ELISA Pro: Human ApoM
3716-1HP-1 | 3716-1HP-2 | 3716-1HP-10

Datasheet & Protocol

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Table of Contents
Introduction ����������������������������������������������������������������������������� 4
Shipping and storage �������������������������������������������������������������� 4
Contents ������������������������������������������������������������������������������������ 5
Preparation ������������������������������������������������������������������������������� 6Protocol ������������������������������������������������������������������������������������ 8
Performance ��������������������������������������������������������������������������� 10

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Introduction

Mabtech’s carefully validated ELISA Pro kits provide all the necessary reagents toconveniently quantify analytes in serum, plasma, and cell culture supernatants in arobust, sensitive, and specific manner. For Research Use Only (RUO). Not for use indiagnostic procedures.

ELISA assay principle

ELISA Pro kits are supplied with ELISA strip
plates precoated with monoclonal antibody(mAb). Analyte in the sample is captured by thecoated mAb and detected by the biotinylated
detection mAb followed by Streptavidin-HRP(SA-HRP). Addition of TMB substrate will resultin a colored substrate product. The reaction
is stopped with sulfuric acid and the opticaldensity can be quantified using an ELISA
plate reader. The concentration of analyte isdetermined by comparison to a serial dilutionof the ELISA standard analyzed in parallel.

HRPSAB

Analysis of serum and plasma samplesThe ELISA Pro kits include Apo ELISA buffer,a buffer that prevents false-positive signals.
The buffer blocks heterophilic antibodies
from cross-linking the assay antibodies.
Heterophilic antibodies are commonly foundin human serum/plasma and can also be present in other species. The buffer has beenvalidated using serum/plasma samples from healthy human blood donors.

Shipping and storage

The kit is shipped at ambient temperature. All reagents should be stored at 4-8 °C uponreceipt, except the standard, which should be stored at -20 °C. The expiry date indicates
how long unopened products, stored according to instructions, are recommended foruse. Do not combine components from different kit batches or components from othersuppliers.

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ContentsComponent

1-plate kit

2-plate kit

10-plate kit

Pre-coated ELISA strip plate:Anti-apoM mAb M5

1 x 96 wells

2 x 96 wells

10 x 96 wells

Purified human apoM ELISA standard

1 vial1 vial2 vials

Detection mAb M7, biotin(0.5 mg/ml)

15 μl

25 μl

125 μlStreptavidin-HRP

15 μl

25 μl

125 μlStandard reconstitution buffer A81 ml

1 ml

2 x 1 ml

Wash buffer concentrate

120 ml120 ml5 x 120 ml

Apo ELISA buffer concentrate 5x60 ml

60 ml

5 x 60 ml

Streptavidin-HRP diluent

15 ml

25 ml

120 mlTMB substrate

15 ml

25 ml

120 mlStop solution

15 ml

25 ml

120 mlAdhesive plate covers

3630

To ensure total recovery of the stated quantity, bottles and vials have been overfilled.

Materials required but not supplied• Microplate reader capable of reading at 450 nm• ELISA plate washer; automated or manual (e.g., multipipette or squirt bottle)• Precision pipettes, tips, and graduated cylinders• Tubes for standard and sample dilutions• Distilled or deionized water

Safety information

The Stop solution, 0.18 M H2SO 4 (< 1%), is irritating to eyes and skin and should be handledwith care. The standard should also be handled carefully as the effects of exposure areunknown. Buffers and reagents in solution contain the preservative Kathon CG (0.002%),a potential allergen that may cause sensitization through skin contact. Human and animal
samples should be treated as potentially hazardous biologic material. All material should
be disposed of in accordance with local regulations. For further information please
consult the Safety Data Sheet on our website.

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5Preparation• Allow the plates and assay reagents to reach room temperature before starting theassay (except for the TMB substrate which should preferably be used cold).

• Plan the plate layout to include a standard curve, samples, and an assay backgroundcontrol, all in duplicate. The volume per well should not exceed 100 μl. Include a plate
blank (wells with only Substrate and Stop solution) to be used for subtraction beforeanalysis.

Wash buffer

Add 50 ml Wash buffer concentrate to 950 ml distilled or deionized water (sufficient for
all washing steps of 1 plate). If crystals have formed in the 20x concentrate, bring toroom temperature and mix gently to dissolve.

Apo ELISA buffer

Prepare the required volume of Apo ELISA buffer by diluting Apo ELISA bufferconcentrate 5-fold with distilled or deionized water. For each plate, add 30 ml Apo ELISA
buffer concentrate to 120 ml water.

Samples

All samples should be diluted at least 2-fold in Apo ELISA buffer. Remove visible
precipitates and dilute in tubes/plates, buffer should be added prior to the samples.
Strongly hemolyzed and hyperlipemic samples may give inaccurate quantifications.
Samples containing high levels of analyte exceeding the standard range of the assay will
require further dilution.

Dilution guidelines for human serum/plasma

We recommend a dilution factor of 20,000X based on repeated analyses of fasting healthy
subjects. Precise pipetting is important, change tips between dilution steps and use
freshly made dilutions. Indicated volumes are sufficient for duplicates.
10 μl

10 μl

15 μl

100 μl/wellSerum/plasma6Apo ELISA buﬀer:

990 μl90 μl285 μlDilution:

100x

1000X

20,000X

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ELISA standard

Reconstitute the ELISA standard to a stock solution of 0.5 μg/ml by adding 260 μl
Standard reconstitution buffer. Do not stir. Allow the standard to dissolve for 20 minutes
and mix thoroughly. The standard should be kept in aliquots at -20 °C. Avoid repeatedfreeze-thaw cycles.

Preparation of standard curve

Dilute the standard stock solution to create a standard curve as shown. The indicatedvolumes are sufficient for duplicates. The last vial is used as an assay background control,i.e., the standard should be omitted. Prepare the standard curve within 30 minutes of use.

20 μl

150 μl150 μl150 μl150 μl150 μl150 μlNo standard

Standard stock
0.5 μg/ml

Apo ELISA buﬀer:

480 μl300 μl300 μl300 μl300 μl300 μl300 μl300 μlConcentration:

20ng/ml

6.7
ng/ml

2.2ng/ml

0.74
ng/ml

0.25
ng/ml

0.08
ng/ml

0.03
ng/ml

0Standard curve

Detection antibody

Dilute the detection mAb in Apo ELISA buffer to a concentration of 0.5 μg/ml within 15
minutes of use. For each plate, add 12 μl detection mAb to 12 ml Apo ELISA buffer.

Streptavidin-HRP

Dilute the Streptavidin-HRP 1:1000 in Streptavidin-HRP diluent within 15 minutes of use.
For each plate, add 12 μl Streptavidin-HRP to 12 ml Streptavidin-HRP diluent.

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Protocol

Prepare the reagents, standard curve, and samples as described in the Preparation
section. Assemble the required number of strips in the plate frame and label the top ofeach strip. Store the remaining strips in the foil bag containing the desiccant at 4-8 °C.

1.	 Wash the plate 5 times with wash buffer, 300 μl per well. After the final wash,

invert and tap the plate firmly against absorbent paper. Immediately proceed tothe next step.

2.	 Add 100 μl per well of samples (diluted at least 2-fold), standard, and assay

background control. Mix by tapping the plate. Cover the plate with an adhesiveplate cover and incubate for 2 hours at room temperature.

3.	 Wash as in step 1.

4.	 Add 100 μl per well of detection mAb. Cover the plate and incubate for 1 hour atroom temperature.

5.	 Wash as in step 1.

6.	 Add 100 μl per well of Streptavidin-HRP. Cover the plate and incubate for 1 hour atroom temperature.

7.	 Wash as in step 1.

8.	 Add 100 μl of TMB substrate to each well. Incubate at room temperature,protected from direct light for 15 minutes.

9.	 Add 100 μl of Stop solution to each well to stop the color development.

10.	 Measure absorbance at 450 nm within 15 minutes. Preferably use a reader
capable of subtracting a reference wavelength between 570 and 650 nm.

We recommend the use of an ELISA software utilizing a 4- or 5-parameter curvefit. Subtract the mean absorbance value of the blank from the samples, standard
and assay background control prior to creating the standard curve and analyzing
the results.

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Wash
Add samples,standard, and assay
background control
Incubate 2 hours
Wash
B

Add detection
antibody
Incubate 1 hour

Wash
HRPSA

Add Streptavidin-HRP
Incubate 1 hour

Wash
Add TMB substrate
Incubate 15 minutes

Add Stop solution+www.mabtech.com

Measure absorbanceat 450 nm within
15 minutes

9Performance

Human ApoM

A450 - 650 nm

101

Representative standard curve

0.1

0.01
0.01

0.1

1

10100

Standard concentration (ng/ml)

Standard range 0.027-20 ng/ml
Sensitivity 0.01 ng/ml

The lowest concentration that can be detected, but not necessarily quantified with
precision and accuracy. This was determined by adding 5 standard deviations to the mean
OD of background wells.

Calibration

No international standard exists for calibration.

Precision
Intra-assay
Samplen12Inter-assay
312

3

8883

3

3

Mean (ng/ml)

1.87

0.97

0.55

2.03

0.99

0.54

SD

0.07

0.03

0.01

0.08

0.03

0.02

CV%

3.9

2.7

2.4

3.8

3.2

3.9

Intra-assay and inter-assay precision were determined at 3 different concentrations ofanalyte (8 replicates per concentration in 3 assays).

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Linearity of Dilution
Conc. ng/ml

PlasmaDilution X

Average recovery% (range)

2.0 ng/ml 20,000X

102 (90-106)

1.0 ng/ml 40,000X

100 (96-106)

0.5 ng/ml 80,000X

104 (96-109)

Three different dilutions of human plasma. Eight replicates per dilution were tested in 3assays.

Specificity
The kit is based on a matched pair of mAbs specific for human ApoM.

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Updated on 2022/12/02
Developed and manufactured by MABTECH
AB, Sweden, whose quality management
system complies with the standards
ISO 9001:2015 & ISO 13485:2016.

CERTIFIED
ISO 9001

CERTIFIED
ISO 13485

Quality management systems

Medical devices
Quality management systems

The products are for research use only.
MABTECH shall not be liable for the use or handling of the product orfor consequential, special, indirect or incidental damages there from.
Mabtech AB (Head Office)SwedenTel: +46 8 716 27 00
mabtech@mabtech.com

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Mabtech, Inc.
USA
Tel: +1 513 871-4500
mabtech.usa@mabtech.com