A HA / HI detection kit for rapid detection of BCoV titer and neutralizing antibodies

Abstract

The present invention belongs to the field of cell engineering and specifically discloses a HA / HI detection kit for rapidly detecting BCoV titers and neutralizing antibodies. The kit comprises the following components: 1 mL of inactivated BCoV virus; 30 mL of PBS buffer containing 0.1% bovine serum albumin (BSA); a positive serum control: bovine serum with positive BCoV antibodies; a negative serum control: bovine serum with negative BCoV antibodies; and 1% fresh Chinese hamster erythrocytes or a 1% aldehyded Chinese hamster erythrocyte suspension. The kit is simple to use, saving time, and can produce results within one hour. It provides a convenient and rapid means for identifying BCoV and evaluating the immune efficacy of vaccines.

Description

Technical Field

[0001] The invention relates to the field of cell engineering, in particular to an HA / HI detection kit for rapidly detecting BCoV titer and neutralizing antibodies. Background Art

[0002] Bovine coronavirus (BCoV) is a causative agent of bovine diarrhea discovered in the 1970s. Clinically, it presents as diarrhea and bloody stool in newborn calves, and severe watery diarrhea (sometimes with blood and mucus) in adult cattle during winter. The disease is primarily transmitted through the digestive and respiratory tracts and has a high morbidity rate. Once infected, it can easily cause herd-wide infection and induce secondary infections, resulting in significant economic losses for the intensive cattle farming industry. Currently, inactivated and live attenuated vaccines are the primary clinical prevention and control options. However, these vaccines pose biohazard risks and genetic variations that can lead to loss of efficacy. Therefore, evaluation of vaccine efficacy is crucial. Currently, there are no effective, rapid HA / HI assays in China for detecting bovine coronavirus titers and neutralizing antibodies. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides an HA / HI detection kit for rapid detection of BCoV titer and neutralizing antibodies. It is easy to operate, saves time, and the experimental operation is simple and the results can be obtained within 1 hour, providing a convenient and fast means for the identification of BCoV and the evaluation of vaccine immune effects.

[0004] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0005] A HA / HI test kit for rapid detection of BCoV titer and neutralizing antibodies, consisting of the following components:

[0006] Inactivated BCoV virus, 1 mL;

[0007] PBS buffer containing 0.1% bovine serum albumin (BSA), 30 mL;

[0008] Positive serum control: bovine BCoV antibody-positive serum;

[0009] Negative serum control: bovine BCoV antibody-negative serum;

[0010] Red blood cells: 1% fresh Chinese hamster erythrocytes or 1% aldehyde-treated Chinese hamster erythrocyte suspension.

[0011] Furthermore, the virus titer of the inactivated BCoV virus is 10 8.0 TCID 50 / mL.

[0012] Furthermore, the pH of the PBS buffer is 7.2.

[0013] Furthermore, the HI titer of the bovine BCoV antibody-positive serum is 1:64.

[0014] Furthermore, the HI titer of the bovine BCoV antibody-negative serum is less than or equal to 1:2.

[0015] Furthermore, the 1% fresh Chinese hamster erythrocytes or the 1% hydroformylation Chinese hamster erythrocyte suspension is obtained by the following method:

[0016] 1) Preparation of Chinese hamster erythrocytes

[0017] Fresh anticoagulated blood was collected from healthy adult Chinese hamsters; then suspended in an equal volume of physiological saline, centrifuged at 3000 rpm for 5 minutes, and the supernatant and white blood cells were discarded. Repeat this process three times to obtain Chinese hamster red blood cells; then the Chinese hamster red blood cells were mixed with Aldrich's solution and stored at 4°C for later use.

[0018] 2) Aldehydation of Chinese hamster erythrocytes

[0019] Chinese hamster erythrocytes were centrifuged and the supernatant discarded. The cells were then washed and centrifuged three times with 10-20 times the volume of packed Chinese hamster erythrocytes using normal saline. 0.1 mL of packed Chinese hamster erythrocytes was then mixed with 2.5 mL of PBS buffer (pH 7.2) and shaken thoroughly. 0.1 mL of 2.5% glutaraldehyde was then added dropwise while shaking the cells. The cells were shaken on a shaker at room temperature for 60 minutes. The cells were then washed three times with PBS buffer (pH 7.2) and twice with distilled water. Finally, a 10% Chinese hamster erythrocyte suspension was prepared in normal saline with 0.01% thimerosal and stored in a refrigerator at 4°C until use.

[0020] 3) Prepare 1% fresh Chinese hamster erythrocytes or 1% aldehyde-treated Chinese hamster erythrocytes

[0021] Take 9 mL of PBS buffer containing 0.1% bovine serum albumin (BSA), add 0.1 mL of the above-mentioned fresh Chinese hamster erythrocytes or 10% Chinese hamster erythrocyte suspension, mix well, and prepare 1% fresh Chinese hamster erythrocytes or aldehyded 1% Chinese hamster erythrocytes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The present invention uses hamster red blood cells. The BCoV HA / HI test has high sensitivity and strong specificity, and the red blood cell agglutination phenomenon is easy to observe and has the best effect;

[0024] 2) The present invention saves costs. Compared with PCR or ELISA, the required reagents and materials are easy to obtain and low in cost.

[0025] 3) The present invention saves time, the experimental operation is simple and the results can be obtained within 1 hour, which saves time.

[0026] 4) The kit of the present invention is easy to use and does not require specialized technicians. It can be operated according to the instructions and does not require special equipment. The results are intuitively visible and easy to determine. It is suitable for clinical and laboratory testing of small amounts or dion samples.

[0027] 5) The present invention can replace the TCID 50 The virus neutralization test is simple to operate and provides a convenient and fast means for the identification of BCoV and the evaluation of vaccine immune effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are the results of the HA test between BCoV strains and hamster erythrocytes.

[0029] Figure 2 TCID is the HA titer of BCoV-HLJ002 strain and the virus titer 50 Relationship diagram.

[0030] Figure 3 This is a graph showing the relationship between the HI value of the BCoV-HLJ002 strain and the neutralizing antibody titer. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] The strains and serum sources used in the following examples are: BCoV-HLJ001, BCoV-HLJ002, BCoV-HLJ323, and BCoV-HLJ325 strains were isolated, identified, and preserved in this laboratory (the isolation and identification methods refer to the master's thesis of this laboratory: Gao Guoqiang. Isolation and Identification of Bovine Coronavirus and Establishment of an Indirect ELISA Diagnostic Method for Recombinant N Protein [D]. Heilongjiang Bayi Agricultural University, 2018). BCoV-positive serum was prepared from cattle immunized with BCoV-HLJ002 strain, and BCoV-negative serum was collected from newborn calves that did not eat colostrum.

[0033] Example 1

[0034] Red blood cell preparation

[0035] Collect fresh anticoagulated blood from healthy adult chickens, mice, Chinese hamsters, and piglets. Resuspend the blood in an equal volume of normal saline and centrifuge at 3000 rpm for 5 minutes. Discard the supernatant and white blood cells. Repeat the above steps to wash the red blood cells three times. Then, mix the red blood cells with Aldrich's solution and store at 4°C until ready for use.

[0036] Aldehydation of erythrocytes

[0037] Centrifuge the pellet of Chinese hamster erythrocytes (RBCs), discard the supernatant, and then wash and centrifuge three times with a volume of physiological saline equivalent to 10-20 times the packed RBC volume. Mix 0.1 mL of packed RBCs with 2.5 mL of PBS (phosphate buffered saline) at pH 7.2 and shake thoroughly. Add 0.1 mL of 2.5% glutaraldehyde dropwise while shaking. Continue shaking on a shaker at room temperature for 60 minutes. Wash three times with pH 7.2 PBS and twice with distilled water. Finally, prepare a 10% RBC suspension in physiological saline with 0.01% thimerosal and store in a refrigerator at 4°C until use.

[0038] 1% fresh red blood cells and aldehyde red blood cells

[0039] 0.1 mL of the above-mentioned fresh red blood cells or aldehyde-treated red blood cell pellet was added to 9 mL of 0.1% BSA PBS solution, and the mixture was mixed evenly to prepare a 1% red blood cell suspension.

[0040] HA experimental operation

[0041] (1) Add 30 μL of PBS diluent (containing 0.1% BSA) to each well of a V-bottom 96-well microtiter plate, add 30 μL of BCoV solution to the first well, pipette evenly, and then perform 2-fold serial dilutions, i.e., 1:2, 1:2. 2 , 1:2 3 , 1:2 4 , 1:2 5 …1:2 10 Discard 30 μL of virus solution from the last well, and do not add virus solution to the 11th well as a control for red blood cell precipitation.

[0042] (2) Add 30 μL of 1% red blood cells to each well, repeat 3 times for each sample, and then place the microplate on a microplate oscillator and shake for 30 seconds to mix the red blood cells and virus evenly.

[0043] (3) Place the microplate in a sealed bag, seal it, and place it in a constant temperature incubator at 37°C for 1 hour.

[0044] (4) When the red blood cells in the control wells without virus solution are completely precipitated, observe the red blood cell agglutination in the wells of each experimental group, and determine the highest virus dilution at which the red blood cells are completely agglutinated as the virus hemagglutination titer or virus toxicity titer.

[0045] The results of the screening test of BCoV's agglutination effect on red blood cells of different animals showed that BCoV had no agglutination effect on piglet and sheep red blood cells, but had agglutination effect on Balb / c mouse, C57 mouse and chicken red blood cells. However, the agglutination phenomenon was not obvious, incomplete agglutination occurred, the agglutination endpoint was difficult to determine, and the hemagglutination titer was lower than that of Chinese hamster red blood cells. BCoV can produce a significant agglutination effect on Chinese hamster red blood cells, and the agglutination endpoint is easy to determine. Figure 1 The HA titers of erythrocytes from different animal sources and different BCoV strains vary greatly, as shown in Table 1. Based on the above results, Chinese hamster erythrocytes were selected as the erythrocytes for the BCoV HA test.

[0046] Table 1

[0047]

[0048] Therefore, this example uses Chinese hamster erythrocytes to prepare a HA / HI detection kit for rapid detection of BCoV titer and neutralizing antibodies, and the kit consists of the following components:

[0049] The virus titer was 10 8.0 TCID 50 / mL of inactivated BCoV virus, 1mL;

[0050] PBS buffer (pH 7.2) containing 0.1% bovine serum albumin (BSA), 30 mL;

[0051] Positive serum control: bovine BCoV antibody positive serum with an HI titer of 1:64;

[0052] Negative serum control: bovine BCoV antibody negative serum with HI titer less than 1:2;

[0053] Red blood cells: 1% fresh Chinese hamster erythrocytes or 1% aldehyde-treated Chinese hamster erythrocyte suspension.

[0054] In order to determine the HA titer and virus titer of BCoV (TCID 50 ) whether there is a connection, using the HA / HI detection kit for rapid detection of bovine coronavirus titer and neutralizing antibodies of this embodiment, the HA price was 1:2 8 The BCoV solution was diluted to 1:2 4 , 1:2 5 , 1:2 6 , 1:2 7 and 1:2 8 , and then the virus titer of the above hemagglutination value was measured respectively. Each group of experiments was repeated 3 times, and then the curve equation of BCoV hemagglutination value and virus titer was drawn.

[0055] The virus titer of BCoV with different hemagglutination values ​​was obtained by measuring the virus titer, as shown in Table 2. HA and TCID were fitted by EXCELL. 50 The equation between them is y=0.9497x+0.9673(R 2 =0.999, x is log2 HI, y is log 10 TCID 50 ),See Figure 2 . Established the BCoV and HA and TCID 50 The connection between them provides a simple HA method for the determination of BCoV toxicity.

[0056] Table 2

[0057]

[0058] Example 2

[0059] The HA / HI test kit for rapid detection of bovine coronavirus titer and neutralizing antibodies of this example was used to perform the HI test.

[0060] First, prepare the unit virus: according to the BCoV hemagglutination titer results, prepare 4 units of virus in PBS with 0.1% BSA and keep at 4℃ for later use;

[0061] Treatment of serum to be tested: Inactivate the bovine serum to be tested in a water bath at 56°C for 30 minutes, mix once every 10 minutes, and use it for HI test to simplify the treatment steps;

[0062] HI operation:

[0063] (1) Add 30 μL of PBS diluent (containing 0.1% BSA) to each well of a V-bottom 96-well microtiter plate, add 30 μL of the serum to be tested to the first well, pipette evenly, and perform 2-fold serial dilutions, i.e., 1:2, 1:2. 2 , 1:2 3 , 1:2 4 , 1:2 5 …1:2 10 Discard 30 μL of serum diluent from the last well, and add 30 μL of PBS without serum to the 11th and 12th wells as controls for red blood cell sedimentation and agglutination.

[0064] (2) Then add 30 μL of 4 units of virus solution to each well, and do not add virus solution to the 12th well.

[0065] (3) Finally, add 30 μL of 1% red blood cells to each well and place the microplate on a microplate shaker for 30 seconds.

[0066] (4) Place the microplate in a sealed bag, seal it, and place it in a constant temperature incubator at 37°C for 1 hour.

[0067] (5) When the red blood cells in the control wells without virus solution were completely precipitated, and the wells with only 4 units of virus but no serum added showed obvious red blood cell agglutination, the red blood cell precipitation in the wells of each experimental group was observed, and the highest serum dilution factor at which the red blood cells were completely precipitated was determined as the sample hemagglutination inhibition titer or antibody titer.

[0068] (6) Each serum sample to be tested was repeated three times.

[0069] In order to explore the correlation between the HI value of BCoV positive serum and the neutralizing antibody titer, this example selected a HI value of 1:2 5 , 1:2 6 , 1:2 7 and 1:2 8 Three samples of BCoV positive bovine serum were collected and their neutralizing antibody titers were tested respectively. The relationship curve between BCoV HI titer and neutralizing antibody titer was drawn based on the test results, as shown in Figure 2. Figure 3 shown.

[0070] According to the HI test results of anti-BCoV antibodies in bovine serum samples as shown in Table 3, the relationship between the HI value of BCoV positive serum samples and the neutralizing antibody titer is y=x+2(R 2 =1), y is log2NT, and x is log2HI. It can be seen that there is a linear relationship between the HI value of BCoV-positive serum samples and the neutralizing antibody titer.

[0071] Table 3

[0072] Serum number HI price Neutralization titer 1-1 <![CDATA[1:2 5 ]]> <![CDATA[1:2 3 ]]> 1-2 <![CDATA[1:2 5 ]]> <![CDATA[1:2 3 ]]> 1-3 <![CDATA[1:2 5 ]]> <![CDATA[1:2 3 ]]> 2-1 <![CDATA[1:2 6 ]]> <![CDATA[1:2 4 ]]> 2-2 <![CDATA[1:2 6 ]]> <![CDATA[1:2 4 ]]> 2-3 <![CDATA[1:2 6 ]]> <![CDATA[1:2 4 ]]> 3-1 <![CDATA[1:2 7 ]]> <![CDATA[1:2 5 ]]> 3-2 <![CDATA[1:2 7 ]]> <![CDATA[1:2 5 ]]> 3-3 <![CDATA[1:2 7 ]]> <![CDATA[1:2 5 ]]> 4-1 <![CDATA[1:2 8 ]]> <![CDATA[1:2 6 ]]> 4-2 <![CDATA[1:2 8 ]]> <![CDATA[1:2 6 ]]> 4-3 <![CDATA[1:2 8 ]]> <![CDATA[1:2 6 ]]>

[0073] Furthermore, fresh Chinese hamster erythrocytes and aldehyde-treated Chinese hamster erythrocytes were compared in terms of HA and HI:

[0074] HA tests were performed with 1% aldehyded Chinese hamster erythrocytes instead of 1% fresh Chinese hamster erythrocytes, and HI tests were performed with BCoV-HLJ001, BCoV-HLJ002, BCoV-HLJ323, and BCoV-HLJ325 strains. The results were compared with the corresponding tests using 1% fresh hamster erythrocytes to determine the differences between the two.

[0075] The results of the BCoV HA assay (see Table 4) and HI assay (omitted) using 1% hydroformylated hamster erythrocytes instead of 1% fresh hamster erythrocytes were identical, with no visible differences. This indicates that hydroformylated hamster erythrocytes can completely replace fresh hamster erythrocytes in the BCoV HA / HI assay and can extend the shelf life of the kit.

[0076] Table 4

[0077]

[0078] Summary, HA value and virus titer TCID of BCoV of the present invention 50 There is a correlation between HI titer and neutralizing antibody titer, which makes the determination of BCoV virus titer and the evaluation of neutralizing antibody levels in immunized cattle or infected cattle easier and faster. It is suitable for serological epidemiological surveys of BCoV infection and the evaluation of BCoV vaccine immune effects. It can also provide a reference standard for the establishment of rapid antigen or antibody detection methods in the future.

[0079] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A HA / HI detection kit for rapid detection of BCoV titer and neutralizing antibodies, characterized in that: It is composed of the following components: The inactivated virus titer was 10 8.0 TCID 50 / mL of BCoV virus, 1mL; PBS buffer (pH 7.2) containing 0.1% bovine serum albumin (BSA), 30 mL; Positive serum control: bovine BCoV antibody positive serum with an HI titer of 1:64; Negative serum control: bovine BCoV antibody-negative serum with an HI titer of less than or equal to 1:2; Red blood cells: 1% fresh Chinese hamster erythrocytes or 1% aldehyde-treated Chinese hamster erythrocyte suspension.

2. The HA / HI detection kit for rapid detection of BCoV titer and neutralizing antibodies according to claim 1, characterized in that: The 1% fresh Chinese hamster erythrocytes or the 1% aldehyde-formulated Chinese hamster erythrocyte suspension is obtained by the following method: 1) Preparation of Chinese hamster erythrocytes Fresh anticoagulated blood was collected from healthy adult Chinese hamsters. The blood was then suspended in an equal volume of physiological saline and centrifuged at 3000 rpm for 5 min. The supernatant and white blood cells were discarded. This process was repeated three times to obtain Chinese hamster red blood cells. The red blood cells were then mixed with Aldrich's solution and stored at 4°C until ready for use. 2) Aldehydation of Chinese hamster erythrocytes Take the Chinese hamster erythrocytes and centrifuge the pellet, discard the supernatant, and then wash and centrifuge three times with 10-20 times the volume of packed Chinese hamster erythrocytes in normal saline. Then, mix 0.1 mL of packed Chinese hamster erythrocytes with 2.5 mL of PBS buffer (pH 7.2) and shake well. Then, add 0.1 mL of 2.5% glutaraldehyde dropwise while shaking. Continue shaking on a shaker at room temperature for 60 minutes. Wash three times with PBS buffer (pH 7.2) and twice with distilled water. Finally, prepare a 10% Chinese hamster erythrocyte suspension in 0.01% thimerosal-containing normal saline and store in a refrigerator at 4°C until use. 3) Prepare 1% fresh Chinese hamster erythrocytes or 1% aldehyde-treated Chinese hamster erythrocytes Take 9 mL of PBS buffer containing 0.1% bovine serum albumin (BSA), add 0.1 mL of the above-mentioned fresh Chinese hamster erythrocytes or 10% Chinese hamster erythrocyte suspension, mix well, and prepare 1% fresh Chinese hamster erythrocytes or aldehyded 1% Chinese hamster erythrocytes.

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