Zika MAC ELISA. For Use Under an Emergency Use Authorization Only Instructions for Use. IgMAntibody Capture Enzyme Linked ImmunoSorbent Assay

Transcription

1 Zika MAC ELISA For Use Under an Emergency Use Authorization Only Instructions for Use IgMAntibody Capture Enzyme Linked ImmunoSorbent Assay Detects presence of IgM antibody to Zika virus in serum and CSF specimens 3 days to perform o Day 1: Coat plates with anti human IgM antibody o Day 2: Addition of patient sample and antigen o Day 3: Addition of conjugate and substrate; read. 1

2 NOTICE: Modifications of these assays (i.e., use of platforms or chemistries other than those described) is not permitted. These assays should not be further distributed without the explicit consent of the CDC.

3 ACCEPTABLE SPECIMENS: Acute and convalescent human serum NOTE: Serum should be collected in a serum separator tube. Tube should be centrifuged and serum decanted prior to shipment to avoid hemolysis. Cerebrospinal fluid (CSF) specimens CSF may only be tested when submitted alongside a patientmatched serum specimen

4 MATERIALS PROVIDED B CDC: Normal Vero E6 Antigen (CDC catalog #AV0001); Lyophilized normal antigen Zika Vero E6 Tissue Culture Antigen (CDC catalog #AV0002 or AV0003; these are antigens produced from different Zika strains; only one is required for the assay); Lyophilized Zika antigen (inactivated) prepared for use in Zika IgM ELISA. Flavivirus IgM positive control (CDC catalog #AV0004): Chimeric monoclonal antibody specific for Flavivirus; lyophilized. NOTE: These materials will be provided by CDC, Ft. Collins, CO. To request these reagents, please Dr. Barbara Johnson at bfj9@cdc.gov

5 Dilutions: We recommend you use the dilutions given below when you first run the test. However, users should optimize the dilutions in their individual laboratories. See additional information in Assay Standardization on page 13 of protocol. Goat anti-human IgM: diluted 1:2000 in coating buffer (titration may be required depending on the vendor) Flavivirus IgM positive control: Flavivirus IgM positive control diluted 1:3000 in wash buffer Patient serum: diluted 1:400 in wash buffer (no titration required) Negative control: Normal human sera diluted to 1:400 (no titration required) Zika Vero E6 antigen: diluted 1:160 in wash buffer Normal Vero E6 antigen: diluted 1:160 in wash buffer Detecting antibody conjugate (use vendor recommended dilution; titration may be required)

6 PREPARING THE PLATE: Determine the number of ELISA plates needed. Using a finetipped permanent marker, number and label the 96-well plates. Identify the location of each clinical specimen (S1-S8) by using a corresponding template. To keep timing of reagent addition consistent, process plates in the order that they are numbered during all steps of the procedure.

7 EACH SERUM SPECIMEN IS TESTED IN TRIPLICATE ON BOTH VIRAL (blue) AND NORMAL VERO E6 (yellow) ANTIGENS. EIGHT (8) TEST SPECIMENS CAN BE ANALZED PER PLATE. Plates should be kept in an enclosed, humidified environment during all incubation times with the exception of the coating step. A large Ziploc-type bag containing a moist paper towel works well for this purpose.

8 COATING THE PLATES: Dilute goat anti-human IgM 1:2000 in coating buffer (or titrated dilution). Coat the inner 60 wells of the 96 well plate with 75 μl per well of diluted goat anti-human IgM. (Leave outer rows/columns empty). Incubate at 2-8 C overnight. Note: Plates should remain at 2-8 C until needed for testing, up to one week. Antibody structure = Anti human IgM antibody coated testing wells

9 BLOCKING THE PLATES: After overnight incubation, dump out the coating antibody. Blot plates on paper towels or other absorbent material. Block plates with 200 μl blocking buffer per well. Incubate at room temperature for 30 minutes. Blocking buffer

10 ADDITION OF SAMPLE/CONTROLS: Wash wells 5X with wash buffer by using an automatic plate washer. Dilute patient s serum 1:400 in wash buffer. Add 50 μl per well of the diluted patient's serum (S) to a block of 6 wells. Add 50 μl Flavivirus IgM positive control (into the Ref labeled wells) diluted in wash buffer 1:3000 or according to a previously determined titration. Dilute negative human serum control (N) 1:400 in wash buffer. Add 50 μl diluted (1:400 in wash buffer) negative human serum control (N) to the block of 6 wells labeled N. Incubate plates for 1 hour at 37 C in a humidified chamber. IgM antibody structure = 5 x All IgM antibody in patient specimen is captured in the well by the anti human IgM antibody, not just Zikaspecific IgM

11 ADDITION OF ANTIGEN: Dilute Zika Vero E6 antigen in wash buffer 1:160 or according to a previously performed titration. Dilute Normal Vero E6 antigen in wash buffer to the same concentration as the Zika Vero E6 antigen. Wash wells 5X with wash buffer by using an automatic plate washer. Add 50 μl per well of diluted Zika Vero E6 antigen to the left three wells of each serum block Add 50 μl per well of diluted Normal Vero E6 antigen to the right three wells of each block. Incubate plates overnight at 2-8 C in a humidified chamber. Inactivated Zika virus (antigen) binds to anti Zika IgM antibodies

12 ADDITION OF CONJUGATE: Wash the plate with a series of 5 washes. Dilute horseradish peroxidase-conjugated monoclonal antibody in blocking buffer according to a previously performed titration. Add 50 μl per well of diluted horseradish peroxidase-conjugated monoclonal antibody Incubate plates for 1 hour at 37 C in a humidified chamber. Bound antigen is detected by peroxidase labeled IgG antiflavivirus antibody

13 ADDITION OF SUBSTRATE: Wash the plate using a series of 10 washes. With the plate at room temperature (20-25 C), add 75 μl per well of TMB substrate to all well Immediately cover plates to block out light. Incubate at room temperature for 10 minutes. A blue color will develop in antibody-positive wells. TMB substrate is then added to induce a color change with the labeled IgG antibody

14 ADDITION OF STOP SOLUTION: Add 50 μl per well of stop solution to all wells, including the outer rows of wells on the plate. NOTE: The plate reader should be set to zero itself on some of these wells The wells that were blue will now change to a yellow color. Allow plates to sit at room temperature for 1 minute. Read plates in microtiter plate reader by using a 450 nm filter. Stop solution is then added to induce a color change to yellow

15 TEST VALIDIT DETERMINATION Before the results can be calculated for each clinical specimen, the test must be determined to be valid. For a test to be valid, the mean OD of the negative control must be <0.2 AND the following ratio must be greater than or equal to 2.0. This is the P/N of the positive control. Test validity must be determined for each plate. Results for clinical specimens may only be determined if the test is valid. If the test is not valid, then that plate must be repeated. If the validity tests for the plate still fails after a repeat, then one or more of the reagent or test parameters was likely in error, and troubleshooting should be performed.

16 DETERMINATION OF SPECIMEN P/N : To determine whether the clinical specimens (S1-S8) contain IgM to the Zika virus (which would indicate recent infections with that virus) the following must be calculated: Mean OD of the test specimen reacted with Zika Vero E6 antigen (P) Mean OD of the normal human serum reacted with Zika Vero E6 antigen (N) This is the P/N of the test specimen. All specimens for which Specimen P/N is < 2, report as negative. No further analysis is required.

17 Interpreting Test Results: SPECIMEN BACKGROUND EVALUATION For each specimen with a Specimen P/N 2, determine whether non-specific background is being generated. Perform the following calculation: Mean OD of specimen reacted on Zika antigen Mean OD of specimen reacted on Normal antigen If the product of this calculation is <2, non-specific background is being generated, and the result MUST be reported as inconclusive. Inconclusive specimens should be retested. If repeat testing also yields inconclusive results, forward specimen for further analysis and/or request collection of additional serum for analysis. If requirement is met, proceed with specimen result interpretation.

18 ANALSIS OF POSITIVE AND EQUIVOCAL RESULTS: All test specimen P/N values greater than or equal to 3.0 should be reported as presumptive IgM-positive (see table below), as long as they meet the requirements listed above. P/N values 2.0 P/N<3.0 should be considered equivocal. Further tests should be performed to determine the status of these specimens (see Table 2 below).

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20 Example plate: good positive control, good negative control, one positive sample

21 Example plate results sheet: good positive control, good negative control, one positive sample

22 Example plate results sheet: good positive control, good negative control, one sample with high background (specimen 4 = inconclusive)

23 Titration of conjugate: 1. Using all the other reagents at the suggested working dilutions, titrate the conjugate. Begin a test with 4 columns of 6 wells as follows: positive control/viral antigen; positive control normal antigen; negative control/viral antigen; negative control/normal antigen. 2. Starting at 1:500 (suggested), make a series of 2-fold dilutions down the plate (1:500, 1000, 2000, 4000, 8000, 16000) 3. Start with 100 ul of 1:500 conjugate in the first well of the series. Add 50 ul/well wash buffer to all the other wells. 4. Transfer 50 ul out of the first wells in the series into the 2 nd wells, mix, transfer to the successive wells and repeat the operation. Discard the last 50 ul. 5. Proceed with the remainder of the test as per the protocol.

24 Titration results: 1. Identify the dilution which gives an OD of the positive control on the viral antigen to as close to 1.0 as possible. 2. Make sure that the OD of the negative control on the viral antigen at this dilution is < Also make sure that the OD of the controls on the normal antigen is < This is your new working dilution for the conjugate. 5. If an OD of 1.0 lies somewhere in between two dilutions, choose a dilution part way between. 6. Test out the new working dilution in the usual MAC-ELISA format to make sure it works well. Adjust if necessary. The OD of the positive control doesn t need to be exactly If background is an issue it may be necessary to re-titrate some of the other reagents.

25 Notes: In the event that an early acute CSF or serum is negative by this test, a convalescent serum specimen must be requested and tested before that patient is reported as negative for serological evidence of recent viral infection. Without testing of a convalescent specimen, a negative result may reflect testing of an acute-phase specimen obtained before antibody has risen to detectable levels. All positive results should be reported to CDC via ArboNET.

26 Notes: The MAC-ELISA should be standardized and verified prior to use in the laboratory. It is recommended that the mean optical density of the positive control serum reacted with the Zika Vero E6 antigen be set to approx The negative control serum reacted with the Zika Vero E6 antigen should be <0.2 (usually but can vary). The standardization of reagents is normally achieved via titration, always comparing the optical densities of the reagents when reacted on viral and Normal Vero E6 antigen. Standardization and re-standardization may be confirmed by testing verification panels.

27 Notes continued CSF should be used undiluted or 1:5 at the most. A known negative CSF must be used for calculating the P/N. CSF s should be tested singly on viral and normal antigen.

28 Further information: For information regarding Zika testing algorithm, please refer to CDC guidance for state and local public health laboratories: For specimen referral instructions, please refer to: Questions or comments about this procedure may be directed to the Laboratory Response Network (LRN) helpdesk: