Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (8): 1676-1684.doi: 10.3864/j.issn.0578-1752.2022.08.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Method Improvement and Its Application of Micro Complement Fixation Test for Brucellosis

JIANG Hui1(),FENG Yu2,QIN YuMing2,ZHU LiangQuan2,FAN XueZheng1,DING JiaBo1()   

  1. 1Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193
    2National/OIE Reference Laboratory for Brucellosis, China Institute of Veterinary Drug Control, Beijing 100081
  • Received:2021-04-12 Accepted:2021-05-31 Online:2022-04-16 Published:2022-05-11
  • Contact: JiaBo DING E-mail:15011216921@163.com;dingjiabo@126.com

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Abstract:

【Objective】 The complement fixation test is used as a confirmatory test for the serodiagnosis of brucellosis. The complement fixation tests are divided into constant complement fixation test (CFT) and micro complement fixation test (mCFT) in diagnostic techniques for animal brucellosis (GB/T 18646-2018). However, there are differences in the related technical parameters and judgment of the result between them, which lead to the inconsistency of CFT and mCFT results. In this study, By the improvement and optimization of the parameters of mCFT, this study made the detection results of mCFT equivalent to classical CFT, and achieved the high-throughput and accurate diagnosis of CFT for brucellosis. 【Method】 According to classical CFT reaction conditions and diagnostic criteria, the reaction time, diluent, titer determination, result judgment and other related parameters for the current national standard of mCFT were optimized and improved. Compared with the original method, the dilution of the tested serum was changed from 1:2-1:64 to 1:10. The dilution method was the same as classical CFT so that the detection critical value was consistent with the constant method. The OD600 value was read by the microplate reader to determine the hemolysis intensity, which reduced the error caused by visual judgment and improved the efficiency and accuracy of result judgment. The reaction time was shortened from 30 min to 20 min in water bath at 37℃, which improved the detection efficiency without affecting the results. The diluent was changed from barbital buffer to normal saline, which used the same diluent of classical CFT and achieved satisfactory results. The 409 clinical bovine and sheep serum samples (163 bovine serum samples and 246 sheep serum samples) from Inner Mongolia, Shandong, Beijing and Jiangsu were detected by improved mCFT, the national standard mCFT, and classical CFT, respectively. The coincidence rate was analyzed and compared. 【Result】By detecting the 409 clinical samples, the result showed that 53 samples were positive, 11 samples were suspicious and 345 were negative by improved mCFT. The 53 positive samples, 9 suspicious samples and 347 negative samples were detected by classical CFT. Compared with the results of the two methods, only two bovine serum samples were suspicious by the improved mCFT, which were negative by classical CFT. The results of the other 407 samples were consistent. The coincidence rate of bovine serum samples was 98.77%, which of sheep serum samples was 100%, and the total coincidence rate was 99.51% between improved mCFT and classical CFT. However, the positive criteria of national standard mCFT were lower than that of classical CFT, and no suspicious interval was set. The results showed that 97 samples were positive and 312 samples were negative. Compared with the results of classical CFT, the coincidence rate of bovine serum samples was 88.34%, which of sheep serum samples was 89.84%; the total coincidence rate was 89.24%, which was far lower than 99.51% of improved mCFT and classical CFT. 【Conclusion】 By optimizing the parameters of mCFT, the mCFT method was established, which was highly consistent with the criterion and the results of the classical CFT.

Key words: brucellosis, micro complement fixation test, method improvement, application

Table 1

Preparation of standard colorimetric hole (μL)"

组分
Components		 标准溶血孔Standard hemolytic hole
1 2 3 4 5 6 7 8 9 10 11
100%溶血液100% hemolytic solution 100 90 80 70 60 50 40 30 20 10 0
100%不溶血液100% no hemolytic solution 0 10 20 30 40 50 60 70 80 90 100
溶血程度Hemolysis degree 100 90 80 70 60 50 40 30 20 10 0
判定符号Symbol - + + ++ ++ ++ +++ +++ +++ ++++ ++++
判定标准Standard 阴性Negative 可疑Suspicious 阳性Positive

Table 2

Dilution of the hemolysin (mL)"

EP管号
EP tube No.		 1 2 3 4 5 6 7 8 9 10
1﹕500稀释溶血素
1﹕500 hemolysin		 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
生理盐水Saline 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
稀释度Dilution 1﹕1000 1﹕1500 1﹕2000 1﹕2500 1﹕3000 1﹕3500 1﹕4000 1﹕4500 1﹕5000 1﹕5500

Table 3

Determination of the hemolysin titer (μL)"

孔号 Hole No. 1 2 3 4 5 6 7 8 9 10
各稀释度溶血素
Dilution of the hemolysin		 1﹕1000
25		 1﹕1500
25		 1﹕2000
25		 1﹕2500
25		 1﹕3000
25		 1﹕3500
25		 1﹕4000
25		 1﹕4500
25		 1﹕5000
25		 1﹕5500
25
2.5%红细胞
2.5% SRBC		 25 25 25 25 25 25 25 25 25 25
1﹕10补体
1﹕10 Complement		 25 25 25 25 25 25 25 25 25 25
生理盐水 Saline 50 50 50 50 50 50 50 50 50 50
37℃水浴20 min 37℃ water bath for 20 min

Table 4

Determination of the complement titer (μL)"

组分
Components		 列数Column number
1 2 3 4 5 6 7 8 9 10 11 12
1﹕10补体
1﹕10 Complement		 5 6.5 8 9.5 11 12.5 14 15.5 17 18.5 20 0
A：抗原/B：生理盐水
A:Antigen/B:Saline		 25 25 25 25 25 25 25 25 25 25 25 25
生理盐水Saline 45 43.5 42 40.5 39 37.5 36 34.5 33 31.5 30 50
37℃水浴20 min 37℃ water bath for 20 min
2个单位溶血素
2 units of hemolysin		 25 25 25 25 25 25 25 25 25 25 25 25
2.5%红细胞 2.5% SRBC 25 25 25 25 25 25 25 25 25 25 25 25
37℃水浴20min 37℃ water bath for 20min

Table 5

Determination of the antigen titer (μL)"

组分
Components		 列数Column number 对照
Control
1 2 3 4 5 6 7 8 9 10
各稀释度抗原
Dilution of the antigen		 1﹕10
25		 1﹕50
25		 1﹕75
25		 1﹕100
25		 1﹕150
25		 1﹕200
25		 1﹕250
25		 1﹕300
25		 1﹕400
25		 1﹕500
25		 25
各稀释度阴/阳性血清
Dilution of the negative/positive serum		 25 25 25 25 25 25 25 25 25 25 0
工作补体Working complement 25 25 25 25 25 25 25 25 25 25 25
生理盐水Saline 0 0 0 0 0 0 0 0 0 0 25
37℃水浴20 min 37℃ water bath for 20min
2个单位溶血素
2 units of hemolysin		 25 25 25 25 25 25 25 25 25 25 25
2.5%红细胞 2.5% SRBC 25 25 25 25 25 25 25 25 25 25 25
37℃水浴20 min 37℃ water bath for 20 min

Table 6

Detection of the clinical samples (μL)"

组分
Components		 临床样品组
Clinical sample group		 对照组
Control group
检测孔
Detection hole		 对照孔
Control hole		 阳性血清
Positive serum		 阴性血清
Negative serum		 补体
Complement		 抗原
Antigen		 溶血素
Hemolysin
1﹕10血清稀释液
1﹕10 dilution of the serum		 25 25 25 25 25 25 0 0 0
工作抗原Working antigen 25 0 25 0 25 0 0 25 0
工作补体Working complement 25 25 25 25 25 25 25 25 0
生理盐水Saline 0 25 0 25 0 25 50 25 75
37℃水浴20 min 37℃ water bath for 20 min
2个单位溶血素
2 units of hemolysin		 25 25 25 25 25 25 25 25 25
2.5%红细胞2.5% SRBC 25 25 25 25 25 25 25 25 25
37℃水浴20 min 37℃ water bath for 20 min

Table 7

The result of brucellosis complement fixation test of the clinical samples"

检测方法
Detection method		 血清种类
Serum type		 总数
Total number		 阳性数（阳性率）
Positive number (Positive rate)		 可疑数（可疑率）
Suspicious number (Suspicious rate)		 阴性数（阴性率）
Negative number (Negative rate)		 与常量法CFT符合率
Coincidence rate with the classical CFT
常量法CFT
Classical CFT		 牛血清Bovine serum 163 30 (18.41%) 7 (4.29%) 126 (77.3%) /
羊血清Sheep serum 246 23 (9.35%) 2 (0.81%) 221 (89.84%) /
合计Total 409 53 (12.96%) 9 (2.2%) 347 (84.84%) /
国标mCFT
National standard mCFT		 牛血清Bovine serum 163 49 (30.06%) 0 114 (69.94%) 88.34%
羊血清Sheep serum 246 48 (19.51%) 0 198 (80.49%) 89.84%
合计Total 409 97 (23.72%) 0 312 (76.28%) 89.24%
改进mCFT
Improved mCFT		 牛血清Bovine serum 163 30 (18.41%) 9 (5.52%) 124 (76.07%) 98.77%
羊血清Sheep serum 246 23 (9.35%) 2 (0.81%) 221 (89.84%) 100%
合计Total 409 53 (12.96%) 11 (2.69%) 345 (84.35%) 99.51%
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