A high dilution ratio liquid phase blocking ELISA test method for foot-and-mouth disease type O and type A antibodies

Abstract

This invention relates to the field of experimental reagent dosage technology, specifically a liquid-phase blocking ELISA detection method for foot-and-mouth disease (FMD) type O and A antibodies. The detection method steps are as follows: 1. Take rabbit anti-coated ELISA plates: one rabbit anti-coated ELISA plate can detect up to 10 samples. A box contains five rabbit anti-coated ELISA plates. Select the corresponding number of rabbit anti-coated ELISA plates according to the number of samples; 2. Set the values; 3. Finalize the total number of samples for the prepared liquid volume; 4. Prepare PBST working solution, FMD type O and A virus antigen working solution, FMD type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. This method has the advantages of being simple, reducing actual waste, rationally planning actual usage, reducing experimental errors, quickly completing experimental value calculations, saving time, and improving experimental efficiency.

Description

Technical Field

[0001] This invention relates to the field of experimental reagent dosage technology, specifically a high-dilution ELISA method for detecting foot-and-mouth disease type O and type A antibodies using liquid phase blocking, which is simple, reduces actual waste, and allows for reasonable planning of actual usage. Background Technology

[0002] As is well known, the improved version of the Foot-and-Mouth Disease (FMD) O and A antibody liquid phase blocking ELISA detection kit is a standard kit used in this experiment. It requires the measurement of FMD O and A virus antigens, 25-fold PBST concentrate, FMD O and A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution. The following problems exist during the reagent handling process: 1. Laboratory personnel generally prepare reagents according to the instructions for use of the kit, without a standardized execution method; 2. The experimenters prepared the reagents according to the instructions of the kit. However, because the theoretical volume of reagents prepared was less than the actual volume required, the experiment could not be completed. Therefore, the experimenters prepared more reagents than the theoretical volume, resulting in varying degrees of waste.

[0003] 3. The experimenters prepared the reagents according to the instructions of the kit. Before each step of the experiment, they had to calculate the required volume of reagents, which was quite tedious.

[0004] This results in long testing times, low work efficiency, waste of reagents due to excessive reagent volume, and an inability to quickly calculate test values. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a simple, efficient, and reasonable method for detecting foot-and-mouth disease type O and type A antibodies using liquid-phase blocking ELISA with high dilution ratios.

[0006] The technical solution adopted by this invention to solve its technical problem is: A liquid-phase blocking ELISA method for detecting foot-and-mouth disease type O antibodies at high dilutions, characterized by the following steps: 1. Obtaining Rabbit Anti-Coated ELISA Plates: One rabbit anti-coated ELISA plate can detect up to 10 samples. One box contains five rabbit anti-coated ELISA plates. Select the corresponding number of rabbit anti-coated ELISA plates according to the number of samples. II. Setting values: The actual number of samples to be tested is set as n, the number of rabbit anti-coated ELISA plates with 10 samples is set as x, and the number of samples on the rabbit anti-coated ELISA plate that does not have 10 samples is set as y, where x is an integer and y is in the range of 0-9. The relationship between n, x, and y is: n = 10 × x + y. 3. The total number of samples for the final preparation of the volumetric liquid: set the total number of samples for the final preparation of the volumetric liquid as n', set the number of samples to be added virtually as z, n'=n+z, since n=10×x+y, we can deduce: n'=10×x+y+z, (1) when n is 1-9, when y is 1-4 samples, add 2 samples virtually, when y is 5-9 samples, add 1 sample virtually; (2) when n is 10*x, that is, when y is 0 samples, add x samples virtually respectively; (3) when n is composed of whole plate test samples and less than 10 samples, when y is 1-4 samples, add 2+x samples virtually, when y is 5-9 samples, add 1+x samples virtually. IV. Preparation of PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution: 1. When y is 1-4 samples, the calculation for virtually adding 2 samples is as follows: (1) Calculation of the required amount of PBST working solution: a. Serum dilution volume Q1 = {[(y+2)×8+2×8] wells-4 wells}×50ul+[(y+2) wells×25ul]; b. The amount of material used for washing the plate is Q2 = [(y+2)×8+2×8]holes×300ul×6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+2)×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+2)×8+2×8] wells×50ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+2)×8+2×8]hole×50ul÷2, where T is in ul; (5) Calculation of the required amount of stop solution: The required amount of stop solution is U = [(y+2)×8+2×8]hole×50ul, where U is in ul; 2. When y is 5-9 samples, the calculation for virtually adding 1 sample is as follows: (1) Calculation of the required amount of PBST working solution: a. Serum dilution volume Q1 = {[(y+1)×8+2×8] wells-4 wells}×50ul+[(y+1) wells×25ul]; b. The amount of material used for washing the plate is Q2 = [(y+1)×8+2×8]holes×300ul×6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+1)×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+1)×8+2×8] wells×50ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+1)×8+2×8]hole×50ul÷2, where T is in ul. (5) Calculation of the required amount of stop solution: The required amount of stop solution is U = [(y+1)×8+2×8]hole×50ul, where U is in ul; 3. When y is 0 samples, it means the number of rabbit anti-coated ELISA plates filled with 10 samples is x. The number of rabbit anti-coated ELISA plates is x = n ÷ 10. This is calculated by virtually adding x samples. Each rabbit anti-coated ELISA plate is used to test 10 samples. The required PBST working solution, foot-and-mouth disease type O and A virus antigen working solutions, foot-and-mouth disease type O and A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution are calculated based on adding 1 sample. (1) Calculation of the amount of PBST working solution required for each rabbit anti-coated ELISA plate to detect 10 samples: a. Serum dilution volume Q1 = {[11×8+2×8] wells - 4 wells}×50ul + [11 wells×25ul]; b. The amount of material used for washing the plate is Q2 = [11×8+2×8] wells × 300ul × 6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the amount of working solution required for testing 10 samples of foot-and-mouth disease type O and type A virus antigens per rabbit anti-coated ELISA plate: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[11×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the amount of working solution required for each rabbit anti-coated ELISA plate to detect 10 samples of foot-and-mouth disease type O and type A enzyme-labeled antibodies: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [11 × 8 + 2 × 8] wells × 50 μL, where S is in μL. (4) Calculation of the amount of TMB substrate A solution or TMB substrate B solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of TMB substrate A solution or TMB substrate B solution is T = [11 × 8 + 2 × 8] wells × 50 μL ÷ 2, where T is in μL. (5) Calculation of the amount of stop solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of stop solution, U, is calculated as follows: U = [11 × 8 + 2 × 8] orifices × 50 μL, where U is in μL. Multiplying the actual quantities required above by x will give the final required quantities for each.

[0007] In step four of this invention, the PBST working solution and foot-and-mouth disease type O and type A virus antigen working solutions mentioned in steps 1, 2, and 3 are actually concentrated solutions. One part of PBST concentrated solution requires 24 parts of distilled water for dilution. The foot-and-mouth disease type O and type A virus antigen concentrated solutions are diluted according to the dilution ratio indicated on the bottle containing the foot-and-mouth disease type O and type A virus antigen concentrated solutions. The specific method is as follows: A. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. We can get Q3 = Q ÷ 25. The corresponding amount of distilled water is Q4 = 24 × Q3, where Q4 is ul. B. The dilution factor required for 1 part of foot-and-mouth disease type O and type A virus antigen concentrate is m, and the foot-and-mouth disease type O and type A virus antigen concentrate is R1. R1 = R ÷ m. The value of m is marked on the bottle containing the foot-and-mouth disease type O and type A virus antigen concentrate.

[0008] Because of the above-described structure, this invention has the advantages of simple method, reduced actual waste, and reasonable planning of actual usage. Detailed Implementation

[0009] The present invention will be further described below: A liquid-phase blocking ELISA method for detecting foot-and-mouth disease type O and type A antibodies at high dilutions, characterized by the following steps: 1. Obtaining Rabbit Anti-Coated ELISA Plates: One rabbit anti-coated ELISA plate can detect up to 10 samples. One box contains five rabbit anti-coated ELISA plates. Select the corresponding number of rabbit anti-coated ELISA plates according to the number of samples. II. Setting values: The actual number of samples to be tested is set as n, the number of rabbit anti-coated ELISA plates with 10 samples is set as x, and the number of samples on the rabbit anti-coated ELISA plate that does not have 10 samples is set as y, where x is an integer and y is in the range of 0-9. The relationship between n, x, and y is: n = 10 × x + y. 3. The total number of samples for the final preparation of the volumetric liquid: set the total number of samples for the final preparation of the volumetric liquid as n', set the number of samples to be added virtually as z, n'=n+z, since n=10×x+y, we can deduce: n'=10×x+y+z, (1) when n is 1-9, when y is 1-4 samples, add 2 samples virtually, when y is 5-9 samples, add 1 sample virtually; (2) when n is 10*x, that is, when y is 0 samples, add x samples virtually respectively; (3) when n is composed of whole plate test samples and less than 10 samples, when y is 1-4 samples, add 2+x samples virtually, when y is 5-9 samples, add 1+x samples virtually. IV. Preparation of PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution: 1. When y is 1-4 samples, the calculation for virtually adding 2 samples is as follows: (1) Calculation of the required amount of PBST working solution: a. Serum dilution volume Q1 = {[(y+2)×8+2×8] wells-4 wells}×50ul+[(y+2) wells×25ul]; b. The amount of material used for washing the plate is Q2 = [(y+2)×8+2×8]holes×300ul×6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+2)×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+2)×8+2×8] wells×50ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+2)×8+2×8]hole×50ul÷2, where T is in ul; (5) Calculation of the required amount of stop solution: The required amount of stop solution is U = [(y+2)×8+2×8]hole×50ul, where U is in ul.

[0010] 2. When y is 5-9 samples, the calculation for virtually adding 1 sample is as follows: (1) Calculation of the required amount of PBST working solution: a. Serum dilution volume Q1 = {[(y+1)×8+2×8] wells-4 wells}×50ul+[(y+1) wells×25ul]; b. The amount of material used for washing the plate is Q2 = [(y+1)×8+2×8]holes×300ul×6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+1)×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+1)×8+2×8] wells×50ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+1)×8+2×8]hole×50ul÷2, where T is in ul. (5) Calculation of the required amount of stop solution: The required amount of stop solution is U = [(y+1)×8+2×8]hole×50ul, where U is in ul; 3. When y is 0 samples, it means the number of rabbit anti-coated ELISA plates filled with 10 samples is x. The number of rabbit anti-coated ELISA plates is x = n ÷ 10. This is calculated by virtually adding x samples. Each rabbit anti-coated ELISA plate is used to test 10 samples. The required PBST working solution, foot-and-mouth disease type O and A virus antigen working solutions, foot-and-mouth disease type O and A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution are calculated based on adding 1 sample. (1) Calculation of the amount of PBST working solution required for each rabbit anti-coated ELISA plate to detect 10 samples: a. Serum dilution volume Q1 = {[11×8+2×8] wells - 4 wells}×50ul + [11 wells×25ul]; b. The amount of material used for washing the plate is Q2 = [11×8+2×8] wells × 300ul × 6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the amount of working solution required for testing 10 samples of foot-and-mouth disease type O and type A virus antigens per rabbit anti-coated ELISA plate: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[11×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the amount of working solution required for each rabbit anti-coated ELISA plate to detect 10 samples of foot-and-mouth disease type O and type A enzyme-labeled antibodies: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [11 × 8 + 2 × 8] wells × 50 μL, where S is in μL. (4) Calculation of the amount of TMB substrate A solution or TMB substrate B solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of TMB substrate A solution or TMB substrate B solution is T = [11 × 8 + 2 × 8] wells × 50 μL ÷ 2, where T is in μL. (5) Calculation of the amount of stop solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of stop solution, U, is calculated as follows: U = [11 × 8 + 2 × 8] orifices × 50 μL, where U is in μL. Multiplying the actual quantities required above by x will give the final required quantities for each.

[0011] In step four of this invention, the PBST working solution and foot-and-mouth disease type O and type A virus antigen working solutions mentioned in steps 1, 2, and 3 are actually concentrated solutions. One part of PBST concentrated solution requires 24 parts of distilled water for dilution. The foot-and-mouth disease type O and type A virus antigen concentrated solutions are diluted according to the dilution ratio indicated on the bottle containing the foot-and-mouth disease type O and type A virus antigen concentrated solutions. The specific method is as follows: A. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. We can get Q3 = Q ÷ 25. The corresponding amount of distilled water is Q4 = 24 × Q3, where Q4 is ul. B. The dilution factor required for one batch of foot-and-mouth disease type O and type A virus antigen concentrate is m, and the foot-and-mouth disease type O and type A virus antigen concentrate is R1, where R1 = R ÷ m. The value of m is marked on the bottle containing the foot-and-mouth disease type O and type A virus antigen concentrate.

[0012] The above scheme was obtained using the following design method: The experimental procedures in this method strictly followed the instructions for use of the Foot-and-Mouth Disease Type O and Type A Antibody Liquid Phase Blocking ELISA Detection Kit (Modified) (Lanzhou Veterinary Research Biotechnology Co., Ltd.). The sample layout consisted of one rabbit anti-coated ELISA plate testing 10 samples. The serum samples were diluted only to 1:1024 to save reagents, reduce experimental error, quickly complete numerical calculations, save time, and improve experimental efficiency.

[0013] A rabbit anti-coated ELISA plate has 8 rows horizontally and 12 columns vertically, with a total of 96 wells. The first 10 columns are for 10 sample test positions, and the last 2 columns are for control. In this protocol, each sample is divided into 8 dilutions per column.

[0014] I. Setting Theoretical Values: By testing 10 samples using one rabbit anti-coated ELISA plate, the scientifically reasonable volumes of the working solutions for foot-and-mouth disease type O and type A virus antigens, PBST working solution, foot-and-mouth disease type O and type A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution were calculated. This yielded the required volumes of these components for one sample and were set as theoretical values.

[0015] (a) Calculation method for the theoretical value of PBST concentrate required for 1 sample One rabbit anti-coated ELISA plate was used to test 10 samples. PBST working solution was used to dilute the serum and wash the plate.

[0016] The serum dilution volume is (96-4) wells × 50ul + 10 wells × 25ul = 4850ul; the plate washing volume is 96 wells × 300ul × 6 times = 172800ul; (Results from long-term and large-scale experiments: In the experiment, there are 2 plate washing steps, and each step requires 3 washes) Summing the results of the two equations above, we can calculate that the total volume of PBST working solution used is 4850ul + 172800ul = 177650ul = 177.65ml. Since 1 part of PBST concentrate requires 24 parts of distilled water for dilution, let 177.65ml of PBST working solution correspond to 177.65ml of PBST concentrate as x, we can calculate x = 177.65ml ÷ 25 = 7.106ml, and the corresponding volume of distilled water used is 24x = 170.544ml.

[0017] In actual experimental operations, the values ​​of 7.106 ml and 170.544 ml are not compatible with the volume range of the pipette and measuring instrument. We followed the principle of ensuring the measured values ​​were compatible with the volume range of the pipette and measuring instrument, while also rounding to the nearest whole number, by increasing these two values ​​to obtain scientifically reasonable values. Assuming x = 7.2, the corresponding distilled water volume is 24x = 172.8 ml; assuming x = 7.3, the corresponding distilled water volume is 24x = 175.2 ml; assuming x = 7.4, the corresponding distilled water volume is 24x = 177.6 ml; assuming x = 7.5, the corresponding distilled water volume is 24x = 180 ml. After calculation, we obtained a scientifically reasonable value for PBST concentrate: 7.5 ml, and for distilled water: 180 ml. Therefore, the amount of PBST working solution measured for testing 10 samples is 7.5 ml + 180 ml = 187.5 ml.

[0018] One sample requires 7.5 ml ÷ 10 = 0.75 ml of PBST concentrate. In summary, the theoretical value of PBST concentrate required for one sample is 0.75 ml.

[0019] (II) Calculation method for the theoretical value of foot-and-mouth disease type O and type A virus antigen required for 1 sample For an experiment testing 10 samples using a rabbit anti-coated ELISA plate, the working solution volume for foot-and-mouth disease (FMD) type O and A virus antigens is (96 wells - 4 wells) × 50 μL + 400 μL = 5000 μL. Assuming 5000 μL of FMD type O and A virus antigen working solution corresponds to x μL of FMD type O and A virus antigens, we can obtain x + (dilution factor - 1)x = 5000 μL from the dilution of the FMD type O and A virus antigens. Using the above formula, we can calculate x = (5000 ÷ dilution factor) μL. Therefore, one sample requires (500 ÷ dilution factor) μL of FMD type O and A virus antigens. In summary, the theoretical value of FMD type O and A virus antigens required for one sample is (500 ÷ dilution factor) μL.

[0020] (III) Calculation method for the theoretical value of enzyme-labeled antibody required for one sample: For an experiment testing 10 samples on one rabbit anti-coated ELISA plate, the volume of working solution for foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies is 96 wells × 50 μL = 4800 μL. Therefore, one sample requires 4800 μL ÷ 10 = 480 μL of working solution for FMD type O and A enzyme-labeled antibodies. In summary, the theoretical value of working solution for FMD type O and A enzyme-labeled antibodies required for one sample is 480 μL.

[0021] (iv) Calculation method for the theoretical values ​​of TMB substrate A solution and TMB substrate B solution required for one sample: For an experiment testing 10 samples using one rabbit anti-coated ELISA plate, the volume of TMB substrate A solution and TMB substrate B solution required is (96 wells × 50 μL) ÷ 2 = 2400 μL. Therefore, one sample requires 2400 μL of TMB substrate A solution and 240 μL of TMB substrate B solution, respectively. In summary, the theoretical value of TMB substrate A solution and TMB substrate B solution required for one sample is 240 μL each.

[0022] (V) Calculation method for the theoretical value of stop solution required for 1 sample: For an experiment testing 10 samples on 1 rabbit anti-coated ELISA plate, the stop solution volume is 96 wells × 50ul = 4800ul. The stop solution required for 1 sample is 4800ul ÷ 10 = 480ul. Summary: The theoretical value of stop solution required for 1 sample is 480ul.

[0023] Conclusion 1. To test 10 samples using one rabbit anti-coated ELISA plate, the following are required: 7500 μL of 25x PBST concentrate, μL of foot-and-mouth disease type O and type A virus antigens (5000 ÷ antigen dilution factor), 4800 μL of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies, 2400 μL of TMB substrate A solution, 4800 μL of TMB substrate B solution, and 4800 μL of stop solution.

[0024] Conclusion 2. When testing 10 samples with one rabbit anti-coated ELISA plate, one sample requires 750 μL of 25x PBST concentrate, 500 μL of foot-and-mouth disease type O and A virus antigens (500 ÷ antigen dilution factor), 480 μL of working solution for foot-and-mouth disease type O and A enzyme-labeled antibodies, 240 μL of TMB substrate A solution, 480 μL of TMB substrate B solution, and 480 μL of stop solution, respectively.

[0025] II. Application of theoretical values ​​in experimental operation: Taking the actual number of samples to be tested as the dependent variable, the theoretical amounts of PBST working solution, foot-and-mouth disease type O and A virus antigen working solutions, foot-and-mouth disease type O and A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution are calculated using the theoretical value rules. Then, the actual amounts of the above working solutions required in the experimental operation are calculated, and the two values ​​are compared. The deviation is corrected by increasing the number of virtual samples to complete the experiment.

[0026] (a) Detecting one sample with one rabbit anti-coated ELISA plate: 1. Compare the values ​​of PBST working solution to correct for sample quantity deviation. (1) Calculation of theoretical value of PBST working solution: 1 sample requires 750ul of PBST concentrate and 750ul of distilled water × 24 = 18000ul. The theoretical value of PBST working solution is 750ul + 18000ul = 18750ul.

[0027] (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (24 wells - 4 wells) × 50ul + (1 well × 25ul) = 1025ul. b. Wash volume used during plate washing: 24 wells × 300ul × 6 times = 43200ul.

[0028] The sum of the two results above is the actual value of the PBST working solution: 1025ul + 43200ul = 44225ul. The actual value of 44225ul is greater than the theoretical value of 18750ul, so the experiment cannot be performed. Let's add one sample, assuming the number of samples to be tested is 2, and calculate the theoretical value. Following the above calculation method, the theoretical value of the PBST working solution for 2 samples is 37500ul. The actual value of 44225ul is greater than the theoretical value of 37500ul, so the experiment cannot be performed. Let's add another sample, assuming the number of samples to be tested is 3, and calculate the theoretical value. Following the above calculation method, the theoretical value of the PBST working solution for 3 samples is 56250ul. The actual value of 44225ul is less than the theoretical value of 56250ul, so the experiment can be performed. In summary: One rabbit anti-coated ELISA plate can be used to test one sample. Following the theoretical value rule, adding two samples virtually to prepare the PBST working solution allows the experiment to be performed.

[0029] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, correcting for sample quantity deviations. (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigen: 1 sample requires foot-and-mouth disease type O and type A virus antigen (500 ÷ antigen dilution factor) ul, antigen diluent (antigen dilution factor - 1) × (500 ÷ antigen dilution factor) ul. Assuming the antigen dilution factor is 40 (of course, it can also be set to 50, 60, 70, 80), the foot-and-mouth disease type O and type A virus antigen is (500 ÷ 40) ul = 12.5 ul, and the antigen diluent is (40 - 1) × (500 ÷ 40) ul = 487.5 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigen is 12.5 ul + 487.5 ul = 500 ul.

[0030] (2) Calculation of actual value of working solution for foot-and-mouth disease type O and type A virus antigen: The actual value is (24 wells - 4 wells) × 50ul + 400ul = 1400ul.

[0031] The actual value of 1400ul is greater than the theoretical value of 500ul, so the experiment cannot be conducted. A virtual sample is added, assuming the number of samples to be tested is 2, and the theoretical value is calculated. Using the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 2 samples is 1000ul. The actual value of 1400ul is greater than the theoretical value of 1000ul, so the experiment cannot be conducted. Another virtual sample is added, assuming the number of samples to be tested is 3, and the theoretical value is calculated. Using the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 3 samples is 1500ul. The actual value of 1400ul is less than the theoretical value of 1500ul, so the experiment can be conducted.

[0032] In summary, one rabbit anti-coated ELISA plate can be used to test one sample. Following the theoretical value rule, two additional samples can be virtually added to prepare working solutions for foot-and-mouth disease type O and type A virus antigens, which can then be used for testing.

[0033] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviation: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​of foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 1 sample are 480ul, 240ul, 240ul, and 480ul, respectively.

[0034] (2) Calculation of actual values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 1200ul, 600ul, 600ul, and 1200ul, respectively. The actual value of 1200ul is greater than the theoretical value of 480ul, and the actual value of 600ul is greater than the theoretical value of 240ul, so the experiment cannot be performed. One sample is added virtually, assuming the number of samples to be tested is 2, and the theoretical values ​​are calculated. Following the above calculation method, the theoretical values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution for a sample quantity of 2 are 960ul, 480ul, 480ul, and 960ul, respectively. The actual value of 1200ul is greater than the theoretical value of 960ul, and the actual value of 600ul is greater than the theoretical value of 480ul, so the experiment cannot be performed. One more sample is added virtually, assuming the number of samples to be tested is 3, and the theoretical values ​​are calculated. Following the above calculation method, the theoretical values ​​of the working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for a sample quantity of 3 were calculated to be 1440 μL, 720 μL, 720 μL, and 1440 μL, respectively. The actual values ​​(1200 μL < theoretical value 1440 μL) and (600 μL < theoretical value 720 μL) indicate that the experiment can proceed.

[0035] In summary, one rabbit anti-coated ELISA plate can be used to test one sample. Following the theoretical value rule, two additional samples can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for the experiment.

[0036] Conclusion: One rabbit anti-coated ELISA plate can be used to test one sample. Following the theoretical value rule, two additional samples can be added virtually to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment can be completed smoothly.

[0037] (II) Detection of 2 samples with 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation (1) Calculation of theoretical value of PBST working solution Two samples require 750ul × 2 = 1500ul of PBST concentrate and 1500ul × 24 = 36000ul of distilled water. Therefore, the theoretical value of the PBST working solution is 1500ul + 36000ul = 37500ul.

[0038] (2) Calculation of the actual value of PBST working solution: a. Diluent serum volume (32 wells - 4 wells) × 50ul + (2 wells × 25ul) = 1450ul. b. Washing volume: 32 wells × 300ul × 6 times = 57600ul. The sum of the above two results is the actual value of PBST working solution: 1450ul + 57600ul = 59050ul. The actual value 59050ul > the theoretical value 37500ul, so the experiment cannot be performed. Add one sample virtually, assuming the number of samples to be tested is 3, and calculate the theoretical value. According to the above calculation method, the theoretical value of PBST working solution for 3 samples to be tested is 56250ul. The actual value 59050ul > the theoretical value 56250ul, so the experiment cannot be performed. Add one more sample virtually, assuming the number of samples to be tested is 4, and calculate the theoretical value. Based on the above calculation method, the theoretical value of PBST working solution for testing 4 samples is 75000 μL. The actual value of 59050 μL is less than the theoretical value of 75000 μL, so the experiment can proceed.

[0039] In summary, one rabbit anti-coated ELISA plate was used to test two samples. Following the theoretical value pattern, two additional samples were virtually added to prepare the PBST working solution for testing.

[0040] 2. Comparison of working solutions for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solutions for foot-and-mouth disease type O and type A virus antigens: 2 samples require 2 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and (antigen dilution factor - 1) × 2 × (500 ÷ antigen dilution factor) ul of antigen diluent. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 2 × (500 ÷ 40) ul = 25 ul, and the value of antigen diluent is (40 - 1) × 2 × (500 ÷ 40) ul = 975 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 25 ul + 975 ul = 1000 ul. (2) Calculation of actual value of working solution for foot-and-mouth disease type O and type A virus antigens: The actual value is (32 wells - 4 wells) × 50 ul + 400 ul = 1800 ul. The actual value of 1800 μL is greater than the theoretical value of 1000 μL, so the experiment cannot be conducted. Let's add one more sample, assuming the total number of samples to be tested is 3, and calculate the theoretical value. Using the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 3 samples is 1500 μL. The actual value of 1800 μL is greater than the theoretical value of 1500 μL, so the experiment cannot be conducted. Let's add another sample, assuming the total number of samples to be tested is 4, and calculate the theoretical value. Using the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 4 samples is 2000 μL. The actual value of 1800 μL is less than the theoretical value of 2000 μL, so the experiment can be conducted.

[0041] In summary, one rabbit anti-coated ELISA plate can be used to test two samples. Following the theoretical value rule, two additional samples can be virtually added to prepare working solutions for foot-and-mouth disease type O and type A virus antigens, which can then be used for testing.

[0042] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​of the working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 2 samples are 960ul, 480ul, 480ul, and 960ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 1600ul, 800ul, 800ul, and 1600ul, respectively. The actual value of 1600ul is greater than the theoretical value of 960ul, and the actual value of 800ul is greater than the theoretical value of 480ul, so the experiment cannot be performed. Adding one sample virtually, assuming the total number of samples to be tested is 3, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 3 samples to be tested are 1440 μL, 720 μL, 720 μL, and 1440 μL, respectively. The actual values ​​(1600 μL > theoretical value 1440 μL) and (800 μL > theoretical value 720 μL) are not suitable for the experiment. Adding another sample virtually, assuming the total number of samples to be tested is 4, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 4 samples to be tested are 1920 μL, 960 μL, 960 μL, and 1920 μL, respectively. If the actual value is 1600ul < the theoretical value is 1920ul, and the actual value is 800ul < the theoretical value is 960ul, then the experiment can be conducted.

[0043] In summary, one rabbit anti-coated ELISA plate was used to test two samples. Following the theoretical value rule, two additional samples were virtually added to prepare working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for the experiment.

[0044] Conclusion: One rabbit anti-coated ELISA plate can be used to detect two samples. Following the theoretical value rule, two additional samples can be added virtually to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment can be completed smoothly.

[0045] (III) Detection of 3 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 3 samples require 750ul × 3 = 2250ul of PBST concentrate and 2250ul × 24 = 54000ul of distilled water. The theoretical value of PBST working solution is 2250ul + 54000ul = 56250ul. (2) Calculation of actual value of PBST working solution: a. Amount of serum diluted (40 wells - 4 wells) × 50ul + (3 wells × 25ul) = 1875ul. b. Amount used for washing the plate: 40 wells × 300ul × 6 times = 72000ul. The sum of the results of the above two formulas is the actual value of PBST working solution: 1875ul + 72000ul = 73875ul. The actual value 73875ul > the theoretical value 56250ul, so the experiment cannot be performed. Adding one sample virtually, assuming a total of 4 samples need to be tested, we calculate the theoretical value. Following the above calculation method, the theoretical value of the PBST working solution for 4 samples is 75000 μL. The actual value of 73850 μL is less than the theoretical value of 75000 μL, so the experiment can proceed.

[0046] In summary, one rabbit anti-coated ELISA plate was used to test three samples. Following the theoretical value pattern, one additional sample was added virtually to prepare the PBST working solution for testing.

[0047] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 3 samples require 3 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and antigen diluent (antigen dilution factor - 1) × 3 × (500 ÷ antigen dilution factor) ul. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 3 × (500 ÷ 40) ul = 37.5 ul, and the value of antigen diluent is (40 - 1) × 3 × (500 ÷ 40) ul = 1462.5 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 37.5 ul + 1462.5 ul = 1500 ul. (2) Calculation of actual value of foot-and-mouth disease type O and type A virus antigen working solution: The actual value is (40 wells - 4 wells) × 50ul + 400ul = 2200ul. The actual value of 2200ul > the theoretical value of 1500ul, so the test cannot be carried out. Add one sample virtually, assuming that the number of samples to be tested is 4, and calculate the theoretical value. According to the above calculation method, the theoretical value of foot-and-mouth disease type O and type A virus antigen working solution for 4 samples to be tested is 2000ul. The actual value of 2200ul > the theoretical value of 2000ul, so the test cannot be carried out. Add one more sample virtually, assuming that the number of samples to be tested is 5, and calculate the theoretical value. According to the above calculation method, the theoretical value of foot-and-mouth disease type O and type A virus antigen working solution for 5 samples to be tested is 2500ul. The actual value of 2200ul < the theoretical value of 2500ul, so the test can be carried out.

[0048] In summary, one rabbit anti-coated ELISA plate can be used to test three samples. Following the theoretical value pattern, two additional samples can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A virus antigens, which can then be used for testing.

[0049] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​of the working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 3 samples are 1440ul, 720ul, 720ul, and 1440ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 2000ul, 1000ul, 1000ul, and 2000ul, respectively. The actual value 2000ul > the theoretical value 1440ul, and the actual value 1000ul > the theoretical value 720ul, so the experiment cannot be performed. Adding one sample virtually, assuming the total number of samples to be tested is 4, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 4 samples to be tested are 1920 μL, 960 μL, 960 μL, and 1920 μL, respectively. The actual values ​​(2000 μL > theoretical value 1920 μL) and (1000 μL > theoretical value 960 μL) are not suitable for the experiment. Adding another sample virtually, assuming the total number of samples to be tested is 5, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 5 samples to be tested are 2400 μL, 1200 μL, 1200 μL, and 2400 μL, respectively. If the actual value is 2000ul < the theoretical value is 2400ul, and the actual value is 1000ul < the theoretical value is 1200ul, then the experiment can be conducted.

[0050] In summary, one rabbit anti-coated ELISA plate can be used to test three samples. Following the theoretical value rule, two additional samples can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution, which can then be used for experiments.

[0051] Conclusion: One rabbit anti-coated ELISA plate can detect 3 samples, following the theoretical value. To uniformly increase the number of samples, we added 2 samples to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment can be completed smoothly.

[0052] (IV) Detection of 4 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 4 samples require 750ul × 4 = 3000ul of PBST concentrate and 3000ul × 24 = 72000ul of distilled water. The theoretical value of PBST working solution is 3000ul + 72000ul = 75000ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (48 wells - 4 wells) × 50ul + (4 wells × 25ul) = 2300ul. b. Washing volume 48 wells × 300ul × 6 times = 86400ul. The sum of the above two results is the actual value of PBST working solution 2300ul + 86400ul = 88700ul. The actual value 88700ul > the theoretical value 75000ul, so the experiment cannot be performed. Adding one sample virtually, assuming the total number of samples to be tested is 5, the theoretical value is calculated. Following the above calculation method, the theoretical value of PBST working solution for 5 samples is 93750 μL. The actual value is 88700 μL < the theoretical value of 93750 μL, therefore the experiment can proceed.

[0053] In summary, one rabbit anti-coated ELISA plate was used to test four samples. Following the theoretical value pattern, one additional sample was added virtually to prepare the PBST working solution for testing.

[0054] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 4 samples require 4 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and (antigen dilution factor - 1) × 4 × (500 ÷ antigen dilution factor) ul of antigen diluent. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 4 × (500 ÷ 40) ul = 50 ul, and the value of antigen diluent is (40 - 1) × 4 × (500 ÷ 40) ul = 1950 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 50 ul + 1950 ul = 2000 ul. (2) Calculation of actual value of working solution for foot-and-mouth disease type O and type A virus antigens: The actual value is (48 wells - 4 wells) × 50 ul + 400 ul = 2600 ul. The actual value of 2600 μL is greater than the theoretical value of 2000 μL, so the experiment cannot be conducted. Let's add one more sample, assuming the total number of samples to be tested is 5, and calculate the theoretical value. Using the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 5 samples is 2500 μL. The actual value of 2600 μL is greater than the theoretical value of 2500 μL, so the experiment cannot be conducted. Let's add another sample, assuming the total number of samples to be tested is 6, and calculate the theoretical value. Using the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 6 samples is 3000 μL. The actual value of 2600 μL is less than the theoretical value of 3000 μL, so the experiment can be conducted.

[0055] In summary, one rabbit anti-coated ELISA plate can be used to test four samples. Following the theoretical value pattern, two additional samples can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A virus antigens, which can then be used for testing.

[0056] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​of the working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 4 samples are 1920ul, 960ul, 960ul, and 1920ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 2400ul, 1200ul, 1200ul, and 2400ul, respectively. The actual value 2400ul > the theoretical value 1920ul, and the actual value 1200ul > the theoretical value 960ul, so the experiment cannot be carried out. Adding one sample virtually, assuming a total of 5 samples needing testing, calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 5 samples are calculated to be 2400 μL, 1200 μL, 1200 μL, and 2400 μL, respectively. The actual values ​​of 2400 μL and 1200 μL are consistent with the theoretical values, respectively, and the experiment can proceed.

[0057] In summary, one rabbit anti-coated ELISA plate can be used to test four samples. Following the theoretical value pattern, one additional sample can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution, which can then be used for experiments.

[0058] Conclusion: One rabbit anti-coated ELISA plate can detect 4 samples, following the theoretical value. To ensure consistency, two additional samples were added to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment can be completed smoothly.

[0059] (V) Detection of 5 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation (1) Calculation of theoretical value of PBST working solution: 5 samples require 750ul × 5 = 3750ul of PBST concentrate and 3750ul × 24 = 90000ul of distilled water. The theoretical value of PBST working solution is 3750ul + 90000ul = 93750ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (56 wells - 4 wells) × 50ul + (5 wells × 25ul) = 2725ul.

[0060] b. Washing volume: 56 wells × 300 μL × 6 washes = 100800 μL. The sum of the above two results is the actual value of the PBST working solution: 2725 μL + 100800 μL = 103525 μL. The actual value (103525 μL) > the theoretical value (93750 μL), therefore the experiment cannot be performed. Let's add one sample, assuming the number of samples to be tested is 6, and calculate the theoretical value. Following the above calculation method, the theoretical value of the PBST working solution for 6 samples is 112500 μL. The actual value (103525 μL) < the theoretical value (112500 μL), therefore the experiment can be performed.

[0061] In summary, one rabbit anti-coated ELISA plate was used to test five samples. Following the theoretical value pattern, one additional sample was added virtually to prepare the PBST working solution for testing.

[0062] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 5 samples require 5 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and antigen diluent (antigen dilution factor - 1) × 5 × (500 ÷ antigen dilution factor) ul. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 5 × (500 ÷ 40) ul = 62.5 ul, and the value of antigen diluent is (40 - 1) × 5 × (500 ÷ 40) ul = 2437.5 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 62.5 ul + 2437.5 ul = 2500 ul. (2) Calculation of actual value of foot-and-mouth disease type O and type A virus antigen working solution: The actual value is (56 wells - 4 wells) × 50ul + 400ul = 3000ul. The actual value of 3000ul > the theoretical value of 2500ul, so the experiment cannot be carried out. One sample is added virtually, assuming the number of samples to be tested is 6, and the theoretical value is calculated. According to the above calculation method, the theoretical value of the foot-and-mouth disease type O and type A virus antigen working solution for a sample quantity of 6 is 3000ul. The actual value of 3000ul = the theoretical value of 3000ul, so the experiment can be carried out.

[0063] In summary, one rabbit anti-coated ELISA plate can be used to test five samples. Following the theoretical value pattern, one additional sample can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A virus antigens, which can then be used for testing.

[0064] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 5 samples are 2400ul, 1200ul, 1200ul, and 2400ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 2800ul, 1400ul, 1400ul, and 2800ul, respectively. The actual value 2800ul > the theoretical value 2400ul, and the actual value 1400ul > the theoretical value 1200ul, so the experiment cannot be carried out. Adding one sample virtually, assuming a total of 6 samples to be tested, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 6 samples to be tested are 2880 μL, 1440 μL, 1440 μL, and 2880 μL, respectively. The actual values ​​(2800 μL < theoretical value 2880 μL) and (1400 μL < theoretical value 1440 μL) are acceptable for testing. In summary, one rabbit anti-coated ELISA plate can be used to test 5 samples. Following the theoretical value pattern, adding one sample virtually to prepare the working solutions of FMD type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution allows for testing.

[0065] Conclusion: One rabbit anti-coated ELISA plate can be used to test 5 samples. Following the theoretical value, adding one sample to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution can successfully complete the experiment.

[0066] (VI) Detection of 6 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 6 samples require 750ul × 6 = 4500ul of PBST concentrate and 4500ul × 24 = 108000ul of distilled water. The theoretical value of PBST working solution is 4500ul + 108000ul = 112500ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (64 wells - 4 wells) × 50ul + (6 wells × 25ul) = 3150ul. b. Washing volume 64 wells × 300ul × 6 times = 115200ul. The sum of the above two results is the actual value of PBST working solution 3150ul + 115200ul = 118350ul. The actual value (118350 μL) is greater than the theoretical value (112500 μL), so the experiment cannot be performed. A virtual sample is added, assuming the number of samples to be tested is 7, and the theoretical value is calculated. Following the above calculation method, the theoretical value of the PBST working solution for 7 samples is 131250 μL. The actual value (118350 μL) is less than the theoretical value (131250 μL), so the experiment can be performed. In summary: One rabbit anti-coated ELISA plate can be used to test 6 samples. Following the theoretical value pattern, a virtual sample is added to prepare the PBST working solution, and the experiment can be performed.

[0067] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 6 samples require 6 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and (antigen dilution factor - 1) × 6 × (500 ÷ antigen dilution factor) ul of antigen diluent. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 6 × (500 ÷ 40) ul = 75 ul, and the value of antigen diluent is (40 - 1) × 6 × (500 ÷ 40) ul = 2925 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 75 ul + 2925 ul = 3000 ul. (2) Calculation of actual value of working solution for foot-and-mouth disease type O and type A virus antigens: The actual value is (64 wells - 4 wells) × 50 ul + 400 ul = 3400 ul. The actual value (3400 μL) is greater than the theoretical value (3000 μL), so the experiment cannot be performed. A virtual sample is added, assuming the number of samples to be tested is 7, and the theoretical value is calculated. Following the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 7 samples is 3500 μL. The actual value (3400 μL) is less than the theoretical value (3500 μL), so the experiment can be performed. In summary: One rabbit anti-coated ELISA plate can be used to test 6 samples. Following the theoretical value pattern, a virtual sample is added to prepare the FMD type O and A virus antigen working solution, and the experiment can be performed.

[0068] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 6 samples are 2880ul, 1440ul, 1440ul, and 2880ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 3200ul, 1600ul, 1600ul, and 3200ul, respectively. The actual value of 3200ul is greater than the theoretical value of 2880ul, and the actual value of 1600ul is greater than the theoretical value of 1440ul, so the experiment cannot be carried out. Adding one sample virtually, assuming a total of 7 samples to be tested, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 7 samples to be tested are 3360 μL, 1680 μL, 1680 μL, and 3360 μL, respectively. The actual values ​​(3200 μL < theoretical value 3360 μL) and (1600 μL < theoretical value 1680 μL) are acceptable for testing. In summary, one rabbit anti-coated ELISA plate can be used to test 6 samples. Following the theoretical value pattern, adding one sample virtually to prepare the working solutions of FMD type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution allows for testing.

[0069] Conclusion: One rabbit anti-coated ELISA plate can be used to test 6 samples. Following the theoretical value, adding one sample to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution can successfully complete the experiment.

[0070] (VII) Detection of 7 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 7 samples require 750ul × 7 = 5250ul of PBST concentrate and 5250ul × 24 = 126000ul of distilled water. The theoretical value of PBST working solution is 5250ul + 126000ul = 131250ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (72 wells - 4 wells) × 50ul + (7 wells × 25ul) = 3575ul. b. Washing volume 72 wells × 300ul × 6 times = 129600ul. The sum of the above two results is the actual value of PBST working solution 3575ul + 129600ul = 133175ul. The actual value (133175 μL) is greater than the theoretical value (131250 μL), so the experiment cannot be performed. A virtual sample of 8 samples is added, and the theoretical value is calculated. Following the above calculation method, the theoretical value of the PBST working solution for 8 samples is 150000 μL. The actual value (133175 μL) is less than the theoretical value (150000 μL), so the experiment can be performed. In summary: One rabbit anti-coated ELISA plate can be used to test 7 samples. Following the theoretical value pattern, a virtual sample of 1 additional sample is added to prepare the PBST working solution, and the experiment can be performed.

[0071] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 7 samples require 7 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and antigen diluent (antigen dilution factor - 1) × 7 × (500 ÷ antigen dilution factor) ul. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 7 × (500 ÷ 40) ul = 87.5 ul, and the value of antigen diluent is (40 - 1) × 7 × (500 ÷ 40) ul = 3412.5 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 87.5 ul + 3412.5 ul = 3500 ul. (2) Calculation of actual value of foot-and-mouth disease type O and type A virus antigen working solution: The actual value is (72 wells - 4 wells) × 50ul + 400ul = 3800ul. The actual value of 3800ul > the theoretical value of 3500ul, so the experiment cannot be carried out. Virtually add 1 sample, assuming that the number of samples to be tested is 8, and calculate the theoretical value. According to the above calculation method, the theoretical value of foot-and-mouth disease type O and type A virus antigen working solution with 8 samples to be tested is 4000ul. The actual value of 3800ul < the theoretical value of 4000ul, so the experiment can be carried out. Summary: 1 rabbit anti-coated ELISA plate can be used to test 7 samples. Following the theoretical value rule, 1 sample can be virtually added to prepare foot-and-mouth disease type O and type A virus antigen working solution, so the experiment can be carried out.

[0072] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 7 samples are 3360ul, 1680ul, 1680ul, and 3360ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 3600ul, 1800ul, 1800ul, and 3600ul, respectively. The actual value of 3600ul is greater than the theoretical value of 3360ul, and the actual value of 1800ul is greater than the theoretical value of 1680ul, so the experiment cannot be completed. Adding one sample virtually, assuming a total of 8 samples to be tested, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 8 samples to be tested are 3840 μL, 1920 μL, 1920 μL, and 3840 μL, respectively. The actual values ​​(3600 μL < theoretical value 3840 μL) and (1800 μL < theoretical value 1920 μL) are acceptable for testing. In summary, one rabbit anti-coated ELISA plate can be used to test 7 samples. Following the theoretical value pattern, adding one sample virtually to prepare the working solutions of FMD type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution allows for testing.

[0073] Conclusion: One rabbit anti-coated ELISA plate was used to test 7 samples. Following the theoretical value, one additional sample was added to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment could be completed successfully.

[0074] (VIII) Detection of 8 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 8 samples require 750ul × 8 = 6000ul of PBST concentrate and 6000ul × 24 = 144000ul of distilled water. The theoretical value of PBST working solution is 6000ul + 144000ul = 150000ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (80 wells - 4 wells) × 50ul + (8 wells × 25ul) = 4000ul. b. Washing volume: 80 wells × 300ul × 6 times = 144000ul. The sum of the above two results is the actual value of PBST working solution: 4000ul + 144000ul = 148000ul. The actual value of 148,000 μL is less than the theoretical value of 150,000 μL, so the experiment can proceed. In summary: One rabbit anti-coated ELISA plate was used to test 8 samples. Following the theoretical value pattern, 0 samples were added virtually to prepare the PBST working solution, and the experiment can proceed.

[0075] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 8 samples require 8 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and (antigen dilution factor - 1) × 8 × (500 ÷ antigen dilution factor) ul of antigen diluent. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 8 × (500 ÷ 40) ul = 100 ul, and the value of antigen diluent is (40 - 1) × 8 × (500 ÷ 40) ul = 3900 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 100 ul + 3900 ul = 4000 ul. (2) Calculation of actual value of working solution for foot-and-mouth disease type O and type A virus antigens: The actual value is (80 wells - 4 wells) × 50 ul + 400 ul = 4200 ul. The actual value of 4200ul is greater than the theoretical value of 4000ul, so the experiment cannot be conducted. A virtual sample is added, assuming the number of samples to be tested is 9, and the theoretical value is calculated. Following the above calculation method, the theoretical value of the foot-and-mouth disease (FMD) type O and A virus antigen working solution for 9 samples is 4500ul. The actual value of 4200ul is less than the theoretical value of 4500ul, so the experiment can be conducted. In summary: One rabbit anti-coated ELISA plate can be used to test 8 samples. Following the theoretical value pattern, a virtual sample is added to prepare the FMD type O and A virus antigen working solution, and the experiment can be conducted.

[0076] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 8 samples are 3840ul, 1920ul, 1920ul, and 3840ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 4000ul, 2000ul, 2000ul, and 4000ul, respectively. The actual value 4000ul > the theoretical value 3840ul, and the actual value 2000ul > the theoretical value 1920ul, so the experiment cannot be carried out. Adding one sample virtually, assuming a total of 9 samples to be tested, we calculate the theoretical values. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 9 samples to be tested are 4320 μL, 2160 μL, 2160 μL, and 4320 μL, respectively. The actual values ​​(4000 μL < theoretical value 4320 μL) and (2000 μL < theoretical value 2160 μL) are acceptable for testing. In summary, one rabbit anti-coated ELISA plate can be used to test 8 samples. Following the theoretical value pattern, adding one sample virtually to prepare the working solutions of FMD type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution allows for testing.

[0077] Conclusion: One rabbit anti-coated ELISA plate can detect 8 samples, following the theoretical value. To ensure consistency, one additional sample was added to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment can be completed smoothly.

[0078] (ix) Detection of 9 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 9 samples require 750ul × 9 = 6750ul of PBST concentrate and 6750ul × 24 = 162000ul of distilled water. The theoretical value of PBST working solution is 6750ul + 162000ul = 168750ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (88 wells - 4 wells) × 50ul + (9 wells × 25ul) = 4425ul. b. Washing volume (88 wells × 300ul × 6 times) = 158400ul. The sum of the above two results is the actual value of PBST working solution: 4425ul + 158400ul = 162825ul. The actual value of 162,825 μL is less than the theoretical value of 168,750 μL, so the experiment can proceed. In summary, one rabbit anti-coated ELISA plate was used to test 9 samples, following the theoretical value pattern. Virtually adding 0 samples to prepare the PBST working solution allows for further experimentation.

[0079] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 9 samples require 9 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and antigen diluent (antigen dilution factor - 1) × 9 × (500 ÷ antigen dilution factor) ul. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 9 × (500 ÷ 40) ul = 112.5 ul, and the value of antigen diluent is (40 - 1) × 9 × (500 ÷ 40) ul = 4387.5 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 112.5 ul + 4387.5 ul = 4500 ul. (2) Calculation of actual value of foot-and-mouth disease type O and type A virus antigen working solution: The actual value is (88 wells - 4 wells) × 50ul + 400ul = 4600ul. The actual value of 4600ul > the theoretical value of 4500ul, so the experiment cannot be carried out. Virtually add 1 sample, assuming that the number of samples to be tested is 10, and calculate the theoretical value. According to the above calculation method, the theoretical value of foot-and-mouth disease type O and type A virus antigen working solution for 10 samples to be tested is 5000ul. The actual value of 4600ul < the theoretical value of 5000ul, so the experiment can be carried out. Summary: 1 rabbit anti-coated ELISA plate can be used to test 9 samples. Following the theoretical value rule, 1 sample can be virtually added to prepare foot-and-mouth disease type O and type A virus antigen working solution, so the experiment can be carried out.

[0080] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 9 samples are 4320ul, 2160ul, 2160ul, and 4320ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 4400ul, 2200ul, 2200ul, and 4400ul, respectively. The actual value 4400ul > the theoretical value 4320ul, and the actual value 2200ul > the theoretical value 2160ul, so the experiment cannot be carried out. A virtual sample was added, assuming the total number of samples to be tested was 10, and the theoretical values ​​were calculated. Following the above calculation method, the theoretical values ​​for the working solutions of foot-and-mouth disease (FMD) type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 10 samples to be tested were 4800 μL, 2400 μL, 2400 μL, and 4800 μL, respectively. The actual values ​​(4400 μL < theoretical value 4800 μL) and (2200 μL < theoretical value 2400 μL) were found to be within acceptable limits for testing. In summary, one rabbit anti-coated ELISA plate can be used to test 9 samples. Following the theoretical value pattern, a virtual sample was added to prepare the working solutions of FMD type O and A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution, which are then suitable for testing.

[0081] Conclusion: One rabbit anti-coated ELISA plate was used to test 9 samples, following the theoretical value. To ensure consistency, one additional sample was added to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment was successfully completed.

[0082] III. Comparison and analysis of the actual usage and measured quantity of each reaction solution in the kit (I) Actual amount of each reaction solution used The actual volumes of PBST working solution used for testing samples 1-9 were 44225ul, 59050ul, 73875ul, 88700ul, 103525ul, 118350ul, 133175ul, 148000ul, and 162825ul, respectively.

[0083] For samples 1-9, the actual amounts of working solution used for foot-and-mouth disease type O and type A virus antigens were 1400ul, 1800ul, 2200ul, 2600ul, 3000ul, 3400ul, 3800ul, 4200ul, and 4600ul, respectively.

[0084] For testing samples 1-9, the actual amounts of enzyme-labeled antibody working solutions for foot-and-mouth disease type O and type A were 1200ul, 1600ul, 2000ul, 2400ul, 2800ul, 3200ul, 3600ul, 4000ul, and 4400ul, respectively.

[0085] For testing 1-9 samples, the actual amounts of TMB substrate A or B solution used were 600ul, 800ul, 1000ul, 1200ul, 1400ul, 1600ul, 1800ul, 2000ul, and 2200ul, respectively.

[0086] For samples 1-9, the actual amounts of stop solution used were 1200ul, 1600ul, 2000ul, 2400ul, 2800ul, 3200ul, 3600ul, 4000ul, and 4400ul, respectively.

[0087] (II) Quantity of each reaction solution For samples 1-9, the quantities of PBST working solution were 56250ul, 75000ul, 93750ul, 112500ul, 112500ul, 131250ul, 150000ul, 168750ul, and 187500ul, respectively.

[0088] For testing 1-9 samples, the working solutions for foot-and-mouth disease type O and type A virus antigens were measured in quantities of 1500ul, 2000ul, 2500ul, 3000ul, 3000ul, 3500ul, 4000ul, 4500ul, and 5000ul, respectively.

[0089] For testing 1-9 samples, the working solutions of enzyme-labeled antibodies for foot-and-mouth disease type O and type A were measured in quantities of 1440ul, 1920ul, 2400ul, 2880ul, 2880ul, 3360ul, 3840ul, 4320ul, and 4800ul, respectively.

[0090] For testing 1-9 samples, the quantities of TMB substrate A or B solution were 720ul, 960ul, 1200ul, 1440ul, 1440ul, 1680ul, 1920ul, 2160ul, and 2400ul, respectively.

[0091] For testing samples 1-9, the stop solution volumes were 1440ul, 1920ul, 2400ul, 2880ul, 2880ul, 3360ul, 3840ul, 4320ul, and 4800ul, respectively.

[0092] (III) The difference between the measured quantity of working solution for each reaction solution in the kit and the actual quantity used. For samples 1-9, the differences between the measured quantity and the actual amount of PBST working solution used were 12025ul, 15950ul, 19875ul, 23800ul, 8975ul, 12900ul, 16825ul, 20750ul, and 24675ul, respectively.

[0093] For samples 1-9, the differences between the measured quantity and the actual amount of foot-and-mouth disease type O and type A virus antigen working solution used were 100ul, 200ul, 300ul, 400ul, 0ul, 100ul, 200ul, 300ul, and 400ul, respectively.

[0094] For samples 1-9, the differences between the measured quantity and the actual amount of foot-and-mouth disease type O and type A enzyme-labeled antibody working solution were 240ul, 320ul, 400ul, 480ul, 80ul, 160ul, 240ul, 320ul, and 400ul, respectively.

[0095] For testing 1-9 samples, the difference between the measured quantity of TMB substrate A or B solution and the actual amount used is 120ul, 160ul, 200ul, 240ul, 40ul, 80ul, 120ul, 160ul, and 200ul, respectively.

[0096] For samples 1-9, the differences between the measured quantity of stop solution and the actual amount used were 240ul, 320ul, 400ul, 480ul, 80ul, 160ul, 240ul, 320ul, and 400ul, respectively.

[0097] (iv) The difference between the measured quantity of PBST working solution and the actual amount used. The difference between the measured quantity and the actual usage of foot-and-mouth disease type O and type A virus antigen working solution, and the corresponding concentrated solution (antigen diluent) value. 1. For testing 1-9 samples, the difference between the measured amount of PBST working solution and the actual amount used corresponds to the values ​​of 25x PBST concentrate: 481ul, 638ul, 795ul, 952ul, 359ul, 516ul, 673ul, 830ul, and 987ul.

[0098] 2. Test 1-9 samples. The difference between the measured quantity and the actual amount of foot-and-mouth disease type O and type A virus antigen working solution is the corresponding value of foot-and-mouth disease type O and type A virus antigen, respectively (set antigen dilution factor as 40): 2.5ul, 5ul, 7.5ul, 10ul, 0ul, 2.5ul, 5ul, 7.5ul, 10ul.

[0099] 3. Test 1-9 samples. The difference between the measured quantity and the actual amount of foot-and-mouth disease type O and type A virus antigen working solution and the corresponding values ​​of foot-and-mouth disease type O and type A virus antigen dilution solution are (set antigen dilution factor as 40): 97.5ul, 195ul, 292.5ul, 390ul, 0ul, 97.5ul, 195ul, 292.5ul, 390ul.

[0100] (v) The following contents are included in the kit: 25x PBST concentrate, foot-and-mouth disease type O and type A virus antigen, antigen dilution solution, foot-and-mouth disease type O and type A enzyme-labeled antibody working solution, TMB substrate A or B solution, and stop solution volumes of 60ml, 6ml, 30ml, 30ml, 15ml, and 30ml, respectively. (vi) The volume ratio of the concentrated solution (reaction solution) to the total volume corresponding to the difference between the measured quantity of working solution and the actual quantity used for each reaction solution in the kit. 1. For samples 1-9, the volume ratio of 25x PBST concentrate to the total volume corresponding to the difference between the measured amount of PBST working solution and the actual amount used is 0.8%, 1%, 1.3%, 1.58%, 0.598%, 0.86%, 1.12%, 1.38%, and 1.64%, respectively.

[0101] 2. For 1-9 samples, the volume ratio of the virus antigen corresponding to the difference between the measured quantity and the actual amount used in the working solution of foot-and-mouth disease type O and type A virus antigens is 0.04%, 0.08%, 0.12%, 0.16%, 0%, 0.04%, 0.08%, 0.12%, and 0.16%, respectively (assuming an antigen dilution factor of 40).

[0102] 3. For samples 1-9, the volume ratio of the difference between the measured quantity and the actual amount of foot-and-mouth disease (FMD) type O and A virus antigen working solution to the total volume of the corresponding FMD type O and A virus antigen dilution solution is as follows (assuming an antigen dilution factor of 40): 0.325%, 0.65%, 0.975%, 1.3%, 0%, 0.325%, 0.65%, 0.975%, 1.3%.

[0103] 4. For 1-9 samples, the volume ratio of the difference between the measured quantity and the actual amount of foot-and-mouth disease type O and type A enzyme-labeled antibody working solution to the total volume is 0.8%, 1.06%, 1.33%, 1.6%, 0.26%, 0.53%, 0.8%, 1.06%, and 1.33%, respectively.

[0104] 5. For testing 1-9 samples, the volume ratio of the difference between the measured quantity of TMB substrate A or B solution and the actual amount used to the total volume is 0.8%, 1.06%, 1.33%, 1.6%, 0.26%, 0.53%, 0.8%, 1.06%, and 1.33%, respectively.

[0105] 6. For testing 1-9 samples, the volume ratio of the difference between the measured amount of stop solution and the actual amount used to the total volume is 0.8%, 1.06%, 1.33%, 1.6%, 0.26%, 0.53%, 0.8%, 1.06%, and 1.33%, respectively.

[0106] Therefore, the maximum values ​​of the volume ratios of the working solutions (concentrates) corresponding to the differences between the measured quantities and the actual quantities of the following components for testing samples 1-9 are: 25x PBST concentrate, foot-and-mouth disease type O and A virus antigen, antigen dilution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A or B solution, and stop solution. These values ​​are 1.64%, 0.16%, 1.3%, 1.6%, 1.6%, and 1.6%, respectively.

[0107] Through years of extensive testing and verification, it has been shown that the volume and proportion of the additional reaction solutions measured in the above kit have no impact on the experiment and can be ignored.

[0108] (VII) Detection of 10 samples on 1 rabbit anti-coated ELISA plate: 1. Comparison of PBST working solution values ​​and correction of sample quantity deviation: (1) Calculation of theoretical value of PBST working solution: 10 samples require 750ul × 10 = 7500ul of PBST concentrate and 7500ul × 24 = 180000ul of distilled water. The theoretical value of PBST working solution is 7500ul + 180000ul = 187500ul. (2) Calculation of actual value of PBST working solution: a. Dilute serum volume (96 wells - 4 wells) × 50ul + (10 wells × 25ul) = 4850ul. b. Washing volume (96 wells × 300ul × 6 times) = 172800ul. The sum of the above two results is the actual value of PBST working solution: 4850ul + 172800ul = 177650ul. The actual value of 177650ul is less than the theoretical value of 187500ul, so the experiment can be conducted.

[0109] In summary, one rabbit anti-coated ELISA plate can be used to test 10 samples, following the theoretical value pattern. By virtually adding 0 samples to prepare the PBST working solution, experiments can be conducted.

[0110] 2. Comparison of working solution values ​​for foot-and-mouth disease type O and type A virus antigens, and correction of sample quantity deviation: (1) Calculation of theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens: 10 samples require 10 × (500 ÷ antigen dilution factor) ul of foot-and-mouth disease type O and type A virus antigens, and (antigen dilution factor - 1) × 10 × (500 ÷ antigen dilution factor) ul of antigen diluent. Assuming the antigen dilution factor is 40, the calculated value of foot-and-mouth disease type O and type A virus antigens is 10 × (500 ÷ 40) ul = 125 ul, and the value of antigen diluent is (40 - 1) × 10 × (500 ÷ 40) ul = 4875 ul. The theoretical value of working solution for foot-and-mouth disease type O and type A virus antigens is 125 ul + 4875 ul = 5000 ul. (2) Calculation of actual value of working solution for foot-and-mouth disease type O and type A virus antigens: The actual value is (96 wells - 4 wells) × 50 ul + 400 ul = 5000 ul. The actual value of 5000ul equals the theoretical value of 5000ul, which is sufficient for the experiment. However, due to residual working solution on the sidewall of the pipette tip, the working solution is insufficient, preventing the experiment from being completed. A virtual sample is added, assuming the number of samples to be tested is 11, and the theoretical value is calculated. Following the above calculation method, the theoretical value of the working solution for foot-and-mouth disease type O and type A virus antigens for 11 samples is 5520ul. (If the antigen result has a decimal, the "round up" principle applies; the antigen value is 138ul). The actual value of 5000ul is less than the theoretical value of 5520ul, so the experiment can be conducted and successfully completed.

[0111] In summary, one rabbit anti-coated ELISA plate can be used to test 10 samples. Following the theoretical value pattern, one additional sample can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A virus antigens, which can then be used for testing.

[0112] 3. Comparison of working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution values, and correction of sample quantity deviations: (1) Calculation of theoretical values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The theoretical values ​​of foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution required for 10 samples are 4800ul, 2400ul, 2400ul, and 4800ul, respectively. (2) Calculation of actual values ​​for working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution: The actual values ​​are 4800ul, 2400ul, 2400ul, and 4800ul, respectively. The actual value of 4800 μL equals the theoretical value of 4800 μL, and the actual value of 2400 μL equals the theoretical value of 2400 μL. The experiment can proceed, but due to residual working solution on the pipette tip sidewall, the working solution is insufficient, preventing the experiment from being completed. A virtual sample is added, assuming the number of samples to be tested is 11, and the theoretical values ​​are calculated. Following the above calculation method, the theoretical values ​​for the working solution of foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution for 11 samples to be tested are 5280 μL, 2640 μL, 2640 μL, and 5280 μL, respectively. The actual value of 4800 μL < the theoretical value of 5280 μL, and the actual value of 2400 μL < the theoretical value of 2640 μL, indicating the experiment can proceed and is successfully completed. In summary, one rabbit anti-coated ELISA plate can be used to test 10 samples. Following the theoretical value rule, one additional sample can be added virtually to prepare working solutions for foot-and-mouth disease type O and type A enzyme-labeled antibodies, TMB substrate A solution, TMB substrate B solution, and stop solution, which can then be used for experiments.

[0113] Conclusion: One rabbit anti-coated ELISA plate can detect 10 samples, following the theoretical value rule. To ensure consistency, one additional sample was added to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution. The experiment can be completed smoothly.

[0114] When 10 samples are tested using one rabbit anti-coated ELISA plate, the same method is used. Through repeated testing and verification over many years, it has been shown that the volume and proportion of the additional reaction solutions in the kit compared to the actual amount have no effect on the experiment and can be ignored.

[0115] In summary, when performing a liquid-phase blocking ELISA assay for foot-and-mouth disease (FMD) type O and A antibodies, with a sample layout of 10 samples tested on one rabbit anti-coated ELISA plate and the serum being tested diluted to only 1:1024, the following parameters can be set for each sample: 750 μL of 25-fold PBST concentrate, μL of FMD type O and A virus antigens (500 ÷ antigen dilution factor), 480 μL of FMD type O and A enzyme-labeled antibody working solution, 240 μL of TMB substrate A solution, 480 μL of TMB substrate B solution, and 480 μL of stop solution.

[0116] When actually testing 1-4 samples, 2 additional samples are virtually added, i.e., the sample quantity is set to 3-6 to prepare PBST working solution, foot-and-mouth disease type O and type A virus antigen working solution, foot-and-mouth disease type O and type A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution.

[0117] When actually testing 5-9 samples, add 1 sample virtually, i.e., set the sample quantity to 6-10 to prepare PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution.

[0118] When actually testing 10, 20, 30, 40, and 50 samples, 1, 2, 3, 4, and 5 samples are virtually added respectively, that is, the sample quantities are set to 11, 22, 33, 44, and 55 to prepare PBST working solution, foot-and-mouth disease type O and type A virus antigen working solution, foot-and-mouth disease type O and type A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution.

[0119] When the actual test sample consists of a whole plate of test samples and less than 10 samples, when y is 1-4 samples, 2+x samples are virtually added; when y is 5-9 samples, 1+x samples are virtually added.

[0120] This method allows researchers to save reagents, reduce experimental errors, quickly complete experimental numerical calculations, save time, and improve experimental efficiency.

[0121] Therefore, the technical solution of this patent is obtained through the above design method.

[0122] If the antigen used in the above scheme is lyophilized antigen, it is generally dissolved in 1 ml of deionized water or distilled water, and then diluted with antigen diluent according to the dilution ratio in the instructions. Antigen dissolution or dilution should be carried out in accordance with the instructions.

[0123] Example 1 (1) The following calculations were performed on 13 samples: Since there are 13 samples, one rabbit anti-coated ELISA plate is needed, and another rabbit anti-coated ELISA plate is needed to test 3 samples. If a rabbit anti-coated ELISA plate tests 10 samples, then the number of rabbit anti-coated ELISA plates that can cover all 10 samples is x. Therefore, the total number of rabbit anti-coated ELISA plates is x = n ÷ 10, where x is 1. The required amount of PBST working solution for each rabbit anti-coated ELISA plate to test 10 samples is calculated as follows: a. The amount of serum used for dilution is Q1 = {[11×8+2×8] wells - 4 wells}×50ul + [11 wells×25ul] = 5275ul.

[0124] b. The amount of material used for washing the plate is Q2 = [11×8+2×8] wells × 300ul × 6 times = 187200ul. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution: Q = Q1 + Q2 = 5275ul + 187200ul = 192475ul = 192.475ml. The units of Q, Q1, and Q2 are all in ul. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. Then Q3 = Q ÷ 25 = 192.475 ml ÷ 25 = 7.699 ml. The corresponding amount of distilled water used is Q4 = 24 × Q3 = 184.776 ml. Q4 is in ul. (2) Calculation of the amount of working solution required for testing 10 samples of foot-and-mouth disease type O and type A virus antigens per rabbit anti-coated ELISA plate: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[11×8+2×8] wells - 4 wells} × 50ul + 4 wells × 100ul = 5400ul; One batch of foot-and-mouth disease type O and type A virus antigen concentrate requires a dilution factor of m, where m is 40, and the foot-and-mouth disease type O and type A virus antigen concentrate is R1, where R1 = R ÷ m = 135ul; (3) Calculation of the amount of working solution required for each rabbit anti-coated ELISA plate to detect 10 samples of foot-and-mouth disease type O and type A enzyme-labeled antibodies: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [11 × 8 + 2 × 8] wells × 50 μL = 5200 μL, where S is in μL. (4) Calculation of the amount of TMB substrate A solution or TMB substrate B solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of TMB substrate A solution or TMB substrate B solution is T = [11 × 8 + 2 × 8] wells × 50 μL ÷ 2 = 2600 μL, where T is in μL. (5) Calculation of the amount of stop solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of stop solution is U = [11×8+2×8]hole×50ul = 5200ul, where U is in ul.

[0125] Therefore, the required quantities for one rabbit anti-coated ELISA plate are: 192.475 ml of PBST working solution, 7.699 ml of PBST concentrate, 184.776 ml of distilled water, 5400 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 135 μL of FMD type O and A virus antigen concentrate, 5200 μL of FMD type O and A enzyme-labeled antibody working solution, 2600 μL of TMB substrate A solution or TMB substrate B solution, and 5200 μL of stop solution.

[0126] The calculation method for detecting 3 samples using another rabbit anti-coated ELISA plate is as follows: Since the three samples fall within the range of y = 1-4 samples, the method for calculating y = 1-4 samples is used: (1) Calculation of the required amount of PBST working solution: a. The amount of serum used for dilution is Q1 = {[(y+2)×8+2×8] wells-4 wells}×50ul+[(y+2) wells×25ul]= (56 wells-4 wells)×50ul+(5 wells×25ul)=2725ul.

[0127] b. The amount of material used for washing the plate is Q2 = [(y+2)×8+2×8] wells×300ul×6 times = 56 wells×300ul×6 times = 100800ul. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution: Q = Q1 + Q2 = 2725ul + 100800ul = 103525ul, where Q, Q1, and Q2 are all in ul. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. Then Q3 = Q ÷ 25 = 4141 ul. The corresponding amount of distilled water used is Q4 = 24 × Q3 = 99384 ul. Q4 is ul (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+2)×8+2×8] wells-4 wells}×50ul+4 wells×100ul=(56 wells-4 wells)×50ul+400ul=3000ul; The dilution factor required for one batch of foot-and-mouth disease type O and type A virus antigen concentrate is m, where m is 40. The concentration of foot-and-mouth disease type O and type A virus antigen concentrate is R1, where R1 = R ÷ m = 3000ul ÷ 40 = 75ul. (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+2)×8+2×8] wells×50ul = 2800ul, where S is inul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+2)×8+2×8]hole×50ul÷2 = 1400ul, where T is in ul. (5) Calculation of the required amount of stop solution: The required amount of stop solution is U = [(y+2)×8+2×8]hole×50ul = 2800ul, where U is in ul.

[0128] Therefore, based on the above, the required quantities for the three samples are: 103,525 μL of PBST working solution, 4,141 μL of PBST concentrate, 3,000 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 75 μL of FMD type O and A virus antigen concentrate, 2,800 μL of FMD type O and A enzyme-labeled antibody working solution, 1,400 μL of TMB substrate A solution or TMB substrate B solution, and 2,800 μL of stop solution.

[0129] One rabbit anti-coated ELISA plate requires 192.475 ml of PBST working solution, 7.699 ml of PBST concentrate, 184.776 ml of distilled water, 5400 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 135 μL of FMD type O and A virus antigen concentrate, 5200 μL of FMD type O and A enzyme-labeled antibody working solution, 2600 μL of TMB substrate A solution or TMB substrate B solution, and 5200 μL of stop solution. Therefore, the required amounts for 13 samples are: 296,000 μL of PBST working solution, 11,840 μL of PBST concentrate, 8,400 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 210 μL of FMD type O and A virus antigen concentrate, 8,000 μL of FMD type O and A enzyme-labeled antibody working solution, 4,000 μL of TMB substrate A solution or TMB substrate B solution, and 8,000 μL of stop solution.

[0130] Example 2 (1) The following calculations were performed on 27 samples: Since there are 27 samples, two rabbit anti-coated ELISA plates are needed. Another rabbit anti-coated ELISA plate will be used to test 7 samples. If two rabbit anti-coated ELISA plates each test 10 samples, and one rabbit anti-coated ELISA plate tests 10 samples, then the number of rabbit anti-coated ELISA plates that completely cover 10 samples is x. The total number of rabbit anti-coated ELISA plates is x = n ÷ 10, where x is 1. The required amount of PBST working solution for each rabbit anti-coated ELISA plate to test 10 samples is calculated as follows: a. The amount of serum used for dilution is Q1 = {[11×8+2×8] wells - 4 wells}×50ul + [11 wells×25ul] = 5275ul.

[0131] b. The amount of material used for washing the plate is Q2 = [11×8+2×8] wells × 300ul × 6 times = 187200ul. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution: Q = Q1 + Q2 = 5275ul + 187200ul = 192475ul = 192.475ml. The units of Q, Q1, and Q2 are all in ul. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. Then Q3 = Q ÷ 25 = 192.475 ml ÷ 25 = 7.699 ml. The corresponding amount of distilled water used is Q4 = 24 × Q3 = 184.776 ml. Q4 is in ul. (2) Calculation of the amount of working solution required for testing 10 samples of foot-and-mouth disease type O and type A virus antigens per rabbit anti-coated ELISA plate: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[11×8+2×8] wells - 4 wells} × 50ul + 4 wells × 100ul = 5400ul; One batch of foot-and-mouth disease type O and type A virus antigen concentrate requires a dilution factor of m, where m is 40, and the foot-and-mouth disease type O and type A virus antigen concentrate is R1, where R1 = R ÷ m = 135ul; (3) Calculation of the amount of working solution required for each rabbit anti-coated ELISA plate to detect 10 samples of foot-and-mouth disease type O and type A enzyme-labeled antibodies: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [11 × 8 + 2 × 8] wells × 50 μL = 5200 μL, where S is in μL. (4) Calculation of the amount of TMB substrate A solution or TMB substrate B solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of TMB substrate A solution or TMB substrate B solution is T = [11 × 8 + 2 × 8] wells × 50 μL ÷ 2 = 2600 μL, where T is in μL. (5) Calculation of the amount of stop solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of stop solution is U = [11×8+2×8]hole×50ul = 5200ul, where U is in ul.

[0132] Therefore, the required reagent volumes for one rabbit anti-coated ELISA plate are as follows: 192.475 ml of PBST working solution, 7.699 ml of PBST concentrate, 184.776 ml of distilled water, 5400 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 135 μL of FMD type O and A virus antigen concentrate, 5200 μL of FMD type O and A enzyme-labeled antibody working solution, 2600 μL of TMB substrate A solution or TMB substrate B solution, and 5200 μL of stop solution. The reagent volumes for the other rabbit anti-coated ELISA plate are the same as above.

[0133] The calculation method for detecting 7 samples using another rabbit anti-coated ELISA plate is as follows: Since the 7 samples fall within the range of 5-9 samples for y, the method for 5-9 samples for y is used for calculation: (1) Calculation of the required amount of PBST working solution: a. The amount of serum used for dilution is Q1 = {[(y+1)×8+2×8] wells-4 wells}×50ul+[(y+1) wells×25ul]= (80 wells-4 wells)×50ul+(8 wells×25ul)=4000ul.

[0134] b. The amount of material used for washing the plate is Q2 = [(y+1)×8+2×8] wells×300ul×6 times = 80 wells×300ul×6 times = 144000ul. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution Q = Q1 + Q2 = 4000ul + 144000ul = 148000ul, where the units of Q, Q1, and Q2 are all in ul. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. Then Q3 = Q ÷ 25 = 5920 ul. The corresponding amount of distilled water used is Q4 = 24 × Q3 = 142080 ul. Q4 is ul (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required amount of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+1)×8+2×8] wells-4 wells}×50ul+4 wells×100ul=(80 wells-4 wells)×50ul+400ul=4200ul; The dilution factor required for one batch of foot-and-mouth disease type O and type A virus antigen concentrate is m, where m is 40. The concentration of foot-and-mouth disease type O and type A virus antigen concentrate is R1, where R1 = R ÷ m = 4200ul ÷ 40 = 105ul. (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+1)×8+2×8] wells×50ul = 4000ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+1)×8+2×8]hole×50ul÷2 = 2000ul, where T is in ul. (5) Calculation of the required amount of stop solution: The required amount of stop solution is U = [(y+1)×8+2×8]hole×50ul = 4000ul, where U is in ul.

[0135] Therefore, based on the above, the required quantities for the 7 samples are: 148,000 μL of PBST working solution, 5,920 μL of PBST concentrate, 142,080 μL of distilled water, 4,200 μL of foot-and-mouth disease type O and type A virus antigen working solution, 105 μL of foot-and-mouth disease type O and type A virus antigen concentrate, 4,000 μL of foot-and-mouth disease type O and type A enzyme-labeled antibody working solution, 2,000 μL of TMB substrate A solution or TMB substrate B solution, and 4,000 μL of stop solution.

[0136] One rabbit anti-coated ELISA plate requires 192.475 ml of PBST working solution, 7.699 ml of PBST concentrate, 184.776 ml of distilled water, 5400 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 135 μL of FMD type O and A virus antigen concentrate, 5200 μL of FMD type O and A enzyme-labeled antibody working solution, 2600 μL of TMB substrate A solution or TMB substrate B solution, and 5200 μL of stop solution.

[0137] For two rabbit anti-coated ELISA plates, the actual volume of PBST working solution is 384.950 ml, PBST concentrate is 15.398 ml, distilled water is 369.552 ml, foot-and-mouth disease type O and type A virus antigen working solution is 10800 μl, foot-and-mouth disease type O and type A virus antigen concentrate is 270 μl, foot-and-mouth disease type O and type A enzyme-labeled antibody working solution is 10400 μl, TMB substrate A solution or TMB substrate B solution is 3200 μl, and stop solution is 10400 μl.

[0138] Therefore, the required quantities for 27 samples are as follows: 532,950 μL of PBST working solution, 21,318 μL of PBST concentrate, 538,630 μL of distilled water, 15,000 μL of foot-and-mouth disease (FMD) type O and A virus antigen working solution, 375 μL of FMD type O and A virus antigen concentrate, 144,000 μL of FMD type O and A enzyme-labeled antibody working solution, 5,200 μL of TMB substrate A solution or TMB substrate B solution, and 14,400 μL of stop solution.

[0139] The above scheme can directly calculate the required amounts of PBST working solution, PBST concentrate, distilled water, foot-and-mouth disease type O and type A virus antigen working solution, foot-and-mouth disease type O and type A virus antigen concentrate, foot-and-mouth disease type O and type A enzyme-labeled antibody working solution, TMB substrate A solution or TMB substrate B solution, and stop solution based on the number of samples tested. Finally, the experimenters should use each reagent according to the instructions. This method can save reagents, reduce experimental errors, avoid over-preparation and waste, quickly complete experimental value calculations, save time, and improve experimental efficiency. The actual quantities can be rounded up to the nearest whole number. This high-dilution foot-and-mouth disease type O and type A antibody liquid-phase blocking ELISA detection method can be applied in teaching experiments, allowing for observation of experimental results across a wide range.

Claims

1. A liquid-phase blocking ELISA method for detecting foot-and-mouth disease type O and type A antibodies at high dilutions, characterized in that... The testing method steps are as follows:

1. Obtaining Rabbit Anti-Coated ELISA Plates: One rabbit anti-coated ELISA plate can detect up to 10 samples. One box contains five rabbit anti-coated ELISA plates. Select the corresponding number of rabbit anti-coated ELISA plates according to the number of samples. II. Setting values: The actual number of samples to be tested is set as n, the number of rabbit anti-coated ELISA plates with 10 samples is set as x, and the number of samples on the rabbit anti-coated ELISA plate that does not have 10 samples is set as y, where x is an integer and y is in the range of 0-9. The relationship between n, x, and y is: n = 10 × x + y.

3. The total number of samples for the final preparation of the volumetric liquid: set the total number of samples for the final preparation of the volumetric liquid as n', set the number of samples to be added virtually as z, n'=n+z, since n=10×x+y, we can deduce: n'=10×x+y+z, (1) when n is 1-9, when y is 1-4 samples, add 2 samples virtually, when y is 5-9 samples, add 1 sample virtually; (2) when n is 10*x, that is, when y is 0 samples, add x samples virtually respectively; (3) when n is composed of whole plate test samples and less than 10 samples, when y is 1-4 samples, add 2+x samples virtually, when y is 5-9 samples, add 1+x samples virtually. IV. Preparation of PBST working solution, foot-and-mouth disease type O and A virus antigen working solution, foot-and-mouth disease type O and A enzyme-labeled antibody working solution, TMB substrate A solution, TMB substrate B solution, and stop solution:

1. When y is 1-4 samples, the calculation for virtually adding 2 samples is as follows: (1) Calculation of the required amount of PBST working solution: a. Serum dilution volume Q1 = {[(y+2)×8+2×8] wells-4 wells}×50ul + [(y+2) wells×25ul], b. The amount of material used for washing the plate is Q2 = [(y+2)×8+2×8]holes×300ul×6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+2)×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+2)×8+2×8] wells×50ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+2)×8+2×8]hole×50ul÷2, where T is in ul; (5) Calculation of the required amount of stop solution: The required amount of stop solution, U, is calculated as follows: U = [(y+2)×8 + 2×8]holes × 50ul, where U is in ul.

2. When y is 5-9 samples, the calculation for virtually adding 1 sample is as follows: (1) Calculation of the required amount of PBST working solution: a. Serum dilution volume Q1 = {[(y+1)×8+2×8] wells-4 wells}×50ul+[(y+1) wells×25ul], b. The amount of material used for washing the plate is Q2 = [(y+1)×8+2×8]holes×300ul×6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the required amount of working solution for foot-and-mouth disease type O and type A virus antigens: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[(y+1)×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the required amount of enzyme-labeled antibody working solution for foot-and-mouth disease type O and type A: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [(y+1)×8+2×8] wells×50ul, where S is in ul. (4) Calculation of the required amount of TMB substrate A solution or TMB substrate B solution: The required amount of TMB substrate A solution or TMB substrate B solution is T = [(y+1)×8+2×8]hole×50ul÷2, where T is in ul. (5) Calculation of the required amount of stop solution: The required amount of stop solution, U, is calculated as follows: U = [(y+1)×8 + 2×8] orifice × 50 μL, where U is in μL.

3. When y is 0 samples, it means the number of rabbit anti-coated ELISA plates filled with 10 samples is x. The number of rabbit anti-coated ELISA plates is x = n ÷ 10. This is calculated by virtually adding x samples. Each rabbit anti-coated ELISA plate is used to test 10 samples. The required PBST working solution, foot-and-mouth disease type O and A virus antigen working solutions, foot-and-mouth disease type O and A enzyme-labeled antibody working solutions, TMB substrate A solution, TMB substrate B solution, and stop solution are calculated based on adding 1 sample. (1) Calculation of the amount of PBST working solution required for each rabbit anti-coated ELISA plate to detect 10 samples: a. Serum dilution volume Q1 = {[11×8+2×8] wells - 4 wells}×50ul + [11 wells×25ul], b. The amount of material used for washing the plate is Q2 = [11×8+2×8] wells × 300ul × 6 times. In the experiment, there are 2 washing steps, and each step is washed 3 times. Therefore, 6 washings are required. The sum of the two results is the actual value of the PBST working solution, Q = Q1 + Q2, where Q, Q1, and Q2 are all in μL. (2) Calculation of the amount of working solution required for testing 10 samples of foot-and-mouth disease type O and type A virus antigens per rabbit anti-coated ELISA plate: The required volume of working solution for foot-and-mouth disease type O and type A virus antigens is R = {[11×8+2×8] wells-4 wells}×50ul+4 wells×100ul; (3) Calculation of the amount of working solution required for each rabbit anti-coated ELISA plate to detect 10 samples of foot-and-mouth disease type O and type A enzyme-labeled antibodies: The required volume of working solution for foot-and-mouth disease type O and type A enzyme-labeled antibodies is S = [11 × 8 + 2 × 8] wells × 50 μL, where S is in μL. (4) Calculation of the amount of TMB substrate A solution or TMB substrate B solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of TMB substrate A solution or TMB substrate B solution is T = [11 × 8 + 2 × 8] wells × 50 μL ÷ 2, where T is in μL. (5) Calculation of the amount of stop solution required for each rabbit anti-coated ELISA plate to detect 10 samples: The required amount of stop solution, U, is calculated as follows: U = [11 × 8 + 2 × 8] orifices × 50 μL, where U is in μL. Multiplying the actual quantities required above by x will give the final required quantities for each.

2. The method for detecting foot-and-mouth disease type O and type A antibodies by liquid phase blocking ELISA at high dilution ratio according to claim 1, characterized in that... In step four, the PBST working solution and foot-and-mouth disease type O and A virus antigen working solutions mentioned in steps 1, 2, or 3 are actually concentrated solutions. One part of PBST concentrate requires 24 parts of distilled water for dilution. The foot-and-mouth disease type O and A virus antigen concentrates are diluted according to the dilution ratio indicated on the bottle containing the foot-and-mouth disease type O and A virus antigen concentrates. The specific method is as follows: A. One part of PBST concentrate requires 24 parts of distilled water for dilution. Let Q ul of PBST working solution correspond to Q3 ul of PBST concentrate. We can get Q3 = Q ÷ 25. The corresponding amount of distilled water is Q4 = 24 × Q3, where Q4 is ul. B. The dilution factor required for one portion of foot-and-mouth disease (FMD) type O and A virus antigen concentrate is m, and the concentration of FMD type O and A virus antigen concentrate is R1, where R1 = R ÷ m. The value of m is indicated on the bottle containing the FMD type O and A virus antigen concentrate.