Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Anti-AAV8 Antibody ELISA Kit
for Human
Code No. 5185/5186

Intended Use................................................ 1
Storage......................................................... 1
Materials Provided....................................... 2
Materials Required but not Provided........... 2
Precautions and Recommendations............. 3
Sample Collection and Storage.................... 4
Summary of Procedure................................ 4
Detailed Protocol......................................... 5-8
Calculations..................…............................ 9
Measurement Range..................................... 9
Quality Control............................................ 9
Troubleshooting........................................... 9Reagent Stability.......................................... 9
Assay Characteristics................................... 10
Example of Test Results.............................. 11-12

Intended Use
Anti-AAV8 Antibody ELISA Kit for Human can be used to measure human anti-AAV8 antibodies insera.
To measure human anti-AAV8 antibody, please use two Anti-AAV8 Antibody ELISA Kits for Human(MBL; Code No. 5185 and 5186) in combination. The two kits are sufficient to produce one 96-well
microplate.
Individual users should determine appropriate conditions when using other types of samples.
This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage• Upon receipt store #5185 components at -80°C, and #5186 components at 2-8°C.
• Don’t expose reagents to excessive light.

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Materials Provided
Code No. 5185 (store at -80°C)
Name

Materials

Quantity

AAV8 empty capsid

Antigen for solid phase to the Microplate

20 μL × 1 vial

Anti-AAV8 antibody standard

Human anti-AAV8 antibodies

200 μL × 1 vial

* The components should be stored at -80°C. Avoid repeated freeze-thaw cycles.
Code No. 5186 (store at 2-8°C)
Name

Materials

Quantity

Microplate

Microwell strips

8-well × 12 strips

Coating buffer

Buffer for coating microwells with AAV8 empty
capsid20 mL × 1 bottle

Blocking buffer

Buffer for blocking microwells (Ready-to-use)

100 mL × 1 bottle

Sample diluent

Buffer for diluting samples (Ready-to-use)

30 mL × 1 bottle

Conjugate diluent
HRP conjugated antibody

Buffer for diluting HRP conjugated antibody(Ready-to-use)
HRP conjugated anti-IgG (Human) polyclonal antibody(100x)

14 mL × 1 bottle
150 L × 1 vial

Wash concentrate (20x)

Buffer for washing microwells (20x)50 mL × 1 bottle

Substrate reagent

TMB/H2O2 solution (Ready-to-use)

20 mL × 1 bottle

Stop solution

0.5N H2SO4 solution (0.25 M) (Ready-to-use)

20 mL × 1 bottle

Plate seals

Plate seals

3 pieces

Materials Required but not Provided• Pipettors (single and multichannel): 2-20 µL, 20-200 µL and 200-1,000 µL precision pipettors withdisposable tips.
• Precision repeating pipettor• Plastic tubes (1.5 mL, 15 mL etc.)• (Optional) Microplate washer: Manual washing is possible.
• Reagent reservoirs• Deionized water of the highest quality• Disposable paper towels• Plate reader: capable of measuring absorbance in 96-well plates at dual wavelengths of 450 nm/620
nm. The plate can also be read at a single wavelength of 450 nm, which will give a somewhat higher
reading.
• (Optional) Software package facilitating data generation and analysis
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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Precautions and Recommendations• All reagents need to be brought to room temperature (18-27°C) before use.
• Do not use kit components beyond the indicated kit expiration date.
• Do not mix reagents with different batches and kits.
• Do not mouth pipette or ingest any of the reagents.
• Fresh samples should be used. Aliquot each sample and store below -20°C if necessary. Avoid repeatedfreezing and thawing. Never store the samples at 4°C, as samples might be affected by storage at thistemperature.
• AAV8 empty capsid is easy to adsorb on polystyrene. When handling AAV8 empty capsid, please use
polypropylene tubes and tips.
• The buffers and reagents in this kit may contain preservatives. Care should be taken to avoid directcontact with these reagents. During disposal, flush with plenty of water and/or handle it according to
the regulations of the facility.
• Dispose tetra-methylbenzidine (TMB) containing solutions in compliance with local regulations.
• Avoid contact with the acidic Stop solution and Substrate reagent, which contains hydrogen peroxide.
Protect eyes and skin and handle with care. In case of contact with the Stop solution and the Substrate
reagent, wash skin thoroughly with water and seek medical attention, when necessary.
• Wear gloves and eye protection when handling immunoassay materials and samples of human origin
and these reagents.
• Biological samples may be contaminated with infectious agents. Do not ingest, expose to open
wounds, or breathe aerosols. Wear protective gloves and dispose of biological samples properly.
• AAV8 empty capsid is a kit component subject to the regulations of the Cartagena, Law
Concerning the Conservation and Sustainable Use of Biological Diversity through Regulations
on the Use of Living Modified Organisms. Please handle it carefully according to the regulationsof each user’s facility.
• AAV8 empty capsid is known as non-infectious against human, however, instruments used in thisassay should be treated according to the regulations of each facility after the assay.
(e.g.)
Soak in 2% glutaraldehyde solution (final concentration) for more than one hour.
Soak in 0.1% sodium hypochlorite solution (available chloric: approximately 1,000 ppm.) for morethan one hour.
Autoclave at 121°C for more than 20 minutes.

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Sample Collection and Storage
Serum: Use a serum separation tube and separate the serum according to the manufacturer's manual.
Fresh samples should be used. Aliquots of serum may also be stored at below -20°C for extended
periods of time. Avoid repeated freeze-thaw cycles.
Other biological samples: Not validated by MBL.

Summary of Procedure
To prepare the antigen-coated wells: Add 100 µL of AAV8 Antigen Solution to the wells
To prepare the antigen-UNCOATED wells: Add 100 µL of Coating Buffer to the wells
Incubate over night at 2-8°C.
Wash the wells with Blocking Buffer

Add 200 µL of Blocking buffer to the wells
Incubate for 2 hours at room temperature (18-27°C).
Add 100 µL of standards and samples to the wells
Incubate for 2 hours at room temperature (18-27°C).
Wash the wells

Add 100 µL of Conjugate Solution
Incubate for 1 hour at room temperature (18-27°C).
Wash the wells

Add 100 µL of Substrate reagent
Incubate for 20 minutes at room temperature (18-27°C).
Add 100 µL of Stop Solution

Measure absorbance at 450 nm

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Detailed Protocol
The Anti-AAV8 Antibody ELISA Kit for Human is provided with removable strips of wells so theassay can be carried out on separate occasions. Experimental conditions may vary. To measure humananti-AAV8 antibody, please use two Anti-AAV8 Antibody ELISA Kits for Human (MBL; Code No.
5185 and 5186) in combination. The two kits are sufficient to produce one 96-well microplate. All
samples and the positive control should be assayed in duplicate. Disposable pipette tips and reagent
troughs should be used for all liquid transfers to avoid cross-contamination of reagents or samples.

<Preparation of Working Solutions>
All reagents need to be brought to room temperature prior to assay. Sample diluent should be inverted
and mixed before use.
1. Wash Buffer
Prepare “Wash Buffer” by adding 50 mL of the Wash concentrate (20x) to 950 mL of deionized(distilled) water (ddH2O). Mix well.
2. AAV8 Antigen Solution
The “AAV8 Antigen Solution” should be used immediately after preparation. Prepare “AAV8Antigen Solution” by diluting AAV8 empty capsid with Coating Buffer according to the attached
lot specific document “Preparation of Antigen”. AAV8 empty capsid should be thawed at room
temperature (18-27°C), and mixed gently. Do not vortex strongly.
*AAV8 empty capsid is easy to adsorb on polystyrene. When handle AAV8 empty capsid, pleaseuse polypropylene tubes and tips.
3. Anti-AAV8 Antibody Standard Solution (Master Standard)
Prepare “Anti-AAV8 Antibody Standard Solution” by diluting Anti-AAV8 antibody standard,
25-fold with Sample diluent by gently mixing (e. g., add 40 L of Anti-AAV8 antibody standard
to 960 L of Sample diluent). After thawing Anti-AAV8 antibody standard, dispense it in smallaliquots (e.g. 50 µL) to plastic micro-centrifuge tubes and store below -80°C to avoid non-specificadsorption to glass surface and multiple freeze-thaw cycles.
The prepared Anti-AAV8 Antibody Standard Solution is referred to as the Master Standard.
* Sample diluent should be inverted and mixed before use.

Prepare Standard dilution series as follows:
Use the Master Standard to produce a dilution series (below). Mix each tube thoroughly before thenext transfer. The Master Standard (Std.1) serves as the highest standard.

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures
Volume of Standard
Sample diluent
Dilution ratio
Std. 11,000 µL of Master Standard
0 µL
x25 (100 U/mL)
Std. 2500 µL of Std. 1
500 µLx50 (50 U/mL)
Std. 3500 µL of Std. 2
500 µLx100 (25 U/mL)
Std. 4500 µL of Std. 3
500 µLx200 (12.5 U/mL)
Std. 5500 µL of Std. 4
500 µLx400 (6.25 U/mL)
Std. 6500 µL of Std. 5500 µLx800 (3.125 U/mL)
Note: Do not use a Repeating pipette. Change tips for every dilution. Wet tip with Sample diluent
before dispensing. The Sample diluent serves as the Blank.
4. Conjugate Solution
Prepare “Conjugate Solution” by diluting HRP conjugated antibody, 100-fold with Conjugate
diluent. Prepare only a sufficient amount of the Conjugate Solution immediately before use (e. g., add
100 L of HRP conjugated antibody to 9,900 µL of Conjugate diluent).
5. Other reagents are ready-to-use.

<Preparation of samples>
Samples require the proper dilution ratio if necessary. As an example, serum samples used in MBL
were diluted 50-fold with Sample diluent. If the measured value falls outside the range of thecalibration curve, we recommend verifying the dilution ratio and retesting. (e. g., add 10 L of eachsample to 490 µL of Sample diluent)* Sample diluent should be inverted and mixed before use.

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures<Assay Procedure>
Coating*Please note that both antigen-coated wells and antigen-UNCOATED wells should be prepared forthe assay.
1. To prepare the antigen-coated wells, pipette 100 µL of AAV8 Antigen Solution to the wells ofMicroplate with a multichannel pipet.
2. To prepare the antigen-UNCOATED wells, pipette 100 µL of Coating buffer to the wells ofMicroplate with a multichannel pipet.
3. Incubate the plate overnight at 2~8°C. Please be careful not to evaporate the solution in the wells.
4. Discard the solution in the wells, and wash the plate twice by filling each well with Blocking Buffer(200 µL) using a multi-channel pipette. Tap the plate on a paper towel to remove any remaining
solution.
*Wash as quickly as possible, do NOT let wells dry up.
An example of using Microwell strips;

Blocking
5. Pipette 200 µL of Blocking buffer to the wells with a multichannel pipet.
6. Incubate the plate for 2 hours at room temperature (18-27°C).
7. Discard the solution in the wells, and tap the plate on a paper towel to remove any remaining solution.

1st Reaction
8. Pipette 100 µL of prepared Standards and diluted samples to the wells, both antigen-coated wells andantigen-UNCOATED wells respectively.
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User’s Manual
For Research Use Only, Not for use in diagnostic procedures
9. Incubate the plate for 2 hours at room temperature (18-27°C).

Washing
10. Discard the solution in the wells.
11. Wash the plate 4 times by filling each well with Wash Buffer using a squirt bottle, multi-channel
pipette, manifold dispenser or microplate washer. Tap the plate on a paper towel to remove any
remaining solution.
* When using an auto-washer, appropriate washing times may vary depending on the instrument.

2nd Reaction
12. Pipette 100 µL of Conjugate Solution to the wells with a multichannel pipet.
13. Incubate the plate for 1 hour at room temperature (18-27°C).

Washing
14. Discard the solution in the wells.
15. Wash the plate 4 times as in step 11.

Enzyme Reaction
16. Pipette 100 µL of Substrate reagent to the wells with a multichannel pipet.
17. Incubate the plate for 20 minutes at room temperature (18-27°C).

Reading
18. Pipette 100 µL of Stop solution to the wells with a multichannel pipet.
19. Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengthsof 450/620 nm. The primary wavelength is 450 nm, and the reference wavelength is 620 nm. Readthe microplate at 450 nm if only a single wavelength can be used. Wells must be read within 30
minutes of adding the Stop solution.

Note-1: Each solution and reagent should be used at room temperature (18-27°C).
Note-2: Use new disposable tips, reservoirs and paper towels at each step to avoid contamination.
Note-3: Once the solution has been poured into the reservoir, never return it to the bottle.
Note-4: Complete removal of liquid at each step is essential to good performance.
Note-5: Ensure that the back of the plate is clean and dry, and that no air bubbles are present on thesurface of the liquid in the wells before reading.
Note-6: The incubation time for color development may vary depending on the environment such astemperature. It can be adjusted according to the coloring intensity.

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Calculations
Anti-AAV8 antibody concentration in each sample can be calculated using a 4-parameter logistic
regression equation.
1) Calculate SPEC O.D. for each well
(O.D. value of antigen-coated wells) – (O.D. value of antigen-UNCOATED wells) = SPEC O.D.
2) Plot the SPEC O.D. values average of the duplicate measurements for each standard except Blank
wells (Y) versus the known concentration (X) of each standard, and draw the best fit curve. Most
microtiter plate readers perform automatic calculations of analyte concentration. At the point ofintersection, extend a vertical line to the x-axis and read the corresponding concentration (U/mL).

Measurement Range
The measurement range is 3.1 U/mL to 100 U/mL. Any sample reading higher than the highest standard
should be diluted with Sample diluent in higher dilution and re-assayed. Dilution factors need to betaken into consideration in calculating the concentration of the sample.

Quality Control
To assure the validity of the results, each assay must include both Blank and the highest standard. The
net O.D. values (450 nm) of these controls must fall within the ranges listed below. If O.D. values do notmeet the requirements, the assay is invalid and requires repeat of the assay.
O.D. values of Blank ≤ 0.09
SPEC O.D. values of the highest standard (Std. 1) ≥ 1.0

Troubleshooting
1. All samples and Blank should be assayed in duplicate, using the protocol described in the Detailed
Protocol. Incubation times or temperatures significantly different from those specified may giveerroneous results.
2. Do not dry the plate as it can adversely affect the assay results. Immediately add each solution step by
step.

Reagent Stability#5185 components must be stored at -80°C, and #5186 components must be stored at 2-8°C. Reagentsshould not be used beyond the stated expiration date.

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Assay Characteristics
1. Precision
Intra-assay Precision (Precision within an assay)
Samples of known concentration were tested in octuplicate on one plate to assess intra-assay precision.
• Intra-assay (Within-Run, n=8)

1
2
3
4
5
6
7
8
Max.
Min.
Mean
SD
CV (%)

Sample 1
74.5
74.5
73.8
71.8
74.6
71.7
73.8
68.5
74.6
68.5
72.9
2.1
2.9

Sample 2
37.5
36.4
35.6
36.7
36.1
37.1
37.8
33.8
37.8
33.8
36.4
1.3
3.5

Sample 3
17.9
17.1
18.4
17.4
17.7
17.0
17.6
16.2
18.4
16.2
17.4
0.7
3.8

Inter-assay Precision (Precision between assays)
Three octuplicate-measurements were performed to assess inter-assay precision. Samples of known
concentration were used.
• Inter-assay (Run-to-Run, n=3)

1
2
3
Mean
SD
CV (%)

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Sample 1
72.9
69.0
59.9
67.2
6.7
9.9

Sample 2
36.4
32.4
28.2
32.3
4.1
12.7

10

Sample 3
17.4
14.3
11.8
14.5
2.8
19.5

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic procedures

Examples of Test Results

Fig.1 Anti-AAV8 antibodies calibration curve

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Anti-AAV8 Antibody ELISA Kit for Human
User’s Manual
For Research Use Only, Not for use in diagnostic proceduresa)b)

Fig.2 The Anti-AAV8 antibody concentration serum derived from healthy adult volunteers (n=14). Thesesamples were diluted 50-fold with Sample diluent.
a) A figure showing distribution of measured values of 14 samples.
b) An enlarged figure of the plot of samples less than 20 U/mL of anti-AAV8 antibodies. (9 out of
14 samples)

MANUFACTURED BY

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD.
URL: https://ruo.mbl.co.jp
E-mail: support@mbl.co.jp
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Anti-AAV8 Antibody ELISA Kit for Human
Preparation of Antigen
For Research Use Only, Not for use in diagnostic procedures

Preparation of Antigen
Lot No. XXXXXX
To prepare “AAV8 Antigen Solution” by diluting AAV8 empty capsid with Coating Buffer.

1. Thaw AAV8 empty capsid at room temperature (18-27°C).
2. Dilute the thawed AAV8 empty capsid X-fold with Coating Buffer.
(e. g., Add the thawed Y L of AAV8 empty capsid to Z µL of Coating Buffer)* AAV8 empty capsid should be mixed with Coating Buffer immediately. The “AAV8 Antigen
Solution” should be used immediately after preparation.
* AAV8 empty capsid is easy to adsorb on polystyrene. When handling AAV8 empty capsid, pleaseuse polypropylene tubes and tips.

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