Hemagglutination test and interferometric titer to determine viral titer

Summary

Certain viruses (such as influenza virus, parainfluenza virus, mumps virus, encephalitis virus, etc.) can selectively agglutinate the erythrocytes of individual animals, a phenomenon known as erythrocyte agglutination, referred to as hemagglutination phenomenon, when these viruses and the corresponding antibody specific binding occurs, the ability to make erythrocytes agglutinate is lost, known as erythrocyte agglutination inhibition. The erythrocyte agglutination and erythrocyte agglutination inhibition tests are simpler, faster, and more economical than the complement binding test, and the hemagglutination inhibition test has the advantages of high sensitivity and high specificity. Source: Virology Testing

Operation method

basic program

Materials and Instruments

Virus specimen to be examined
0.85% sterile saline Alsever's solution Phosphate buffer solution (PBS) Erythrocyte suspension
96-well microhematocrit plate Sterilized syringe Triangular flask Centrifuge tube

Move

1. select the appropriate 96-well microhematocrit plate, V microhematocrit if using chicken or turkey red blood cells or U microhematocrit if using guinea pig or human "O" red blood cells. 2. add 50 µL of physiological saline or PBS to all wells except those in column 1.

2. add 50 µL of saline or PBS to all wells except column 1. 3. add 50 µL of saline or PBS to each well in column 1.

3. Add 100 µL of the virus specimen to be tested to each well in column 1. 4.

4. Pipette 50 µL from each well in column 1 to the corresponding well in column 2 using a multichannel spiker and mix well. Make multiplicative dilutions up to column 11, mix well, and discard 50 µL. 5.

5. The wells in column 12 are used as a red blood cell control. 6.

6. Add 50 µL of 0.5% chicken erythrocyte suspension to each well of the plate using a multichannel spiker, taking care to add sequentially from column 12 to column 1 and changing the pipette tip. 7.

7. Mix and allow to stand at room temperature (22℃~25℃). Allow 30 minutes if using chicken or turkey red blood cells or 60 minutes if using guinea pig or human "O" cells. 8.

8. Determination of Hemagglutination Titers of Specimens

(1) Hematocrit results: indicated by "++++, +++, ++, +, ±, -";

Red blood cells spread evenly on the bottom of the hole is "++++".

erythrocytes spread on the bottom of the well, with a slightly smaller area and irregular edges are "++++".

Erythrocytes forming a ring surrounded by small agglomerates are "+++".

Erythrocytes forming a small cluster with small agglutinated clumps at the edges are "+".

If the red blood cells form a small cluster at the bottom of the hole, with neat and smooth edges and a sense of three-dimensionality, or if the plate is tilted for a few moments, the red blood cells can be seen to be sliding like a tear drop, then it is "-".

(2) Calculation of hemagglutination titer: The result of hemagglutination test is determined as the end point by the inverse of the dilution of + + +, that is, a hemagglutination unit. It indicates that this concentration of virus can cause equal amount of red blood cells to agglutinate, and the inverse of this dilution is the erythrocyte agglutination titer, referred to as hemagglutination titer. Theoretically, the higher dilution of hemagglutination "++" should be "-" and the lower dilution should be "++++" (or "+++"). 4 hemagglutination unit calculation: divide the hemagglutination unit by 4, for example, if a specimen's hemagglutination titer is 1 : 640, then it has four hemagglutination units. For example, if a specimen has a hemagglutination titer of 1 : 640, then its four hemagglutination units would be: 640/4=160, i.e., when the specimen is diluted at 1 : 160, the dilution would contain four hemagglutination units.

Caveat

When two viruses infect the same cell at the same time, the proliferation of one virus inhibits the proliferation of the other, a phenomenon called interference. Sometimes interference can also occur between different types or strains of the same virus. The mechanism of this phenomenon is first considered to be the binding of receptors on the surface of the host cell or changes in the metabolic pathway of the cell after infection by the first virus, thus preventing the adsorption, penetration into the cell or biosynthesis of the other virus. Further studies revealed that the inactivated virus also had an interfering effect, which was difficult to explain by changes in metabolic pathways. It was later discovered that inactivated viruses in cells induced the production of a group of proteins that inhibited viral replication, known as interferons (IFN). The discovery of interferon initiated a series of studies on cellular antiviral effects and virus immunity.The principle of interferon titration is to infect a cell with a virus that does not produce CPE (interferon), incubate the cell for a certain period of time, and then infect the same cell with the corresponding CPE-producing virus and continue to incubate the cell for a certain period of time. If the CPE-producing virus is inhibited from forming CPE, the interfering virus inhibits the proliferation of the CPE-producing virus. Cell culture wells that do not produce CPE are considered positive for the interference titer. The number of positive wells is counted and the titer of the interfering virus is determined by the Karber method.

Common Problems

Alsever (Alsever) Liquid will 20.5 g glucose, 8.0 g sodium citrate ( _Na3C6H5O7 -Na3C6H5O7_2H20 ) Sodium citrate (Na3C6H5O7 - 2H20) 0.55 g citric acid ( _C6H8O7 ) and 4. 2 g NaCl were dissolved in deionized water and fixed to 1,000 ml (pH 7. 1). The 112 C, 20 min autoclaving. 4 Spare.

Phosphate buffer solution (PBS) Dissolve 8.5 g NaCl, 1.1 g _Na2HPO4 and 0.3 g _NaH2PO4-H20 in deionized water and dissolve to 1 000 ml (pH 7.2). Autoclave at 121°C for 15 min and reserve at 4°C.

Erythrocyte suspension Inhale about 3~5 ml of Alsever's solution with a 10 ml or 20 ml sterilized syringe, and then collect 5~10 ml of chicken blood from chicken heart and inject it into a triangular flask containing Alsever's solution quickly, and then shake it well, and the ratio of Alsever's solution to chicken blood was 4:1. Before use, aspirate the erythrocyte suspension into a centrifuge tube, add 5-10 times the volume of saline and wash 3 times, centrifuge the first two times at 2,000 r/min for 5 min, centrifuge the last time at the same speed for 10 min and discard the supernatant, then prepare the erythrocyte suspension with sterile saline at a concentration of 0.5%, and set it aside at 4℃ for not more than 3 days. If guinea pig or human "O" erythrocytes are used, 0.75% erythrocyte suspension should be used.

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