CN111323581A - Influenza hemagglutination inhibition test detection method - Google Patents
Influenza hemagglutination inhibition test detection method Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to the technical field of microbial detection, and discloses a method for detecting an influenza hemagglutination inhibition test, which comprises the steps of pretreating standard reference antiserum to remove non-specific inhibin and non-specific agglutinin in the serum; preparing a red blood cell suspension; determining the hemagglutination titer of the influenza strain; preparing 4 hemagglutination unit antigens for erythrocyte agglutination inhibition test; double-check titration of 4 hemagglutination unit antigens; and (4) identifying the influenza virus and analyzing the antigen by using an HAI method to obtain the serum titer of the detected serum. The detection method of the invention prepares the erythrocyte suspension by taking the chicken erythrocyte, turkey erythrocyte and guinea pig erythrocyte as raw materials, and replaces human erythrocyte with animal erythrocyte, thus solving the difficulty that the experiment can not be carried out due to lack of erythrocyte.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a detection method for influenza hemagglutination inhibition test.
Background
The Hemagglutinin (HA) protein on the surface of influenza virus contains a structure which recognizes and binds to host cell surface receptors and can cause agglutination of some specific erythrocytes. When erythrocytes and viruses are mixed in a proper ratio, erythrocytes agglutinate due to the action of viruses, which is an agglutination phenomenon. Antiserum containing specific HA protein against influenza virus can inhibit hemagglutination, i.e. after binding of antibody to virus, hemagglutinin can not be adsorbed on erythrocyte surface receptor, which is called hemagglutination inhibition (HAI).
The trace erythrocyte agglutination inhibition test is the most commonly used test method for identifying influenza virus types/subtypes and analyzing influenza virus HA antigenic variation at present, and can also be used for detecting the antibody level of general population, evaluating the vaccine effect and the like. However, with the increase of population, the experimental dosage is large, and human red blood cells are rare, so that experimental resources are in short supply.
Disclosure of Invention
In order to solve the above problems of the prior art, the present invention aims to provide a method for detecting a hemagglutination inhibition assay for influenza.
The technical scheme adopted by the invention is as follows:
a method for detecting influenza hemagglutination inhibition assay, comprising the following steps:
s1, pretreating standard reference antiserum to remove non-specific inhibin and non-specific agglutinin in the serum;
s2, preparing a erythrocyte suspension;
s3, determining the hemagglutination titer of the influenza strain;
s4, preparing 4 hemagglutination unit antigens for a erythrocyte agglutination inhibition test;
s5, rechecking and titrating 4 hemagglutination unit antigens;
s6, erythrocyte agglutination inhibition test: and (4) identifying the influenza virus and analyzing the antigen by using an HAI method to obtain the serum titer of the detected serum.
The conventional nonspecific inhibins are α, β and gamma, in addition to those specific to influenza C virus in rat serum, the antibodies of HAI are accurately determined by removing nonspecific inhibin from antiserum in HAI test.
Further, the step S1 includes the following steps:
(1.1) removal of non-specific inhibin from serum: diluting cholera filtrate with 25mL of normal saline; adding 3 volumes of diluted cholera filtrate into 1 volume of serum, and then carrying out water bath for 16-18h at the temperature of 37 ℃; adjusting the temperature to 56 ℃ and carrying out water bath for 30 min; finally, 6 volumes of normal saline is added to obtain serum after RDE treatment;
(1.2) removal of non-specific lectins in serum: adding 1 volume of pure blood spheres into 20 volumes of the RDE-treated serum, uniformly mixing, incubating at the temperature of 2-8 ℃, and uniformly mixing again every 15 min; centrifuging for 5min at 2000r/min after 1h, sucking out supernatant, wherein the supernatant is treated serum; then taking out a 96-well hemagglutination plate, and adding 50uLPBS buffer solution into each well in the first row; 50uL of the treated serum was then removed, diluted 2-fold and added to each well of the first row to obtain treated serum. The added pure blood cell is any one of chicken, turkey and guinea pig pure blood cell.
And adding 50uL of erythrocyte suspension into the treated serum, standing at room temperature for 30-60min, observing test results, and if erythrocytes are deposited, proving that the nonspecific agglutinin in the serum is completely removed.
Further, the step S2 includes the following steps:
(2.1) preparing an aldrin liquid: adding 20.5 parts by weight of glucose, 8 parts by weight of sodium citrate, 0.55 part by weight of citric acid, 4.2 parts by weight of sodium chloride and 1000 parts by weight of deionized water into a container, slightly heating to dissolve to obtain a solution, then adjusting the pH value of the solution to 6.1, carrying out autoclaving for 20min, wherein the pressure in the sterilization process is 8 pounds to obtain an aldrin solution, and storing the prepared aldrin solution in an environment at the temperature of 4 ℃ for later use; the liquid A is called Alsever liquid;
(2.2) pretreatment of the erythrocyte suspension: adding red blood cells into the Ashi solution, and centrifuging for 3-15min at a rotation speed of 1500-2200r per minute to obtain a pretreated red blood cell suspension; if the red blood cells of the turkey or chicken are added, centrifuging for 5min, wherein the rotating speed per minute is 1800r during centrifugation; if human O-type or guinea pig erythrocytes are added, centrifuging for 5min at 2000 r/min;
(2.3 washing the pretreated erythrocyte suspension, namely adding PBS buffer solution with the same volume, fully mixing, centrifuging for 3-15min, removing supernatant fluid at the rotating speed of 1500-2200r per minute, repeatedly washing for 3 times, and removing the supernatant fluid to obtain the washed erythrocyte suspension;
(2.4) preparation of erythrocyte suspension: sucking out the washed erythrocyte suspension, and diluting with PBS buffer solution to obtain erythrocyte suspension with the concentration of 1%.
Further, the step S3 includes the following steps:
(3.1) numbering the wells on the micro hemagglutination plate, wherein the numbering of each column is 1-12 in sequence, and the numbering of each row is A-G in sequence; adding 50 mu L of PBS buffer solution into the 2 nd to 12 th columns of the micro-hemagglutination plate;
(3.2) adding 100. mu.L of virus suspension to the first column (i.e., wells numbered A1-G1);
(3.3) adding 100. mu.L of PBS to the well numbered H1 as a negative control of erythrocytes;
(3.4) adding 50 mu L of erythrocyte suspension into each hole, lightly beating a hemagglutination plate, and uniformly mixing;
(3.5) incubating at room temperature, standing for 20-65min, observing the result, and recording.
The HA test result judging method comprises the following steps:
total red blood cell agglutination was caused to be complete, recorded as "+"; only a portion of the red blood cells agglutinated was recorded as "+/-"; no agglutination was noted "-". The hemagglutination titer is determined by taking the highest dilution at which complete agglutination occurs as an end point, and the reciprocal of the dilution is the hemagglutination titer of the virus.
Further, the step S4 includes the following steps:
(4.1) calculating the total amount of viral antigen required for the hemagglutination inhibition assay: if each serum is diluted in 8 wells and 25. mu.L of antigen is used in each well, 0.2mL of antigen is required for each serum. Calculating the total amount of virus antigen required by the experiment according to the parts of the standard antiserum;
(4.2) calculate the virus dilution: the quotient obtained by dividing the virus HA titre by 8 is the dilution required to formulate 4 hemagglutination units. For example, if the HA titer of a virus is 64, divided by 8 to equal 8, then the virus can be diluted 1:8(1mL virus plus 7mL PBS buffer) to obtain 4 hemagglutination units of antigen.
The 4 hemagglutination units used in the hemagglutination inhibition assay are 4 hemagglutination units per 25. mu.L of virus fluid (equivalent to 8 hemagglutination units in 50. mu.L of virus fluid).
Further, the step S5 includes the following steps:
(5.1) numbering the wells on the micro hemagglutination plate, wherein the numbering of each column is 1-12 in sequence, and the numbering of each row is A-G in sequence; adding 50 mu L of PBS buffer solution into the 2 nd to 12 th columns of the micro-hemagglutination plate;
(5.2) adding 100. mu.L of virus suspension to the first column (i.e., wells numbered A1-G1);
(5.3) adding 100. mu.L of PBS to the well numbered H1 as a negative control of erythrocytes;
(5.4) adding 50 mu L of erythrocyte suspension into each hole, lightly beating a hemagglutination plate, and uniformly mixing;
(5.5) incubating at room temperature, standing for 20-65min, observing the result, and recording.
In order to ensure that the antigen dosage in the erythrocyte agglutination inhibition test is consistent and correct, the newly configured 4 hemagglutination unit antigens need to be rechecked and titrated.
If agglutination occurs in only the first 4 wells, indicating that there are 8 hemagglutination units per 50 μ L of virus, the virus can be diluted accurately and used in the hemagglutination inhibition assay. Agglutination also occurred in well 5, indicating that 16 hemagglutination units per 50 μ L of virus and the antigen had to be diluted in equal amounts. If only the first 3 wells were agglutinated, it was shown that only 4 hemagglutination units per 50. mu.L of virus were present and the virus amount was doubled. Furthermore, 4 hemagglutination unit antigens must be matched at each use.
Further, the step S6 includes the following steps:
(6.1) identification of influenza viruses: identifying influenza virus by using HAI method;
(6.2) influenza virus antigen analysis: influenza virus antigen analysis was performed using the HAI method.
If the result of the 4 hemagglutination units is inaccurate, the 4 hemagglutination units need to be reconfigured until the back drop result is accurate, the 4 hemagglutination units need to be accurately prepared when the antigen analysis is carried out, and the comparability of the antigen analysis result among viruses is ensured.
Further, the step (6.1) includes the steps of:
(6.1.1) performing an experiment by using a 96 hemagglutination plate, wherein the holes on the hemagglutination plate are numbered, the numbers of each column are 1-12 in sequence, and the numbers of each row are A-G in sequence; adding 25 mu LPBS buffer solution into the wells of the 1 st to 5 th and 7 th to 11 th columns of the hemagglutination plate, and adding 50 mu LPBS into the wells of the 6 th and 12 th columns of the hemagglutination plate; then adding 25 muL of anti-A (H1N1) pdm09 influenza virus standard reference serum, anti-seasonal A (H3N2) influenza virus standard reference serum, anti-B-Yamagata system influenza virus standard reference serum and anti-B-Victoria system influenza virus standard reference serum into each hole of A1-A4 and A7-A10 respectively, taking 25 muL of anti-A (seasonal A) pdm09 influenza virus standard reference serum from each hole of the rows A1-A4 and A7-A10 respectively by using a multi-channel pipette, diluting the serum from row A to row H by 2 times, and finally discarding 25 muL in one row;
(6.1.2) adding 25 μ L of 4 hemagglutination unit antigen to be detected 1 to the wells of columns 1 to 4, and adding 25 μ L of 4 hemagglutination unit antigen to be detected 2 to the wells labeled A7 to A10; column 5 and 11 were supplemented with 25 μ LPBS buffer as red blood cell control; 50 mu L of 8 hemagglutination unit antigen 1 and 2 to be detected are respectively added into the first hole of the 6 th and 12 th columns, 2 times dilution is carried out from the line A to the line H, and 50 mu L is discarded in the last line; the aim of the method is to verify the accuracy of the configured 4 units of antigen in the HAI experiment and ensure the accuracy of the experiment result.
(a) Positive control: and detecting the standard reference antigen of each type/subtype influenza virus in the kit according to the steps.
(6.1.3) lightly beating, uniformly mixing, and incubating at room temperature for 30 min;
(6.1.4) adding 50 microliter of prepared 1% erythrocyte suspension into each hole, patting and uniformly mixing, standing at room temperature for 30-60min, and observing hemagglutination inhibition experiment results.
Each 96-well hemagglutination plate can detect two unknown viruses.
Further, the step (6.2) comprises the following steps:
(6.2.1) experiments were performed using 96 hemagglutination plates, with wells numbered on the hemagglutination plate;
(6.2.2) adding 25 mu LPBS buffer solution to each well of the 1 st to 11 th columns of the hemagglutination plate, and adding 50 mu LPBS buffer solution to each well of the 12 th column; adding 25 mu L of reference antiserum and negative control serum for analyzing the influenza virus type/subtype antigen to be detected into each hole of the first row;
(6.2.3) after mixing well from each well in the first row (A1-A11), 25. mu.L of serum is aspirated, 2-fold serial dilutions are made from row 1 to row 8, and 25. mu.L of liquid is discarded from each well in the last row;
(6.2.4) adding 25. mu.L of prepared 4 hemagglutination unit antigen to each well of columns 1-11 of the microplate, adding 50. mu.L of prepared 8 hemagglutination unit antigen to well A12 of column 12, performing 2-fold serial dilution from row 1 to row 4, and discarding 50. mu.L of liquid from each well of the last row; e, F, G, H wells in column 12 were used as red blood cell controls, and the microplate was tapped and incubated at room temperature for 30 min;
(6.2.5) adding 50. mu.L of erythrocyte suspension into each well of the microplate, and patting the microplate gently to mix the erythrocytes with the virus;
(6.2.6) standing at room temperature for 30-60min, and observing the experimental result of hemagglutination inhibition.
And (4) judging a result:
the erythrocyte agglutination inhibition titer refers to the reciprocal of the highest dilution of serum at which hemagglutination is completely inhibited. For example, the serum well diluted at 1:80 showed no agglutination (the agglutination phenomenon was completely inhibited), and the serum well diluted at 1:160 showed agglutination (the agglutination phenomenon was not completely inhibited), and the hemagglutination inhibition titer of the serum against the virus to be measured was 80.
(1) Judgment of the result of influenza virus identification by HAI method
1) The standard reference antiserum can be counted as positive if the inhibition titer of the standard reference antiserum to the antigen to be detected is more than or equal to 20.
2) The antigen to be detected has cross inhibition with the standard reference antiserum, but the inhibition titer with the standard reference antiserum of one type/subtype is more than 4 times that of the standard reference antiserum of other types/subtypes, so that the subtype influenza virus can be judged.
(2) Judgment of results of influenza virus antigen analysis by HAI method
1) When the HAI titer of the virus to be detected and the reference ferret antiserum is less than 8 times (including 8 times) or more than the HAI titer of the reference virus and the reference antiserum per se, the virus to be detected is considered to be a low-reactive strain detected by the reference antiserum; on the other hand, when the difference is within 8 times (8 times is not included), the virus to be tested is considered to be a similar strain of the reference virus.
2) When the antigen is analyzed and tested, the standard reference antigen must be added to detect each time, which is the basis of result interpretation and quality control of the test. The experimental result is ensured to be accurate, and the result is more comparable. The hemagglutination inhibition titer of each positive control fluctuates from the next hole to the next hole, and if the result of the test is not valid beyond the range, the test needs to be repeated.
It is worth noting that: the hemagglutination inhibition test must use 4 hemagglutination units per 25. mu.L of antigen, which must be matched as-is. The preparation of the red blood cell suspension must be standardized. The reagent is correctly stored, and repeated freeze thawing and pollution are avoided. The lyophilized reagents should be dissolved and stored as specified.
The hemagglutination inhibition assay included the following controls:
(1) red blood cell contrast is used for accurately judging the time of reading the result and eliminating the error result caused by the blood cell problem;
(2) negative control sera, to protect against other non-specific antibodies;
(3) positive control: preventing interference of non-specific agglutinin and inhibin; and judging the standard of the result.
The invention has the beneficial effects that:
the detection method of the invention prepares the erythrocyte suspension by taking the chicken erythrocyte, turkey erythrocyte and guinea pig erythrocyte as raw materials, and replaces human erythrocyte with animal erythrocyte, thus solving the difficulty that the experiment can not be carried out due to lack of erythrocyte.
Detailed Description
The present invention is further illustrated below with reference to specific examples.
Example 1:
the embodiment provides a method for detecting influenza hemagglutination inhibition assay, which comprises the following steps:
s1, pretreating standard reference antiserum to remove non-specific inhibin and non-specific agglutinin in the serum;
s2, preparing a erythrocyte suspension;
s3, determining the hemagglutination titer of the influenza strain;
s4, preparing 4 hemagglutination unit antigens for a erythrocyte agglutination inhibition test;
s5, rechecking and titrating 4 hemagglutination unit antigens;
s6, erythrocyte agglutination inhibition test: and (4) identifying the influenza virus and analyzing the antigen by using an HAI method to obtain the serum titer of the detected serum.
The conventional nonspecific inhibins are α, β and gamma, in addition to those specific to influenza C virus in rat serum, the antibodies of HAI are accurately determined by removing nonspecific inhibin from antiserum in HAI test.
The reagents used in the detection method of this example were as follows:
1. standard reference antigens: taking international vaccine strains and domestic epidemic strains as standard reference antigens, and taking newly separated strains as antigens to be detected.
2. Standard reference antisera: antiserum prepared from international vaccine strains and domestic epidemic strains is used as standard reference serum. The sera were subjected to RDE.
The detection method of the embodiment uses the following instruments and materials:
1. a suspension of red blood cells;
PBS buffer at pH 7.4;
3.RDE；
4. water bath (37 ℃, 56 ℃);
5. a bench centrifuge;
6. a plurality of adjustable sample injectors and a single adjustable sample injector;
7. centrifuging the tube;
8.96 hole micro hemagglutination plate (using guinea pig and human red blood cell experiments must use the "U" bottom plate, using chicken red blood cell/turkey red blood cell experiments used "V" bottom plate);
9.200 mu L, 1000 mu LTIP head and water tank;
the conditions of the influenza hemagglutination test are shown in Table 1.
TABLE 1
The step S1 includes the following steps:
(1.1) removal of non-specific inhibin from serum: diluting cholera filtrate with 25mL of normal saline; adding 3 volumes of diluted cholera filtrate into 1 volume of serum, and then carrying out water bath for 16-18h at the temperature of 37 ℃; adjusting the temperature to 56 ℃ and carrying out water bath for 30 min; finally, 6 volumes of normal saline is added to obtain serum after RDE treatment;
(1.2) removal of non-specific lectins in serum: adding 1 volume of pure blood spheres into 20 volumes of the RDE-treated serum, uniformly mixing, incubating at the temperature of 2-8 ℃, and uniformly mixing again every 15 min; centrifuging for 5min at 2000r/min after 1h, sucking out supernatant, wherein the supernatant is treated serum; then taking out a 96-well hemagglutination plate, and adding 50uLPBS buffer solution into each well in the first row; 50uL of the treated serum was then removed, diluted 2-fold and added to each well of the first row to obtain treated serum. The added pure blood cell is any one of chicken, turkey and guinea pig pure blood cell.
And adding 50uL of erythrocyte suspension into the treated serum, standing at room temperature for 30-60min, observing test results, and if erythrocytes are deposited, proving that the nonspecific agglutinin in the serum is completely removed.
The step S2 includes the following steps:
(2.1) preparing an aldrin liquid: adding 20.5 parts by weight of glucose, 8 parts by weight of sodium citrate, 0.55 part by weight of citric acid, 4.2 parts by weight of sodium chloride and 1000 parts by weight of deionized water into a container, slightly heating to dissolve to obtain a solution, then adjusting the pH value of the solution to 6.1, carrying out autoclaving for 20min, wherein the pressure in the sterilization process is 8 pounds to obtain an aldrin solution, and storing the prepared aldrin solution in an environment at the temperature of 4 ℃ for later use; the liquid A is called Alsever liquid;
(2.2) pretreatment of the erythrocyte suspension: adding red blood cells into the Ashi solution, and centrifuging for 3-15min at a rotation speed of 1500-2200r per minute to obtain a pretreated red blood cell suspension; if the red blood cells of the turkey or chicken are added, centrifuging for 5min, wherein the rotating speed per minute is 1800r during centrifugation; if human O-type or guinea pig erythrocytes are added, centrifuging for 5min at 2000 r/min;
(2.3) washing the pretreated red blood cell suspension: washing, adding PBS buffer solution with the same volume, fully mixing, centrifuging for 3-15min at a rotation speed of 1500-;
(2.4) preparation of erythrocyte suspension: sucking out the washed erythrocyte suspension, and diluting with PBS buffer solution to obtain erythrocyte suspension with the concentration of 1%.
The step S3 includes the following steps:
(3.1) numbering the wells on the micro hemagglutination plate, wherein the numbering of each column is 1-12 in sequence, and the numbering of each row is A-G in sequence; adding 50 mu L of PBS buffer solution into the 2 nd to 12 th columns of the micro-hemagglutination plate;
(3.2) adding 100. mu.L of virus suspension to the first column (i.e., wells numbered A1-G1);
(3.3) adding 100. mu.L of PBS to the well numbered H1 as a negative control of erythrocytes;
(3.4) adding 50 mu L of erythrocyte suspension into each hole, lightly beating a hemagglutination plate, and uniformly mixing;
(3.5) incubating at room temperature, standing chicken and turkey red blood cells for 25-30min, and observing human O-type and guinea pig red blood cells for 45-60min, and recording.
The HA test result judging method comprises the following steps:
total red blood cell agglutination was caused to be complete, recorded as "+"; only a portion of the red blood cells agglutinated was recorded as "+/-"; no agglutination was noted "-". The hemagglutination titer is determined by taking the highest dilution at which complete agglutination occurs as an end point, and the reciprocal of the dilution is the hemagglutination titer of the virus. An example of HA titre is shown in Table 2.
TABLE 2
The step S4 includes the following steps:
(4.1) calculating the total amount of viral antigen required for the hemagglutination inhibition assay: if each serum is diluted in 8 wells and 25. mu.L of antigen is used in each well, 0.2mL of antigen is required for each serum. Calculating the total amount of virus antigen required by the experiment according to the parts of the standard antiserum;
(4.2) calculate the virus dilution: the quotient obtained by dividing the virus HA titre by 8 is the dilution required to formulate 4 hemagglutination units. For example, if the HA titer of a virus is 64, divided by 8 to equal 8, then the virus can be diluted 1:8(1mL virus plus 7mL PBS buffer) to obtain 4 hemagglutination units of antigen.
The 4 hemagglutination units used in the hemagglutination inhibition assay are 4 hemagglutination units per 25. mu.L of virus fluid (equivalent to 8 hemagglutination units in 50. mu.L of virus fluid). Example (c): if the total amount of antigen required is 1600 μ L, 4 hemagglutination units are configured as shown in Table 3.
TABLE 3
The step S5 includes the following steps:
(5.1) numbering the wells on the micro hemagglutination plate, wherein the numbering of each column is 1-12 in sequence, and the numbering of each row is A-G in sequence; adding 50 mu L of PBS buffer solution into the 2 nd to 12 th columns of the micro-hemagglutination plate;
(5.2) adding 100. mu.L of virus suspension to the first column (i.e., wells numbered A1-G1);
(5.3) adding 100. mu.L of PBS to the well numbered H1 as a negative control of erythrocytes;
(5.4) adding 50 mu L of erythrocyte suspension into each hole, lightly beating a hemagglutination plate, and uniformly mixing;
(5.5) incubating at room temperature, standing for 20-65min, observing the result, and recording.
In order to ensure that the antigen dosage in the erythrocyte agglutination inhibition test is consistent and correct, the newly configured 4 hemagglutination unit antigens need to be rechecked and titrated.
If agglutination occurs in only the first 4 wells, indicating that there are 8 hemagglutination units per 50 μ L of virus, the virus can be diluted accurately and used in the hemagglutination inhibition assay. Agglutination also occurred in well 5, indicating that 16 hemagglutination units per 50 μ L of virus and the antigen had to be diluted in equal amounts. If only the first 3 wells were agglutinated, it was shown that only 4 hemagglutination units per 50. mu.L of virus were present and the virus amount was doubled. Furthermore, 4 hemagglutination unit antigens must be matched at each use.
The step S6 includes the following steps:
(6.1) identification of influenza viruses: identifying influenza virus by using HAI method;
(6.2) influenza virus antigen analysis: influenza virus antigen analysis was performed using the HAI method.
If the result of the 4 hemagglutination units is inaccurate, the 4 hemagglutination units need to be reconfigured until the back drop result is accurate, the 4 hemagglutination units need to be accurately prepared when the antigen analysis is carried out, and the comparability of the antigen analysis result among viruses is ensured.
The step (6.1) comprises the following steps:
(6.1.1) performing an experiment by using a 96 hemagglutination plate, wherein the holes on the hemagglutination plate are numbered, the numbers of each column are 1-12 in sequence, and the numbers of each row are A-G in sequence; adding 25 mu LPBS buffer solution into the wells of the 1 st to 5 th and 7 th to 11 th columns of the hemagglutination plate, and adding 50 mu LPBS into the wells of the 6 th and 12 th columns of the hemagglutination plate; then adding 25 muL of anti-A (H1N1) pdm09 influenza virus standard reference serum, anti-seasonal A (H3N2) influenza virus standard reference serum, anti-B-Yamagata system influenza virus standard reference serum and anti-B-Victoria system influenza virus standard reference serum into each hole of A1-A4 and A7-A10 respectively, taking 25 muL of anti-A (seasonal A) pdm09 influenza virus standard reference serum from each hole of the rows A1-A4 and A7-A10 respectively by using a multi-channel pipette, diluting the serum from row A to row H by 2 times, and finally discarding 25 muL in one row;
(6.1.2) adding 25 μ L of 4 hemagglutination unit antigen to be detected 1 to the wells of columns 1 to 4, and adding 25 μ L of 4 hemagglutination unit antigen to be detected 2 to the wells labeled A7 to A10; column 5 and 11 were supplemented with 25 μ LPBS buffer as red blood cell control; 50 mu L of 8 hemagglutination unit antigen 1 and 2 to be detected are respectively added into the first hole of the 6 th and 12 th columns, 2 times dilution is carried out from the line A to the line H, and 50 mu L is discarded in the last line; the aim of the method is to verify the accuracy of the configured 4 units of antigen in the HAI experiment and ensure the accuracy of the experiment result.
(a) Positive control: and detecting the standard reference antigen of each type/subtype influenza virus in the kit according to the steps.
(6.1.3) lightly beating, uniformly mixing, and incubating at room temperature for 30 min;
(6.1.4) adding 50 microliter of prepared 1% erythrocyte suspension into each hole, patting and uniformly mixing, standing at room temperature for 30-60min, and observing hemagglutination inhibition experiment results.
Each 96-well hemagglutination plate can detect two unknown viruses.
The step (6.2) comprises the following steps:
(6.2.1) experiments were performed using 96 hemagglutination plates, with wells numbered on the hemagglutination plate;
(6.2.2) adding 25 mu LPBS buffer solution to each well of the 1 st to 11 th columns of the hemagglutination plate, and adding 50 mu LPBS buffer solution to each well of the 12 th column; adding 25 mu L of reference antiserum and negative control serum for analyzing the influenza virus type/subtype antigen to be detected into each hole of the first row;
(6.2.3) after mixing well from each well in the first row (A1-A11), 25. mu.L of serum is aspirated, 2-fold serial dilutions are made from row 1 to row 8, and 25. mu.L of liquid is discarded from each well in the last row;
(6.2.4) adding 25. mu.L of prepared 4 hemagglutination unit antigen to each well of columns 1-11 of the microplate, adding 50. mu.L of prepared 8 hemagglutination unit antigen to well A12 of column 12, performing 2-fold serial dilution from row 1 to row 4, and discarding 50. mu.L of liquid from each well of the last row; e, F, G, H wells in column 12 were used as red blood cell controls, and the microplate was tapped and incubated at room temperature for 30 min;
(6.2.5) adding 50. mu.L of erythrocyte suspension into each well of the microplate, and patting the microplate gently to mix the erythrocytes with the virus;
(6.2.6) standing at room temperature for 30-60min, and observing the experimental result of hemagglutination inhibition.
And (4) judging a result:
the erythrocyte agglutination inhibition titer refers to the reciprocal of the highest dilution of serum at which hemagglutination is completely inhibited. For example, the serum well diluted at 1:80 showed no agglutination (the agglutination phenomenon was completely inhibited), and the serum well diluted at 1:160 showed agglutination (the agglutination phenomenon was not completely inhibited), and the hemagglutination inhibition titer of the serum against the virus to be measured was 80.
(1) Judgment of the result of influenza virus identification by HAI method
1) The standard reference antiserum can be counted as positive if the inhibition titer of the standard reference antiserum to the antigen to be detected is more than or equal to 20.
2) The antigen to be detected has cross inhibition with the standard reference antiserum, but the inhibition titer with the standard reference antiserum of one type/subtype is more than 4 times that of the standard reference antiserum of other types/subtypes, so that the subtype influenza virus can be judged.
(2) Judgment of results of influenza virus antigen analysis by HAI method
1) When the HAI titer of the virus to be detected and the reference ferret antiserum is less than 8 times (including 8 times) or more than the HAI titer of the reference virus and the reference antiserum per se, the virus to be detected is considered to be a low-reactive strain detected by the reference antiserum; on the other hand, when the difference is within 8 times (8 times is not included), the virus to be tested is considered to be a similar strain of the reference virus.
2) When the antigen is analyzed and tested, the standard reference antigen must be added to detect each time, which is the basis of result interpretation and quality control of the test. The experimental result is ensured to be accurate, and the result is more comparable. The hemagglutination inhibition titer of each positive control fluctuates from the next hole to the next hole, and if the result of the test is not valid beyond the range, the test needs to be repeated.
It is worth noting that: the hemagglutination inhibition test must use 4 hemagglutination units per 25. mu.L of antigen, which must be matched as-is. The preparation of the red blood cell suspension must be standardized. The reagent is correctly stored, and repeated freeze thawing and pollution are avoided. The lyophilized reagents should be dissolved and stored as specified.
The hemagglutination inhibition assay included the following controls:
(1) red blood cell contrast is used for accurately judging the time of reading the result and eliminating the error result caused by the blood cell problem;
(2) negative control sera, to protect against other non-specific antibodies;
(3) positive control: preventing interference of non-specific agglutinin and inhibin; and judging the standard of the result.
The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the claims, and which the description is intended to be interpreted accordingly.
Claims (10)
1. A method for detecting an influenza hemagglutination inhibition assay, comprising: the method comprises the following steps:
s1, pretreating standard reference antiserum to remove non-specific inhibin and non-specific agglutinin in the serum;
s2, preparing a erythrocyte suspension;
s3, determining the hemagglutination titer of the influenza strain;
s4, preparing 4 hemagglutination unit antigens for a erythrocyte agglutination inhibition test;
s5, rechecking and titrating 4 hemagglutination unit antigens;
s6, erythrocyte agglutination inhibition test: and (4) identifying the influenza virus and analyzing the antigen by using an HAI method to obtain the serum titer of the detected serum.
2. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: the step S1 includes the following steps:
(1.1) removal of non-specific inhibin from serum: diluting cholera filtrate with 25mL of normal saline; adding 3 volumes of diluted cholera filtrate into 1 volume of serum, and then carrying out water bath for 16-18h at the temperature of 37 ℃; adjusting the temperature to 56 ℃ and carrying out water bath for 30 min; finally, 6 volumes of normal saline is added to obtain serum after RDE treatment;
(1.2) removal of non-specific lectins in serum: adding 1 volume of pure blood spheres into 20 volumes of the RDE-treated serum, uniformly mixing, incubating at the temperature of 2-8 ℃, and uniformly mixing again every 15 min; centrifuging for 5min at 2000r/min after 1h, sucking out supernatant, wherein the supernatant is treated serum; then taking out a 96-well hemagglutination plate, and adding 50uLPBS buffer solution into each well in the first row; 50uL of the treated serum was then removed, diluted 2-fold and added to each well of the first row to obtain treated serum.
3. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: the step S2 includes the following steps:
(2.1) preparing an aldrin liquid: adding 20.5 parts by weight of glucose, 8 parts by weight of sodium citrate, 0.55 part by weight of citric acid, 4.2 parts by weight of sodium chloride and 1000 parts by weight of deionized water into a container, slightly heating to dissolve to obtain a solution, then adjusting the pH value of the solution to 6.1, carrying out autoclaving for 20min, wherein the pressure in the sterilization process is 8 pounds to obtain an aldrin solution, and storing the prepared aldrin solution in an environment at the temperature of 4 ℃ for later use;
(2.2) pretreatment of the erythrocyte suspension: adding red blood cells into the Ashi solution, and centrifuging for 3-15min at a rotation speed of 1500-2200r per minute to obtain a pretreated red blood cell suspension;
(2.3) washing the pretreated red blood cell suspension: washing, adding PBS buffer solution with the same volume, fully mixing, centrifuging for 3-15min at a rotation speed of 1500-;
(2.4) preparation of erythrocyte suspension: sucking out the washed erythrocyte suspension, and diluting with PBS buffer solution to obtain erythrocyte suspension with the concentration of 1%.
4. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: the step S3 includes the following steps:
(3.1) numbering the wells on the micro hemagglutination plate, wherein the numbering of each column is 1-12 in sequence, and the numbering of each row is A-G in sequence; adding 50 mu L of PBS buffer solution into the 2 nd to 12 th columns of the micro-hemagglutination plate;
(3.2) adding 100. mu.L of virus suspension to the first column (i.e., wells numbered A1-G1);
(3.3) adding 100. mu.L of PBS to the well numbered H1 as a negative control of erythrocytes;
(3.4) adding 50 mu L of erythrocyte suspension into each hole, lightly beating a hemagglutination plate, and uniformly mixing;
(3.5) incubating at room temperature, standing for 20-65min, observing the result, and recording.
5. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: the step S4 includes the following steps:
(4.1) calculating the total amount of viral antigen required for the hemagglutination inhibition assay: if each serum is diluted in 8 wells and 25. mu.L of antigen is used in each well, 0.2mL of antigen is required for each serum. Calculating the total amount of virus antigen required by the experiment according to the parts of the standard antiserum;
(4.2) calculate the virus dilution: the quotient obtained by dividing the virus HA titre by 8 is the dilution required to formulate 4 hemagglutination units. For example, if the HA titer of a virus is 64, divided by 8 to equal 8, then the virus can be diluted 1:8(1mL virus plus 7mL PBS buffer) to obtain 4 hemagglutination units of antigen.
6. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: the step S5 includes the following steps:
(5.1) numbering the wells on the micro hemagglutination plate, wherein the numbering of each column is 1-12 in sequence, and the numbering of each row is A-G in sequence; adding 50 mu L of PBS buffer solution into the 2 nd to 12 th columns of the micro-hemagglutination plate;
(5.2) adding 100. mu.L of virus suspension to the first column (i.e., wells numbered A1-G1);
(5.3) adding 100. mu.L of PBS to the well numbered H1 as a negative control of erythrocytes;
(5.4) adding 50 mu L of erythrocyte suspension into each hole, lightly beating a hemagglutination plate, and uniformly mixing;
(5.5) incubating at room temperature, standing for 20-65min, observing the result, and recording.
7. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: the step S6 includes the following steps:
(6.1) identification of influenza viruses: identifying influenza virus by using HAI method;
(6.2) influenza virus antigen analysis: analyzing influenza virus antigen by using HAI method;
(6.3) obtaining the erythrocyte agglutination inhibition titer according to the experimental data.
8. The method for detecting a hemagglutination inhibition assay for influenza according to claim 7, wherein: the step (6.1) comprises the following steps:
(6.1.1) performing an experiment by using a 96 hemagglutination plate, wherein the holes on the hemagglutination plate are numbered, the numbers of each column are 1-12 in sequence, and the numbers of each row are A-G in sequence; adding 25 mu LPBS buffer solution into the wells of the 1 st to 5 th and 7 th to 11 th columns of the hemagglutination plate, and adding 50 mu LPBS into the wells of the 6 th and 12 th columns of the hemagglutination plate; then adding 25 muL of anti-A (H1N1) pdm09 influenza virus standard reference serum, anti-seasonal A (H3N2) influenza virus standard reference serum, anti-B-Yamagata system influenza virus standard reference serum and anti-B-Victoria system influenza virus standard reference serum into each hole of A1-A4 and A7-A10 respectively, taking 25 muL of anti-A (seasonal A) pdm09 influenza virus standard reference serum from each hole of the rows A1-A4 and A7-A10 respectively by using a multi-channel pipette, diluting the serum from row A to row H by 2 times, and finally discarding 25 muL in one row;
(6.1.2) adding 25 μ L of 4 hemagglutination unit antigen to be detected 1 to the wells of columns 1 to 4, and adding 25 μ L of 4 hemagglutination unit antigen to be detected 2 to the wells labeled A7 to A10; column 5 and 11 were supplemented with 25 μ LPBS buffer as red blood cell control; 50 mu L of 8 hemagglutination unit antigen 1 and 2 to be detected are respectively added into the first hole of the 6 th and 12 th columns, 2 times dilution is carried out from the line A to the line H, and 50 mu L is discarded in the last line;
(6.1.3) lightly beating, uniformly mixing, and incubating at room temperature for 30 min;
(6.1.4) adding 50 microliter of prepared 1% erythrocyte suspension into each hole, patting and uniformly mixing, standing at room temperature for 30-60min, and observing hemagglutination inhibition experiment results.
9. The method for detecting a hemagglutination inhibition assay for influenza according to claim 7, wherein: the step (6.2) comprises the following steps:
(6.2.1) experiments were performed using 96 hemagglutination plates, with wells numbered on the hemagglutination plate;
(6.2.2) adding 25 mu LPBS buffer solution to each well of the 1 st to 11 th columns of the hemagglutination plate, and adding 50 mu LPBS buffer solution to each well of the 12 th column; adding 25 mu L of reference antiserum and negative control serum for analyzing the influenza virus type/subtype antigen to be detected into each hole of the first row;
(6.2.3) after mixing well from each well in the first row (A1-A11), 25. mu.L of serum is aspirated, 2-fold serial dilutions are made from row 1 to row 8, and 25. mu.L of liquid is discarded from each well in the last row;
(6.2.4) adding 25. mu.L of prepared 4 hemagglutination unit antigen to each well of columns 1-11 of the microplate, adding 50. mu.L of prepared 8 hemagglutination unit antigen to well A12 of column 12, performing 2-fold serial dilution from row 1 to row 4, and discarding 50. mu.L of liquid from each well of the last row; e, F, G, H wells in column 12 were used as red blood cell controls, and the microplate was tapped and incubated at room temperature for 30 min;
(6.2.5) adding 50. mu.L of erythrocyte suspension into each well of the microplate, and patting the microplate gently to mix the erythrocytes with the virus;
(6.2.6) standing at room temperature for 30-60min, and observing the experimental result of hemagglutination inhibition.
10. The method for detecting a hemagglutination inhibition assay for influenza according to claim 1, wherein: preparing erythrocyte suspension by using any one of chicken erythrocyte, turkey erythrocyte or guinea pig erythrocyte as raw material.
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| CN113533718A (en) * | 2021-06-02 | 2021-10-22 | 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) | Hemagglutination test detection method for avian influenza and newcastle disease |
| CN113504367B (en) * | 2021-06-02 | 2023-08-08 | 威海市环翠区动物疫病预防控制中心(威海市环翠区动物卫生检疫中心) | Hemagglutination inhibition test detection method for avian influenza and newcastle disease |
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