Human Apolipoprotein J (Clusterin)
ELISAPRO kit
Kit for 2 plates
Product code: 3713-1HP-2

TABLE OF CONTENTS

PAGE

Kit description.......................................................................................... 4
Reagents provided.................................................................................... 4
Storage and expiry date............................................................................ 5
Materials required but not supplied......................................................... 5
Safety issues.............................................................................................. 5
Procedural notes....................................................................................... 5
Preparations............................................................................................. 6Assay procedure........................................................................................ 8
Performance of the assay........................................................................... 9
Limitations of the assay.......................................................................... 10
Dilution guidelines................................................................................ 10

KIT DESCRIPTION
Mabtech’s enzyme-linked immunosorbent assay, ELISAPRO, is a complete kit for the quantification of human apolipoprotein J (apoJ) in biological fluids such as serum, plasma and cell culture
supernatants. The assay utilizes ELISA strip plates pre-coated with a capture monoclonal antibody(J29), to which samples are added. Captured apoJ is detected by adding a biotinylated monoclonal
antibody (J84-biotin) followed by streptavidin-horseradish peroxidase (SA-HRP). Addition of theenzyme substrate TMB will result in a colored substrate product with an intensity that is directly
proportional to the concentration of apoJ in the sample. The concentration of the apoJ in the sample is determined by comparison to a serial dilution of purified apoJ standard analyzed in parallel.

Reagents Provided
Component

Quantity

Description/comments

Precoated 96-well strip plate(12 strips x 8 wells)

2 plates

Plate coated with anti-human apoJ (J29).
Supplied in foil bag with desiccant.

apoJ standard

1 vialLyophilized purified apoJ.
See “Preparations” for reconstitution and dilution.

Detection antibody

1 vial (50 μl)

Biotinylated monoclonal antibody,anti-human apoJ (J84-biotin) (0.5 mg/ml).
Dilute before use.

SA-HRP

1 vial (30 μl)

Streptavidin-horseradish peroxidase conjugate.
Dilute before use.

Standard reconstitution buffer
A8 (ready-to-use)

1 vial (1ml)

For reconstitution of lyophilized apoJstandard.

Wash bufferconcentrate (20x)

1 bottle (120 ml)

For all wash steps.
Dilute before use.

Apo ELISA bufferconcentrate 5x

1 bottle (60 ml)

Protein-containing buffer for: 1) dilution of all samples;2) serial dilution of standard; 3) dilution of detection
antibody. Dilute before use.

SA-HRP diluent(ready-to-use)

1 bottle (25 ml)

For dilution of SA-HRP.

TMB substrate(ready-to-use)

1 bottle (25 ml)

3,3’,5,5’ tetramethylbenzidine (TMB) enzyme substratesolution containing hydrogen peroxide.

Stop solution(ready-to-use)

1 bottle (25 ml)

0.18 M H2SO4

Adhesive plate covers

6To cover plates during incubations.

To ensure total recovery of stated quantity, bottles and vials are filled with larger volume than indicated.

STORAGE AND EXPIRY DATE
Shipped at ambient temperature. On arrival all components of the kit, with the exception of
the lyophilized standard, should be stored at 2-8°C. Please note that the lyophilized standard should be kept at -20°C. After reconstitution of the lyophilized standard, it should bealiquoted and kept at -20°C. The expiry date for the unopened kit is indicated on the box.
We recommend to use opened kit components within one month.
MATERIALS REQUIRED BUT NOT SUPPLIED- Microplate reader capable of reading at 450 nm; preferably able of subtracting a reference
wavelength between 570-650 nm.
- ELISA plate washer; automatic (adaptable for ELISA strip plates) or manual (e.g. multipipette or squirt bottle).
- Precision pipettes and tips.
- Beakers, flasks and graduated cylinders necessary for reagent preparations.
- Tubes for standard and sample dilutions.
- Distilled or deionized water.
SAFETY ISSUES- The stop solution, 0.18 M H2SO4(<1%), is irritating to eyes and skin and should be handled with care.
- The standard should be handled with care due to unknown effects of exposure.
- Buffers and reagents in solution contain 0.002% of the preservative Kathon CG, a potential contact allergen which may cause sensitization by skin contact.
- Human and animal samples should be treated as potentially hazardous biological material.
- All material and samples should be disposed of in accordance with local regulations.
PROCEDURAL NOTES- Do not combine components from different kit lots or components from other suppliers.
- Dilutions of standard and samples can be prepared in plastic or glass tubes.
- Total sample or standard volume added per well should not exceed 100 μl.
- The use of duplicates for each dilution of the standard, samples and blank is recommended.
- Prior to adding new reagents to the wells, ensure that there is no residual wash buffer
remaining in the wells. The wells can be emptied by turning the plate upside down followed
by gently tapping the plate against clean tissue paper. Please be careful to avoid that the strips
fall out of the frame e.g. grip the plate by the middle.
- Serum/plasma components present in the sample may have an impact on the performanceof the assay. For this reason, all samples should be diluted at least 2x in ”Apo ELISA buffer”.
- Sodium azide and other nucleophilic substances (often used as preservative in various buffers) interfere with the activity of horseradish peroxidase. Thus, avoid the use of other wash
buffers or solutions that may contain such interfering substances.

PREPARATIONS
PlatesAllow the plates to adjust to room temperature (18-25°C) before opening the bags.
Plan the experiment to include a standard curve and an assay background control (8x 2 wells), blank (2 wells) and sample wells. To the blank wells, only ”TMB substrate” and ”Stop solution” should be added. Before the analysis of absorbance values,
the mean value of the blank wells should be subtracted from the standard, the assay
background control and the sample values.
Assemble the required numbers of strips in the plate frame. The strips used for theexperiment can be marked e.g. with a marker pen. Store the remaining strips in thefoil bag containing the desiccant at 4-8°C.
Wash buffer
Prepare the required volume of wash buffer by diluting ”Wash buffer concentrate” 20xwith distilled or deionized water. For 1 plate, prepare 1000 ml wash buffer by adding
50 ml ”Wash buffer concentrate” to 950 ml distilled or deionized water.
Apo ELISA buffer
Prepare the required volume of Apo ELISA buffer by diluting ”Apo ELISA bufferconcentrate 5x” 5x with distilled or deionized water. For 1 plate, prepare 150 ml Apo
ELISA buffer by adding 30 ml ”Apo ELISA buffer concentrate 5x” to 120 ml distilled or deionized water.
ELISA standard
Reconstitute the apoJ standard in 1 ml of standard reconstitution buffer, do not stir.
It is important to wait 20 minutes before resuspending the liquid. This gives a stock
solution of 1.5 μg/ml which should be used immediately or stored in aliquots at -20°C
for future use. Mix thoroughly and aliquot. Store at -20°C. Avoid repeated freeze-thaw
cycles of the standard aliquotes.
Preparation of standard curve
The standard curve can be made from standard stock solution just reconstituted orfrom thawed aliquotes. Prepare a serial dilution of the standard no more than 30 minprior to the experiment. Duplicate wells for the standard are recommended. Dilutethe standard stock solution to create a standard curve ranging from 0.07-50 ng/ml
according to the scheme below. For the assay background control (0 pg/ml), use only
”Apo ELISA buffer”.

15 µl

150 µl150 µl150 µl150 µl150 µl150 µlNo apoJStandardStockSolution

1.5 µg/ml

50 ng/ml

17.7 ng/ml

5.6 ng/ml

1.9 ng/ml

0.6 ng/ml

0.2 ng/ml

0.07 ng/ml

435 µl300 µl300 µl300 µl300 µl300 µl300 µlStandard Curve

0 pg/ml
300 µlAssay Background
Control

Figure 1. Recommended serial dilution of apoJ standard. The volumes indicated are sufficient for duplicates. The last vial should be 0 pg/ml of standard i.e. the analyte should be omitted.

Samples
It is recommended that visible precipitate in the sample should be removed bycentrifugation. Triton X-treatment of samples, necessary for apoB analysis, will notinterfere with apoJ analysis. Dilute all samples at least 2x in ”Apo ELISA buffer” priorto use, also samples where low analyte levels are anticipated. We recommend the useof duplicate wells for all samples. It is recommended to dilute serum/plasma samples 20,000x to 50,000x. Please see dilution guidelines at page 10. Avoid repeatedfreezing-thawing cycles.
Detection antibody
Dilute the detection antibody ”J84-biotin” 500x in ”Apo ELISA buffer” prior to use.
For 1 plate, dilute 24 μl ”J84-biotin” in 12 ml ”Apo ELISA buffer” which will give afinal ”J84-biotin” concentration of 1 μg/ml.
SA-HRP
Dilute the ”SA-HRP” 1000x in ”SA-HRP diluent” prior to use. For 1 plate, dilute 12μl ”SA-HRP” in 12 ml ”SA-HRP diluent”.

ASSAY PROCEDURE
Throughout the assay all reagents and samples, except the ”TMB substrate”, should beadjusted to room temperature (18-25°C) prior to use. The ”TMB substrate” should preferably be used cold.
1. Assemble the numbers of strips required for the standard curve, assay background control, the blanks and samples in the plate frame. The use of duplicates is recommended.
2. Wash the strips with 5x300 μl/well of diluted wash buffer. The wash buffer should bethoroughly removed in immediate relation to the next step.
3. Add 100 μl/well of each concentration of the diluted apoJ standard and assay background control. For the samples, add 100 μl/well of pre-diluted sample. Dilution guidelines for serum/plasma samples can be found at page 10. Mix by tapping the plate.
Important! Both the serial dilution of the apoJ standard and sample dilutions should bemade in ”Apo ELISA buffer”. Leave the blank wells empty. Cover the plate with adhesiveplate cover and incubate at room temperature for 2 h.
4. Wash the wells as in step 2.
5. Add 100 μl per well of the detection antibody J84-biotin, diluted 500x to a final
concentration of 1 μg/ml in ”Apo ELISA buffer”. Leave the blank wells empty. Cover theplate with adhesive plate cover and incubate at room temp for 60 min.
6. Wash the wells as in step 2.
7. Add 100 μl/well of ”SA-HRP” diluted 1000x in ”SA-HRP diluent”. Leave the blankwells empty. Cover the plate and incubate at room temp for 60 min.
8. Wash the wells as in step 2.
9. Develop by adding 100 μl of ”TMB substrate” to all wells (including the blank wells)
and incubate at room temp in the dark for 15 min.
10. Stop the color development by adding 100 μl of ”Stop solution” to all wells (includingthe blank wells).
11. Measure the absorbance at 450 nm in a microplate reader within 15 min of the addition of the ”Stop solution”. If possible, use a reader capable of subtracting a reference
wavelength between 570-650 nm.
12. The use of ELISA software utilizing e.g. a 4-parameter curve fitting program is recommended for the data analysis. Subtract the mean absorbance value of the blank from the standard,
the assay background control and the sample values prior to creating the standard curve anddetermining the apoJ concentrations in the samples. Note that apoJ values obtained should be
multiplied with the dilution factor used for each sample.

PERFORMANCE OF THE ASSAY
Human apoJ
10A450 nm10,1

0.01
0.01

0.1
110
Standard concentration (ng/ml)

100Figure 2. Display of the standard curve.

Standard range: 0.07-50 ng/ml. The standard range is the range in which determinations of analyte concentration can be done with precision, accuracy and linearity.
Sensitivity: The limit of detection of this assay is 0.04 ng/ml. It is the lowest concentration that is possible to detect but not necessarily quantify with precision and accuracy.
Dilution recovery: Dilution of serum/plasma samples gives a mid-curve recovery of 96103% in repeated experiments.
Precision: The intraassay variation is 4.1%(CV). The interassay variation is 3.7%(CV).

LIMITATIONS OF THE ASSAY
Analysis of samples with high apoJ content
The standard curve should not be extrapolated beyond the recommended standard
range as these parts of the standard curve are non-linear. Samples yielding absorbancevalues exceeding the highest point of the standard range should be re-analyzed at ahigher dilution.
Aberrant sera and plasma
The use of strongly hemolyzed and hyperlipemic serum and plasma samples may
result in erroneous determinations of apoJ concentrations.
Heterophilic antibodies in human serum and plasma
Heterophilic antibodies found in human serum/plasma are capable of binding toboth the capture and detection antibodies used in capture ELISA. Heterophilic
antibodies are found in a majority of human individuals and can, by cross-linking
the assay antibodies used, result in false positive signals in capture ELISA. The ”ApoELISA buffer” provided and used for dilution of samples prevents the heterophilic
antibodies from cross-linking the capture and detection monoclonal antibodies. TheapoJ content of serum/plasma samples can therefore be measured without interference by heterophilic antibodies. The lack of interference by heterophilic antibodies
in this MABTECH kit has been validated using serum/plasma samples from normal
healthy human blood donors. Please note that heterophilic antibody interference insamples from human subjects with various diseases or other conditions have not beenassessed.
DILUTION GUIDELINES
Serum and plasma samples containing EDTA, citrate or heparin may be used. Samples should be aliquoted and frozen. For longer storage, -80°C is recommended. Avoid
repeated freezing-thawing cycles. Before dilution, it is recommended that visible
precipitate in the sample should be removed by centrifugation. Precise pipetting isimportant! Please change pipet tips between the different steps and ensure thoroughmixing. We recommend samples to be assayed in duplicates. Sample dilutions canpreferably be made in polypropylene tubes or plates. Prepare the dilutions as near as
possible to the start of the assay. The indicated volumes are sufficient for duplicates.
The recommended dilutions are based on serum samples from fasting healthy subjects. Samples containing levels of the analyte beyond the standard range will require
other dilutions. Triton X treatment, necessary for apoB analysis, will not interferewith the analysis of other apolipoproteins.

10For analysis of human ApoJ
10 µl

10 µl

15 µl
Use
100 µl/well toapoJ assay

Serum/plasma
Sample dilution: 100X
Apo ELISA buffer: 990 µl

2,500X
240 µl

50,000X
285 µl

It is recommended to dilute the samples 20,000x to 50,000x. Example of dilution;1) 10 μl of plasma to 990 μl Apo ELISA buffer, giving a dilution of 100x.
2) 10 μl 100x dilution to 240 μl Apo ELISA buffer, giving a dilution of 2,500x.
3) 15 μl of 2,500x dilution to 285 μl Apo ELISA buffer, giving a dilution of 50,000x.

When analyzing several apolipoproteins at the same time
100 µl/well to
apoD, apoE, 100 µl/well to
100 µl/well to apoJ, apoM
apoA1, apoH
assaysassaysapoB assay

10 µl

10 µl

30 µl

250 µl

50 µl

Serum/
plasmaSample dilution: 2X
Triton X buffer: 10 µl
Apo ELISA buffer:

200X

5,000X20,000X

200,000X

990 µl

720 µl750 µl450 µl1) 10 μl of plasma to 10 μl of Triton X buffer, giving a dilution of 2x, vortex for 5 seconds.
2) 10 μl of 2x dilution to 990 μl Apo ELISA buffer, giving a dilution of 200x.
3) 30 μl of 200x to 720 μl Apo ELISA buffer, giving a dilution of 5,000x, use 100 μl/well to apoB ELISA
4) 250 μl of 5000x dilution to 750 μl Apo ELISA buffer, giving a dilution of 20,000x, use 100 μl/well toapoE, apoD, apoJ and apoM ELISAs5) 50 μl of 20000x to 450 μl Apo ELISA buffer giving a dilution of 200,000x, use 100 μl/well to apoA1and apoH ELISA

11

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Updated on 2019-04-17

NOTE; for research use only.
MABTECH shall not be liable for theuse or handling of the product or forconsequential, special, indirect or incidental damages therefrom.
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O 90 :2015
ISCTMERT I F I C AT I O

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with the above standards.
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