hospital Medicine, Third Affiliated Hospital of Henan College PLA first fifty-three central hospital, laboratory testing to confirm the PLA AIDS, Beijing CDC five clinical detection means home clinical examination results: (1) second Affiliated hospital of Traditional Chinese Medicine 307 cases of negative samples and assessment of three cases of positive samples, the kit of the present invention, 307 cases of negative samples detected in all positive cases were all detected in sample 3 . 该试剂盒阴性检出率为100%,阳性检出率 The kit negative detection rate of 100%, the positive rate

Claims (3)

1. 一种人类免疫缺陷病毒抗体检测试剂盒,其包括测HIV抗体磁微粒、测HIV抗体示踪结合物、阴性对照、I型阳性对照、II型阳性对照、分析缓冲液,其特征是:所述测HIV抗体磁微粒为分别标记有HIV抗原gp41和HIV抗原gp36的磁性微粒子混合物;测HIV抗体示踪结合物为分别标记有HIV抗原gp41和HIV抗原gp36的异鲁米诺混合物;I型阳性对照为含有HIV-1抗体的人血清/血浆;II型阳性对照为含有HIV-2抗体的兔血清/血浆;分析缓冲液为含牛血清白蛋白的磷酸盐缓冲液。 A human immunodeficiency virus antibody test kit comprising magnetic particles measuring HIV antibody, HIV antibody test tracer conjugate, a negative control, the positive control type I, type II positive controls, assay buffer, wherein: the measured magnetic particles were HIV antibody labeled with gp41 HIV antigen and HIV antigen gp36 magnetic microparticle mixture; HIV antibody test tracer conjugates were labeled with isoluminol mixture was HIV antigen gp41 and gp36 of HIV antigen; type I human positive control containing HIV-1 antibodies in serum / plasma; type II positive control containing HIV-2 antibodies in rabbit serum / plasma; assay buffer is a phosphate buffer containing bovine serum albumin.
2. -种制备权利要求1所述人类免疫缺陷病毒抗体检测试剂盒的制备方法,其特征是具体步骤如下: (1) 制备测HIV抗体磁微粒将带羧基磁微粒与EDC按质量比1 : 2,磁微粒与HIV 抗原gp41的比例为每毫克磁微粒标记2(^g的HIV抗原gp41,磁微粒与HIV抗原gp36的比例为每毫克磁微粒标记25Pg的HIV抗原gp36,在22~26°C条件下进行混匀标记,标记时间1小时;标记后采用甘氨酸封闭多余的位点,使之浓度达到25禮,反应0. 5小时,洗涤三次,加入磁微粒保存液,所述磁微粒保存液为含1%牛血清白蛋白的0. 01M PBS缓冲系统,使之终浓度达到每20 μ L测HIV抗体磁微粒中分别含有6(^g的磁微粒标记HIV抗原gp41和5(^g的磁微粒标记HIV抗原gp36, 2〜8°C保存; (2) 制备测HIV抗体示踪结合物HIV抗原gp41、HIV抗原gp36与异鲁米诺的标记,反应体系为:戊二醛浓度为1. 0-2. 0%,HIV抗原gp41与异鲁米诺的质量比为1 : 5, 2. - The preparation of a human immunodeficiency virus preparation as claimed in claim species antibody detection kit defect, wherein the following steps: (1) Preparation of antibodies to HIV detected magnetic particles magnetic particles having a carboxyl group and the EDC mass ratio of 1: 2, the ratio of the magnetic particles with an HIV antigen gp41 per mg of magnetic particles labeled 2 (^ g HIV antigen gp41, the ratio of the magnetic particles with an HIV antigen gp36 per mg of magnetic particles labeled 25Pg HIV antigen gp36, at 22 ~ 26 ° C under mixing conditions for marking, marking time 1 hour; closed after labeling using the extra glycine site, so as to reach a concentration of 25 ceremony, the reaction 0.5 hours, washed three times preservation solution was added magnetic particles, the magnetic particles stored It was 0. 01M PBS buffer containing 1% bovine serum albumin system, making a final concentration of 6 (^ g magnetic particles labeled HIV antigen gp41 and 5 each measuring 20 μ L of magnetic particles containing HIV antibodies, respectively (^ g magnetic particles labeled HIV antigen gp36, 2~8 ° C stored; (2) preparation of HIV antibody test for HIV antigen tracer binding the gp41, gp36 HIV antigen labeled with isoluminol, reaction system: concentration of glutaraldehyde 1. 0-20% by mass than HIV gp41 antigen as isoluminol and 1: 5, 22〜26°C反应1. 5小时,用pH7. 2-7. 4的0. 01M PBS进行透析,透析后加入等体积甘油-20°C 存放;戊二醛浓度为1. 0-2. 0%,HIV抗原gp36与异鲁米诺的质量比为1 : 5,在22〜26°C反应1. 5小时,用pH7. 2-7. 4的0. 01M PBS进行透析,透析后加入等体积甘油-20°C存放;将异鲁米诺标记HIV抗原用示踪结合物稀释液,所述示踪结合物稀释液为含20%小牛血清的0.01MPBS缓冲系统,分别按稀释比例为异鲁米诺标记gp41抗原1 : 4000稀释,异鲁米诺标记gp36抗原1 : 3000稀释,最终组成测HIV抗体示踪结合物; (3 )制备阴、阳性对照选用5份以上HIV抗体检测为阴性的人血清或血浆混合,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为阴性对照;选用5份以上HIV-1抗体检测为阳性的人血清或血浆混合,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为I型阳性对照;选用HIV-2抗体检测为阳性的兔血 22~26 ° C reaction was 1.5 hours, dialyzed against pH7 2-7 4 of 0. 01M PBS, an equal volume of glycerol was added after dialysis stored -20 ° C;. 1. 0-2 of glutaraldehyde. 0%, and the mass ratio of HIV gp36 antigen as isoluminol of 1: 5, reaction was 1.5 hours at 22~26 ° C, dialyzed against 0. 01M PBS pH7 2-7 4, and after dialysis was added An equal volume of glycerol storage -20 ° C; the HIV antigen labeled with isoluminol conjugate diluent tracer, 0.01MPBS conjugate dilution buffer system containing 20% ​​fetal calf serum of the tracer, respectively dilution ratio a gp41 antigen as isoluminol different: 4000 dilution, isoluminol gp36 antigen 1: 3000 dilution, the final composition of the measured tracer conjugate-HIV antibody; (3) producing a negative, a positive control selection of more than 5 parts by HIV antibody test negative human serum or plasma mixture by 60 ° C, after 1 hour treatment, sterile filtration, storage at 2~8 ° C, as a negative control; selected HIV-1 antibody test is more than 5 parts by positive human sera or mixing plasma by 60 ° C, after 1 hour treatment, sterile filtered and stored at 2~8 ° C, as a positive control type I; selection of HIV-2 antibody positive rabbit blood 或血浆稀释后,经60°C、1小时处理后,除菌过滤,于2〜8°C保存,作为II型阳性对照; (4)制备分析缓冲液磷酸二氢钠0.39g/L、磷酸氢二钠2.68g/L、氯化钠8.50g/L、 牛血清白蛋白l〇g/L、硫柳汞1.0g/L,按上述配方配制稀释液。 Plasma or diluted by 60 ° C, after 1 hour treatment, sterile filtered and stored at 2~8 ° C, type II as a positive control; (4) Preparation of assay buffer Sodium dihydrogen phosphate 0.39g / L, phosphoric acid 2.68 g of disodium hydrogen / L, sodium chloride 8.50 g / L, bovine serum albumin l〇g / L, 1.0 g of thimerosal / L, prepared by dilution of the above formulation.
3. 根据权利要求2所述人类免疫缺陷病毒抗体检测试剂盒的制备方法,其特征在于: 步骤(2)中戊二醛使用浓度为1. 25%。 3. The method for preparing virus antibody test kit according to the defective human immunodeficiency claim 2, wherein: the step (2) using glutaraldehyde concentration 1.25%.
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CN105954510A (en) * 2016-06-30 2016-09-21 深圳市亚辉龙生物科技股份有限公司 Chemiluminescence immunodetection kit for anti-beta 2 glycoprotein I antibodies IgG and preparation method of kit
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