TITLE: The EV71 neutralizing antibody test
AUTHORS: Jian-Te Lee, Ting-Yu Yen, Chun-Yi Lu, Ding-Ping Liu, Yi-Chuan Huang, Luan-Yin Chang, Li-Min Huang, Tzou-Yien Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the process for the neutralizing antibody test of EV71.</div></div>

[STEPS]
?. Inactivate serum (300 μl) at 56℃ for 30 min  ABC1  Sample volumn (μl)
    1x PBS (μl)
    Dilute factor
  2  Ex. 75
    225
    1:4
ABC1  Sample volumn (μl)
    1x PBS (μl)
    Dilute factor
  2  Ex. 75
    225
    1:4
?. Make 2 fold serial dilution of serum from 1:4 to 1:256 with 2% FBS DMEM
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							  ABCDEFGHIJKLM1  sample
    1-1
    1-2
    2-1
    2-2
    3-1
    3-2
    4-1
    4-2
    5-1
    5-2
    6-1
    6-2
  2  A 1:4
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  3  B 1:8
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  4  C 1:16
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  5  D 1:32
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  6  E 1:64
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  7  F 1:128
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  8  G 1:256
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  9  H control
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  a. Add 50 μl 2% FBS DMEM in well B to H
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							b. ● means add 50μl serum sample
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							c. After dilution, every well has 50μl sample
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							d. ex. sample1-1 well H: serum control; sample 1-2 well H: cell control; 1-1 and 1-2 are duplicate
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ABCDEFGHIJKLM1  sample
    1-1
    1-2
    2-1
    2-2
    3-1
    3-2
    4-1
    4-2
    5-1
    5-2
    6-1
    6-2
  2  A 1:4
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  3  B 1:8
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  4  C 1:16
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  5  D 1:32
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  6  E 1:64
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  7  F 1:128
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  8  G 1:256
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  9  H control
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?. Prepare control wells including virus control, serum control and cell control.
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							Virus control well：back titration from 100, 10, 1 to 1/10 100TCID50
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							Serum control：add serum and cell only, no virus
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							Cell control：add cell only, no serum and no virus
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?. Add 50 μl of adjusted virus suspension (100TCID50EV71) onto the well A to well G of microtiterplate, shake reaction well slightly
?. Incubate above reaction wells at 37℃for 2 hours in 5% CO2 incubator
?. Prepare RD cells suspension and adjust cell concentration to 1×105/ml
?. Add 100μl of adjusted RD cell into each reaction well after 2 hours in step5
?. Incubate wells at 37℃in 5% CO2 incubator for 5-6 days until virus control well reach 100TCID50
?. Observe CPE in each well under reverse microscope after 5-6 days incubation
?. Check CPE in control wells
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							Virus control well：100% CPE in 100TCID50 and 10TCID50; 50% CPE in 1TCID50, 50
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							Serum control well：no CPE
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							Cell control well：no CPE
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?. The NT titer was calculated as the reciprocal value of the last dilution of the antiserum that completely inhibited the CPE after incubation.
?. Seropositivity was defined as a reciprocal of the serotiter≧ 8.