Virus Quantification & Neutralization

Introduction The hemagglutinin (HA) protein agglutinates erythrocytes (hence, the derivation of its name.) The hemagglutination inhibition (HAI) The method for identifying influenza field isolates Specific attachment of antibody to the antigenic sites on the HA molecule interferes with the binding between the viral HA and receptors on the erythrocytes. This effect inhibits hemagglutination and is the basis for the test. Originally described by Hirst (1942) and later modified by Salk (1944), is currently performed in microtiter plates. In general, a standardized quantity of HA antigen is mixed with serially diluted antisera, and red blood cells are added to determine specific binding of antibody to the HA molecule.

Virus Quantification The most important property of a virus is its infectivity or ability to invade a cell and parasitize that cell to replicate itself. There are 2 basic types of infectivity assays, Quantal Assays: not count the number of infectious virus particles present in the inoculum but instead obtain a value for the virus titer. Ex) TCID50 (50% Tissue Culture Infective Dose), LD50 (50% Lethal Dose), EID50 (50% Egg Infective Dose) Quantitative Assays: The number of infectious virus particles present in the original suspension can be quantified. Ex) Plaque assays ⇒ We will see this assay result by slide feature of microscope. Other properties Ex) Hemagglutination assay, Hemadsorption

Hemagglutination Assay (HA) Hegagglutination is the aggregation of RBCs in the presence of hemagglutinating virus particles. This phenomenon is due to the presence of outer surface proteins on the hemagglutinating virus that recognize and attach to cellular surface receptors expressed by RBCs. Most common indirect methods for quantifying virus particles in suspension. Not a very sensitive assay since a large minimum number of virus particles are necessary to obtain macroscopic agglutination. It is convenient because of its rapidity and ease of titration of a large number of samples. Some viruses will only agglutinate RBCs of certain animal species

Structure of the influenza A virus particle

Graphic representation of the hemagglutination phenomenon . For the phenomenon of HA to occur, a virion must attach simultaneously to two RBCs, creating a cross-bridge.

Assays to Measure Virus Neutralization Techniques for detection of viral antibody Virus neutralization Hemagglutination-inhibition Imunofluorescence Enzyme-linked immunosorbent assay (ELISA) Complement fixation Immunoelectron microscopy  *There are numerous serological techniques used to diagnose viral infections. These techniques may be used to, Accurately identify an unknown virus isolate utilizing a known reference antibody May utilize a panel of known viral antigens in order to test for viral antibody in a patient suspected of having a disease of viral etiology. Determine the effectiveness of viral vaccine Monitor the incidence of influenza virus throughout the world (WHO)

Hemagglutination-inhibition assay (HI) The HI test may be used for virus identification, to determine the antigenic relationship of different virus isolates, and for the identification and quantification of viral antibody to virus. This assay is used to monitor the antigenic variation of influenza A viruses. Genera or species within the following families of viruses are capable of hemagglutination: adenoviridae, bunyaviridae, coronaviridae, flaviviridae, orthomyxoviridae, paramyxoviridae, parvoviridae, picornaviridae, poxviridae, reoviridae, rhabdoviridae, and togaviridae. Based on the binding of antibody to the viral hemagglutinin with a resultant inhibition of viral hemagglutination. - Widely used for virus identification as well as for identification and quantification of viral antibody to virus.

- Hemagglutination-inhibition assay Viral titration - Plaque assay - Hemagglutination assay Neutralizing viral Ag - Hemagglutination-inhibition assay Round bottom microtiter plate: 조별 2개 (8인 1조)/ 3개 (9인 1조) Influenza virus: 5 ml (8인 1조) / 6 ml (9인 1조) Micropipette: 조별 200㎕/20㎕ 각각 2개 Yellow tip: 1인 40개 PBS: 조별 12 ml 0.5% type O RBC: 조별 16 ml Serum: 조별 1.2 ml

Methods 1. Hemagglutination assay microtiter plate #1 well에 PBS 90㎕, #2~#10 PBS 50 ㎕ Influenza virus 10㎕를 #1에 넣어 혼합 (100 ㎕) 2X stepwise dilution 1번 well의 50㎕를 2번 well의 PBS 50 ㎕ 에 혼합 ▶ 1번 well에 50㎕남고, 2번 well에 100 ㎕ 2번 well의 50㎕를 3번 well의 PBS 50 ㎕ 에 혼합 ▶ 2번 well에 50㎕남고, 3번 well에 100 ㎕ Continue on #9 well에서 남은 희석액 50㎕는 락스통에 discard RBC 부유액 50㎕씩을 분주 실온에서 30분 이상 정치/관찰 Positive control, negative control은 각각 몇 번 well인가?

Phosphate buffered saline ("PBS") Egg allantoic fluid containing influenza virus of unknown titre and antigenic type 0.5% sheep red blood cells in PBS ("RBCs") 96 well plastic microtitre plate:             

#1, PBS 90㎕ #2, PBS 50㎕ #3, PBS 50㎕ #4, PBS 50㎕ #1, PBS 90㎕+virus10㎕ 50㎕

2. Hemagglutination-inhibition assay microtiter plate #2~#10 well까지 PBS 50㎕, #11 well에 100㎕ #1, #2 well에 serum 50㎕씩 2X stepwise dilution : #2~#9 well #9 well에서 남은 50㎕는 락스통에 discard Virus solution 50㎕씩 (#1~#10) 0.5% RBC 100㎕씩 (#1~#10) 잘 섞은 후 실온에서 정치/관찰 Positive control, negative control은 각각 몇 번 well인가?