Virus Quantification & NeutralizationVirus Quantification & Neutralization

IntroductionIntroduction

• The hemagglutinin (HA) protein The hemagglutinin (HA) protein – agglutinates erythrocytes (hence, the derivation of its name.)agglutinates erythrocytes (hence, the derivation of its name.)

• The hemagglutination inhibition (HAI)The hemagglutination inhibition (HAI)– The method for identifying influenza field isolates The method for identifying influenza field isolates – Specific attachment of antibody to the antigenic sites on the HA moleSpecific attachment of antibody to the antigenic sites on the HA mole

cule interferes with the binding between the viral HA and receptors on cule interferes with the binding between the viral HA and receptors on the erythrocytes. This effect inhibits hemagglutination and is the basis the erythrocytes. This effect inhibits hemagglutination and is the basis for the test. for the test.

– Originally described by Hirst (1942) and later modified by Salk (1944), Originally described by Hirst (1942) and later modified by Salk (1944), is currently performed in microtiter plates. is currently performed in microtiter plates.

– In general, a standardized quantity of HA antigen is mixed with seriallIn general, a standardized quantity of HA antigen is mixed with serially diluted antisera, and red blood cells are added to determine specific y diluted antisera, and red blood cells are added to determine specific binding of antibody to the HA molecule.binding of antibody to the HA molecule.

Virus QuantificationVirus Quantification

• The most important property of a virus is its infectivity or abilThe most important property of a virus is its infectivity or ability to invade a cell and parasitize that cell to replicate itself.ity to invade a cell and parasitize that cell to replicate itself.

• There are 2 basic types of infectivity assays,There are 2 basic types of infectivity assays,1)1) Quantal AssaysQuantal Assays: not count the number of infectious virus particles p: not count the number of infectious virus particles p

resent in the inoculum but instead obtain a value for the virus titer.resent in the inoculum but instead obtain a value for the virus titer. Ex) TCID50 (50% Tissue Culture Infective Dose), LD50 (50% Lethal Dose), EID50 Ex) TCID50 (50% Tissue Culture Infective Dose), LD50 (50% Lethal Dose), EID50

(50% Egg Infective Dose)(50% Egg Infective Dose)

2)2) Quantitative AssaysQuantitative Assays: The number of infectious virus particles prese: The number of infectious virus particles present in the original suspension can be quantified.nt in the original suspension can be quantified. Ex) Plaque assays We will see this assay result by slide feature of microscope.⇒Ex) Plaque assays We will see this assay result by slide feature of microscope.⇒

3)3) Other propertiesOther properties Ex) Hemagglutination assay, HemadsorptionEx) Hemagglutination assay, Hemadsorption

Hemagglutination Assay (HA)Hemagglutination Assay (HA)

• Hegagglutination is Hegagglutination is the aggregation of RBCs in the presencthe aggregation of RBCs in the presence of hemagglutinating virus particles.e of hemagglutinating virus particles. This phenomenon is d This phenomenon is due to the presence of outer surface proteins on the hemagglue to the presence of outer surface proteins on the hemagglutinating virus that recognize and attach to cellular surface rutinating virus that recognize and attach to cellular surface receptors expressed by RBCs.eceptors expressed by RBCs.1)1) Most common indirect methods for quantifying virus particles in suspMost common indirect methods for quantifying virus particles in susp

ension.ension.

2)2) Not a very sensitive assay since a large minimum number of virus paNot a very sensitive assay since a large minimum number of virus particles are necessary to obtain macroscopic agglutination.rticles are necessary to obtain macroscopic agglutination.

3)3) It is convenient because of its rapidity and ease of titration of a large It is convenient because of its rapidity and ease of titration of a large number of samples.number of samples.

• Some viruses will only agglutinate RBCs of certain animal sSome viruses will only agglutinate RBCs of certain animal species pecies

Structure of the influenza A virus particleStructure of the influenza A virus particle

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

Assays to Measure Virus NeutralizationAssays to Measure Virus Neutralization

• Techniques for Techniques for detection of viral antibodydetection of viral antibody1)1) Virus neutralizationVirus neutralization2)2) Hemagglutination-inhibitionHemagglutination-inhibition3)3) ImunofluorescenceImunofluorescence4)4) Enzyme-linked immunosorbent assay (ELISA)Enzyme-linked immunosorbent assay (ELISA)5)5) Complement fixationComplement fixation6)6) Immunoelectron microscopy Immunoelectron microscopy 

• *There are numerous serological techniques used to diagno*There are numerous serological techniques used to diagnose viral infections. These techniques may be used to,se viral infections. These techniques may be used to,

1)1) Accurately identify an unknown virus isolate utilizing a known referAccurately identify an unknown virus isolate utilizing a known reference antibodyence antibody

2)2) May utilize a panel of known viral antigens in order to test for viral 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.antibody in a patient suspected of having a disease of viral etiology.

3)3) Determine the effectiveness of viral vaccineDetermine the effectiveness of viral vaccine4)4) Monitor the incidence of influenza virus throughout the world (WHMonitor the incidence of influenza virus throughout the world (WH

O)O)

Hemagglutination-inhibition assay (HI)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.

1.Viral titration

- Plaque assay

- Hemagglutination assay

2.Neutralizing viral Ag

- Hemagglutination-inhibition assay

①Round bottom microtiter plate: 조별 2 개 (8 인 1 조 )/ 3 개 (9 인 1조 )

② Influenza virus: 5 ml (8 인 1 조 ) / 6 ml (9 인 1 조 )

③Micropipette: 조별 200 ㎕ /20 ㎕ 각각 2 개④Yellow tip: 1 인 40 개⑤PBS: 조별 12 ml

⑥0.5% type O RBC: 조별 16 ml

⑦Serum: 조별 1.2 ml

MethodsMethods

1. Hemagglutination assay 1) microtiter plate #1 well 에 PBS 90 ㎕ , #2~#10 PBS 50 ㎕2) Influenza virus 10 ㎕를 #1 에 넣어 혼합 (100 ㎕ )

3) 2X stepwise dilution1)1 번 well 의 50 ㎕를 2 번 well 의 PBS 50 ㎕ 에 혼합

▶ 1 번 well 에 50 ㎕남고 , 2 번 well 에 100 ㎕

2)2 번 well 의 50 ㎕를 3 번 well 의 PBS 50 ㎕ 에 혼합 ▶ 2 번 well 에 50 ㎕남고 , 3 번 well 에 100 ㎕

3)Continue on

4) #9 well 에서 남은 희석액 50 ㎕는 락스통에 discard

5) RBC 부유액 50 ㎕씩을 분주6)실온에서 30 분 이상 정치 / 관찰

Positive control, negative control 은 각각 몇 번 well 인가 ?

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 assay1) microtiter plate #2~#10 well 까지 PBS 50 ㎕ , #11 well 에 100 ㎕2) #1, #2 well 에 serum 50 ㎕씩3) 2X stepwise dilution : #2~#9 well

#9 well 에서 남은 50 ㎕는 락스통에 discard

4) Virus solution 50 ㎕씩 (#1~#10)

5) 0.5% RBC 100 ㎕씩 (#1~#10)

6) 잘 섞은 후 실온에서 정치 / 관찰

Positive control, negative control 은 각각 몇 번 well 인가 ?