Version 1 Last updated 15 April 2020

ab263881
Human AST SimpleStep
ELISA® Kit
(Aspartate
Aminotransferase)

For the quantitative measurement of AST in human serum, plasma,

cell culture supernatant, cell extract, and tissue extract samples.

This product is for research use only and is not intended for
diagnostic use.

Copyright © 2019 Abcam. All rights reserved

Table of Contents

1. Overview 1
2. Protocol Summary 2
3. Precautions 3
4. Storage and Stability 3
5. Limitations 4
6. Materials Supplied 4
7. Materials Required, Not Supplied 5
8. Technical Hints 5
9. Reagent Preparation 7
10. Standard Preparation 8
11. Sample Preparation 9
12. Plate Preparation 12
13. Assay Procedure 13
14. Calculations 15
15. Typical Data 16
16. Typical Sample Values 19
17. Assay Specificity 27
18. Species Reactivity 27
19. Troubleshooting 28
20. Notes 29
Technical Support 30

Copyright © 2019 Abcam. All rights reserved

1. Overview

AST in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay)

kit is designed for the quantitative measurement of AST protein in
human serum, plasma, cell culture supernatant, cell extract, and
tissue extract samples.

The SimpleStep ELISA® employs an affinity tag labeled capture

antibody and a reporter conjugated detector antibody which
immunocapture the sample analyte in solution. This entire complex
(capture antibody/analyte/detector antibody) is in turn immobilized
via immunoaffinity of an anti-tag antibody coating the well. To
perform the assay, samples or standards are added to the wells,
followed by the antibody mix. After incubation, the wells are washed
to remove unbound material. TMB Development Solution is added
and during incubation is catalyzed by HRP, generating blue
coloration. This reaction is then stopped by addition of Stop Solution
completing any color change from blue to yellow. Signal is
generated proportionally to the amount of bound analyte and the
intensity is measured at 450 nm. Optionally, instead of the endpoint
reading, development of TMB can be recorded kinetically at 600
nm.

Cytoplasmic aspartate aminotransaminase (AST), is a key enzyme in

the regulation of glutamate levels. It catalyzes the reversible transfer
of an a-amino group between aspartate and glutamate. Given its
central role in amino acid metabolism, it plays a critical role in many
biological functions. In a clinical context, AST levels in the serum are
used as a marker for liver health. This kit targets the full-length
protein.

ab263881 Human AST SimpleStep ELISA Kit 1

2. Protocol Summary

Prepare all reagents, samples, and standards as instructed

Add 50 µL standard or sample to appropriate wells

Add 50 µL Antibody Cocktail to all wells

Incubate at room temperature for 1 hour

Aspirate and wash each well three times with 350 µL 1X Wash Buffer
PT

Add 100 µL TMB Development Solution to each well and incubate

for 10 minutes.

Add 100 µL Stop Solution and read OD at 450 nm

ab263881 Human AST SimpleStep ELISA Kit 2

3. Precautions

Please read these instructions carefully prior to beginning the assay.

 All kit components have been formulated and quality control
tested to function successfully as a kit.
 We understand that, occasionally, experimental protocols might
need to be modified to meet unique experimental
circumstances. However, we cannot guarantee the
performance of the product outside the conditions detailed in
this protocol booklet.
 Reagents should be treated as possible mutagens and should be
handle with care and disposed of properly. Please review the
Safety Datasheet (SDS) provided with the product for information
on the specific components.
 Observe good laboratory practices. Gloves, lab coat, and
protective eyewear should always be worn. Never pipet by
mouth. Do not eat, drink or smoke in the laboratory areas.
 All biological materials should be treated as potentially
hazardous and handled as such. They should be disposed of in
accordance with established safety procedures.

4. Storage and Stability

Store kit at +4°C immediately upon receipt. Kit has a storage time of
1 year from receipt, providing components have not been
reconstituted.
Refer to list of materials supplied for storage conditions of individual
components.

ab263881 Human AST SimpleStep ELISA Kit 3

5. Limitations

 Assay kit intended for research use only. Not for use in diagnostic
procedures.
 Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.

6. Materials Supplied

Storage
Item Quantity
Condition
Human AST Capture Antibody 10X 600 µL +4ºC
Human AST Detector Antibody 10X 600 µL +4ºC
Human AST Lyophilized Recombinant Protein 2 Vials +4ºC
Antibody Diluent 4BI 6 mL +4ºC
Cell Extraction Buffer PTR 5X 10 mL +4ºC
Cell Extraction Enhancer Solution 50X 1 mL +4ºC
Sample Diluent 25BS 20 mL +4ºC
Sample Diluent NS 12 mL +4ºC
Wash Buffer PT 10X 20 mL +4ºC
TMB Development Solution 12 mL +4ºC
Stop Solution 12 mL +4ºC
SimpleStep Pre-Coated 96-Well Microplate 96 Wells +4ºC
Plate Seal 1 +4ºC

ab263881 Human AST SimpleStep ELISA Kit 4

7. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to
successfully perform this assay:
 Microplate reader capable of measuring absorbance at 450 or
600 nm.
 Method for determining protein concentration (BCA assay
recommended).
 Deionized water.
 Multi- and single-channel pipettes.
 Tubes for standard dilution.
 Plate shaker for all incubation steps.
 Optional: Phenylmethylsulfonyl Fluoride (PMSF) (or other protease
inhibitors).

8. Technical Hints

 Samples generating values higher than the highest standard

should be further diluted in the appropriate sample dilution
buffers.
 Avoid foaming or bubbles when mixing or reconstituting
components.
 Avoid cross contamination of samples or reagents by changing
tips between sample, standard and reagent additions.
 Ensure plates are properly sealed or covered during incubation
steps.
 Complete removal of all solutions and buffers during wash steps
is necessary to minimize background.
 As a guide, typical ranges of sample concentration for
commonly used sample types are shown below in Sample
Preparation (section 11).
 All samples should be mixed thoroughly and gently.
 Avoid multiple freeze/thaw of samples.
 Incubate ELISA plates on a plate shaker during all incubation
steps.
 When generating positive control samples, it is advisable to
change pipette tips after each step.

ab263881 Human AST SimpleStep ELISA Kit 5

 The provided Antibody Diluents and Sample Diluents contain
protease inhibitor aprotinin. Additional protease inhibitors can be
added if required.
 The provided Cell Extraction Buffer 5X contains phosphatase
inhibitors and protease inhibitor aprotinin. Additional protease
inhibitors can be added if required.
 The provided Cell Extraction Enhancer Solution 50X may
precipitate when stored at + 4ºC. To dissolve, warm briefly at
+ 37ºC and mix gently. The Cell Extraction Enhancer Solution 50X
can be stored at room temperature to avoid precipitation.
 To avoid high background always add samples or standards to
the well before the addition of the antibody cocktail.
 This kit is sold based on number of tests. A ‘test’ simply refers to a
single assay well. The number of wells that contain sample,
control or standard will vary by product. Review the protocol
completely to confirm this kit meets your requirements. Please
contact our Technical Support staff with any questions.

ab263881 Human AST SimpleStep ELISA Kit 6

9. Reagent Preparation

 Equilibrate all reagents to room temperature (18-25°C) prior to

use. The kit contains enough reagents for 96 wells. The sample
volumes below are sufficient for 48 wells (6 x 8-well strips); adjust
volumes as needed for the number of strips in your experiment.
 Sample Diluent 25BS may contain precipitate, this is normal. If
precipitate is not dissolved by gentle mixing, the precipitate may
be dissolved by gentle warming and mixing at 37°C for 10
minutes. If precipitate remains, gently spin down and avoid
visible precipitates when pipetting.
 Prepare only as much reagent as is needed on the day of the
experiment. Capture and Detector Antibodies have only been
tested for stability in the provided 10X formulations.

9.1 1X Cell Extraction Buffer PTR (For cell and tissue extracts only):
Prepare 1X Cell Extraction Buffer PTR by diluting Cell Extraction
Buffer PTR 5X and 50X Cell Extraction Enhancer Solution to 1X
with deionized water. To make 10 mL 1X Cell Extraction Buffer
PTR combine 7.8 mL deionized water, 2 mL Cell Extraction
Buffer PTR 5X and 200 µL Cell Extraction Enhancer Solution 50X.
Mix thoroughly and gently. If required protease inhibitors can
be added.
9.2 1X Wash Buffer PT:
Prepare 1X Wash Buffer PT by diluting Wash Buffer PT 10X with
deionized water. To make 50 mL 1X Wash Buffer PT combine 5
mL Wash Buffer PT 10X with 45 mL deionized water. Mix
thoroughly and gently.
9.3 Antibody Cocktail:
Prepare Antibody Cocktail by diluting the capture and
detector antibodies in Antibody Diluent 4BI. To make 3 mL of
the Antibody Cocktail combine 300 µL 10X Capture Antibody
and 300 µL 10X Detector Antibody with 2.4 mL Antibody
Diluent 4BI. Mix thoroughly and gently.

ab263881 Human AST SimpleStep ELISA Kit 7

10. Standard Preparation

 Always prepare a fresh set of standards for every use.

 Discard working standard dilutions after use as they do not store
well.
 The following section describes the preparation of a standard
curve for duplicate measurements (recommended).

10.1 For serum, and plasma samples measurements, reconstitute

the AST protein standard by adding 500 µL of Sample Diluent
25BS.
For cell culture supernatant samples measurements,
reconstitute the AST protein standard by adding 500 µL of
Sample Diluent NS.
For cell and tissue extract samples measurements, reconstitute
the AST protein standard by adding 500 µL of 1X Cell Extraction
Buffer PTR.
Hold at room temperature for 10 minutes and mix thoroughly
and gently. This is the 300,000 pg/mL Stock Standard Solution.
10.1.1 Label eight tubes, Standards 1– 8.
10.1.2 Add 392 μL of appropriate diluent (see step 10.1) into tube
number 1 and 150 μL of appropriate diluent into numbers 2-8.
10.1.3 Use the Stock Standard to prepare the following dilution
series. Standard #8 contains no protein and is the Blank
control:

28 µL
150 µL 150 µL 150 µL 150 µL 150 µL 150 µL 150 µL
µ
µ µ µµ µ µ µ µ

300,000 20,000 10,000 5,000 2,500 1,250 625 312.50 0

pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL

ab263881 Human AST SimpleStep ELISA Kit 8

11. Sample Preparation

Typical Sample Dynamic Range

Sample Type Range
Serum 1.56 – 25%

Plasma – Citrate 1.56 – 25%

Plasma – EDTA 1.56 – 25%

Plasma – Heparin 1.56 – 25%

HepG2 cell culture

3.13 – 50%
supernatant
Human Liver Extract 0.63 – 10 µg/mL

HepG2 Cell Extract 4.69 – 75 µg/mL

11.1 Plasma:
Collect plasma using citrate, EDTA or heparin. Centrifuge
samples at 2,000 x g for 10 minutes. Dilute samples at least 1:4
into Sample Diluent 25BS and assay. Store un-diluted plasma
samples at -20ºC or below for up to 3 months. Avoid repeated
freeze-thaw cycles.
11.2 Serum:
Samples should be collected into a serum separator tube.
After clot formation, centrifuge samples at 2,000 x g for
10 minutes and collect serum. Dilute samples at least 1:4 into
Sample Diluent 25BS and assay. Store un-diluted serum at -20ºC
or below. Avoid repeated freeze-thaw cycles.
11.3 Cell Culture Supernatants:
Centrifuge cell culture media at 2,000 x g for 10 minutes to
remove debris. Collect supernatants and assay. Or dilute
samples at least 1:2 into Sample Diluent NS and assay. Store
un-diluted samples at -20°C or below. Avoid repeated freeze-
thaw cycles.

ab263881 Human AST SimpleStep ELISA Kit 9

11.4 Preparation of extracts from cell pellets:
11.4.1 Collect non-adherent cells by centrifugation or scrape to
collect adherent cells from the culture flask. Typical
centrifugation conditions for cells are 500 x g for 5 minutes at
4ºC.
11.4.2 Rinse cells twice with PBS.
11.4.3 Solubilize pellet at 2x107 cell/mL in chilled 1X Cell Extraction
Buffer PTR.
11.4.4 Incubate on ice for 20 minutes.
11.4.5 Centrifuge at 18,000 x g for 20 minutes at 4°C.
11.4.6 Transfer the supernatants into clean tubes and discard the
pellets.
11.4.7 Assay samples immediately or aliquot and store at -80°C. The
sample protein concentration in the extract may be
quantified using a protein assay.
11.4.8 Dilute samples to desired concentration in 1X Cell Extraction
Buffer PTR.
11.5 Preparation of extracts from adherent cells by direct lysis
(alternative protocol):
11.5.1 Remove growth media and rinse adherent cells 2 times in
PBS.
11.5.2 Solubilize the cells by addition of chilled 1X Cell Extraction
Buffer PTR directly to the plate (use 750 µL - 1.5 mL 1X Cell
Extraction Buffer PTR per confluent 15 cm diameter plate).
11.5.3 Scrape the cells into a microfuge tube and incubate the
lysate on ice for 15 minutes.
11.5.4 Centrifuge at 18,000 x g for 20 minutes at 4°C.
11.5.5 Transfer the supernatants into clean tubes and discard the
pellets.
11.5.6 Assay samples immediately or aliquot and store at -80°C. The
sample protein concentration in the extract may be
quantified using a protein assay.
11.5.7 Dilute samples to desired concentration in 1X Cell Extraction
Buffer PTR.

ab263881 Human AST SimpleStep ELISA Kit 10

11.6 Preparation of extracts from tissue homogenates:
11.6.1 Tissue lysates are typically prepared by homogenization of
tissue that is first minced and thoroughly rinsed in PBS to
remove blood (dounce homogenizer recommended).
11.6.2 Homogenize 100 to 200 mg of wet tissue in 500 µL – 1 mL of
chilled 1X Cell Extraction Buffer PTR. For lower amounts of
tissue adjust volumes accordingly.
11.6.3 Incubate on ice for 20 minutes.
11.6.4 Centrifuge at 18,000 x g for 20 minutes at 4°C.
11.6.5 Transfer the supernatants into clean tubes and discard the
pellets.
11.6.6 Assay samples immediately or aliquot and store at -80°C. The
sample protein concentration in the extract may be
quantified using a protein assay.
11.6.7 Dilute samples to desired concentration in 1X Cell Extraction
Buffer PTR.

ab263881 Human AST SimpleStep ELISA Kit 11

12. Plate Preparation

 The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.
 Unused plate strips should be immediately returned to the foil
pouch containing the desiccant pack, resealed and stored at
4°C.
 For each assay performed, a minimum of two wells must be used
as the zero control.
 For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
 Differences in well absorbance or “edge effects” have not been
observed with this assay.

ab263881 Human AST SimpleStep ELISA Kit 12

13. Assay Procedure

 Equilibrate all materials and prepared reagents to room

temperature prior to use.
 We recommend that you assay all standards, controls and
samples in duplicate.

13.1 Prepare all reagents, working standards, and samples as

directed in the previous sections.
13.2 Remove excess microplate strips from the plate frame, return
them to the foil pouch containing the desiccant pack, reseal
and return to 4ºC storage.
13.3 Add 50 µL of all sample or standard to appropriate wells.
13.4 Add 50 µL of the Antibody Cocktail to each well.
13.5 Seal the plate and incubate for 1 hour at room temperature
on a plate shaker set to 400 rpm.
13.6 Wash each well with 3 x 350 µL 1X Wash Buffer PT. Wash by
aspirating or decanting from wells then dispensing 350 µL 1X
Wash Buffer PT into each well. Wash Buffer PT should remain in
wells for at least 10 seconds. Complete removal of liquid at
each step is essential for good performance. After the last
wash invert the plate and tap gently against clean paper
towels to remove excess liquid.
13.7 Add 100 µL of TMB Development Solution to each well and
incubate for 10 minutes in the dark on a plate shaker set to 400
rpm.
Given variability in laboratory environmental conditions,
optimal incubation time may vary between 5 and 20 minutes.
Note: The addition of Stop Solution will change the color from
blue to yellow and enhance the signal intensity about 3X. To
avoid signal saturation, proceed to the next step before the
high concentration of the standard reaches a blue color of
O.D.600 equal to 1.0.
13.8 Add 100 µL of Stop Solution to each well. Shake plate on a
plate shaker for 1 minute to mix. Record the OD at 450 nm. This
is an endpoint reading.
13.9 Alternative to 13.7 – 13.8: Instead of the endpoint reading at
450 nm, record the development of TMB Substrate kinetically.
Immediately after addition of TMB Development Solution
begin recording the blue color development with elapsed

ab263881 Human AST SimpleStep ELISA Kit 13

 time in the microplate reader prepared with the following
settings:

Mode Kinetic
Wavelength: 600 nm
Time: up to 20 min
Interval: 20 sec - 1 min
Shaking: Shake between readings
 Note: that an endpoint reading can also be recorded at the
completion of the kinetic read by adding 100 µL Stop Solution to
each well and recording the OD at 450 nm.
13.10 Analyze the data as described below.

ab263881 Human AST SimpleStep ELISA Kit 14

14. Calculations

14.1 Calculate the average absorbance value for the blank

control (zero) standards. Subtract the average blank control
standard absorbance value from all other absorbance values.
14.2 Create a standard curve by plotting the average blank
control subtracted absorbance value for each standard
concentration (y-axis) against the target protein
concentration (x-axis) of the standard. Use graphing software
to draw the best smooth curve through these points to
construct the standard curve.
 Note: Most microplate reader software or graphing software will
plot these values and fit a curve to the data. A four-parameter
curve fit (4PL) is often the best choice; however, other algorithms
(e.g. linear, semi-log, log/log, 4-parameter logistic) can also be
tested to determine if it provides a better curve fit to the standard
values.
14.3 Determine the concentration of the target protein in the
sample by interpolating the blank control subtracted
absorbance values against the standard curve. Multiply the
resulting value by the appropriate sample dilution factor, if
used, to obtain the concentration of target protein in the
sample.
14.4 Samples generating absorbance values greater than that of
the highest standard should be further diluted and reanalyzed.
Similarly, samples which measure at an absorbance values less
than that of the lowest standard should be retested in a less
dilute form.

ab263881 Human AST SimpleStep ELISA Kit 15

15. Typical Data

Typical standard curve – data provided for demonstration purposes

only. A new standard curve must be generated for each assay
performed.

10
O.D. (450 nm)

0.1

0.01
100 1000 10000
Human AST (pg/mL)

Standard Curve Measurements

Concentration O.D 450 nm Mean

(pg/mL) 1 2 O.D

0 0.099 0.101 0.100

312.50 0.156 0.143 0.150
625 0.203 0.194 0.198
1,250 0.300 0.305 0.303
2,500 0.495 0.510 0.502
5,000 0.858 0.819 0.839
10,000 1.576 1.558 1.567
20,000 2.558 2.592 2.575
Figure 1. Example of human AST standard curve in Sample Diluent 25BS. The
AST standard curve was prepared as described in Section 10. Raw data
values are shown in the table. Background-subtracted data values (mean
+/- SD) are graphed.

ab263881 Human AST SimpleStep ELISA Kit 16

 10

O.D. (450 nm)

1

0.1

0.01
100 1000 10000
Human AST (pg/mL)

Standard Curve Measurements

Concentration O.D 450 nm Mean

(pg/mL) 1 2 O.D

0 0.113 0.111 0.112

312.50 0.184 0.196 0.190
625 0.261 0.258 0.259
1,250 0.388 0.397 0.393
2,500 0.639 0.653 0.646
5,000 1.096 1.071 1.084
10,000 1.909 1.940 1.925
20,000 3.132 3.161 3.146
Figure 2. Example of human AST standard curve in Sample Diluent NS. The
AST standard curve was prepared as described in Section 10. Raw data
values are shown in the table. Background-subtracted data values (mean
+/- SD) are graphed.

ab263881 Human AST SimpleStep ELISA Kit 17

 10

O.D. (450 nm)

1

0.1

0.01
100 1000 10000
Human AST (pg/mL)

Standard Curve Measurements

Concentration O.D 450 nm Mean

(pg/mL) 1 2 O.D

0 0.123 0.121 .0122

312.50 0.206 0.202 0.204
625 0.297 0.292 0.295
1,250 0.450 0.453 0.451
2,500 0.750 0.749 0.749
5,000 1.284 1.311 1.298
10,000 2.251 2.218 2.234
20,000 3.382 3.345 3.363
Figure 3. Example of human AST standard curve in 1X Cell Extraction Buffer
PTR. The AST standard curve was prepared as described in Section 10. Raw
data values are shown in the table. Background-subtracted data values
(mean +/- SD) are graphed.

ab263881 Human AST SimpleStep ELISA Kit 18

16. Typical Sample Values

SENSITIVITY –
The MDD was determined by calculating the mean of zero standard
replicates and adding 2 standard deviations then extrapolating the
corresponding concentration.

Minimal
Sample Diluent Buffer n=
Detectable Dose
Sample Diluent 25BS 24 43 pg/mL
Sample Diluent NS 32 57 pg/mL
1X Cell Extraction Buffer PTR 24 60 pg/mL

RECOVERY –
Three concentrations of AST recombinant protein were spiked in
duplicate to the indicated biological matrix to evaluate signal
recovery in the working range of the assay.
Average %
Sample Type Range (%)
Recovery

6.25% Serum 101 95 – 114

12.5% Plasma – Citrate 93 89 – 97
12.5% Plasma – EDTA 93 91 – 96
12.5% Plasma – Heparin 103 101 – 104
25% HepG2 cell culture
93 81 - 109
supernatant
2.5 µg/mL Human Liver 99 97 – 103
ExtractCell Extract
75 µg/mL HepG2 92 88 - 94

ab263881 Human AST SimpleStep ELISA Kit 19

Linearity of Dilution
Linearity of dilution is determined based on interpolated values from
the standard curve. Linearity of dilution defines a sample
concentration interval in which interpolated target concentrations
are directly proportional to sample dilution.

Native AST was measured in the following biological samples in a 2-

fold dilution series. Sample dilutions are made in Sample
Diluent 25BS.
25% 25% 25%
25%
Dilution Human Human Human
Interpolated value Human
Factor Plasma Plasma Plasma
Serum
(Citrate) (EDTA) (Heparin)
pg/mL 14,887 9,882 13,683 10,564
Undiluted
% Expected value 100 100 100 100
pg/mL 7,750 4,878 6,409 5,024
2
% Expected value 104 99 94 95
pg/mL 3,907 2,451 3,331 2,607
4
% Expected value 105 99 97 99
pg/mL 2,021 1,147 1,592 1,276
8
% Expected value 109 93 93 97
pg/mL 1,011 608 767 629
16
% Expected value 109 98 90 95

ab263881 Human AST SimpleStep ELISA Kit 20

Native AST was measured in the following biological samples in a 2-
fold dilution series. Sample dilutions are made in Sample Diluent NS.
50%
Dilution
Interpolated value HepG2 Cell Culture
Factor
Supernatant
pg/mL 6,000
Undiluted
% Expected value 100
pg/mL 3,190
2
% Expected value 106
pg/mL 1,656
4
% Expected value 110
pg/mL 841
8
% Expected value 112
pg/mL 424
16
% Expected value 113

Native AST was measured in the following biological samples in a 2-

fold dilution series. Sample dilutions are made in 1X Cell Extraction
Buffer PTR.
10 µg/mL 75 µg/mL
Dilution Interpolated
Human Liver HepG2 Cell
Factor value
Extract Extract

Undilut pg/mL 13,565 14,724

ed % Expected 100 100
value
pg/mL 6,613 7,038
2
% Expected 98 96
value
pg/mL 3,207 3,626
4
% Expected 95 99
value
pg/mL 1,711 1,755
8
% Expected 101 95
value
pg/mL 897 783
16
% Expected 106 85
value

ab263881 Human AST SimpleStep ELISA Kit 21

PRECISION –
Mean coefficient of variations of interpolated values of AST from one
concentration of serum within the working range of the assay.
Intra- Inter-
Assay Assay

n= 8 3
CV (%) 4.2 1.8

ab263881 Human AST SimpleStep ELISA Kit 22

 Undiluted 2X Diluted 4X Diluted
80 8X Diluted 16X Diluted

Human AST (ng/mL)

60

40

20

0
Serum Plasma - Plasma - Plasma -
Citrate EDTA Heparin

Figure 4. Interpolated concentrations of native AST in human serum and

plasma samples. The concentrations of AST were measured in duplicates,
interpolated from the AST standard curves and corrected for sample
dilution. Undiluted samples are as follows: serum 25%, plasma (citrate) 25%,
plasma (EDTA) 25%, and plasma (heparin) 25%. The interpolated dilution
factor corrected values are plotted (mean +/- SD, n=2). The mean AST
concentration was determined to be 63 ng/mL in serum, 39 ng/mL in
plasma (citrate), 52 ng/mL in plasma (EDTA). and 41 ng/mL in plasma
(heparin).

ab263881 Human AST SimpleStep ELISA Kit 23

 Undiluted 2X Diluted 4X Diluted
20 8X Diluted 16X Diluted

Human AST (ng/mL)

15

10

0
HepG2 Supernatant

Figure 5. Interpolated concentrations of native AST in HepG2 cell culture

supernatant samples. The concentrations of AST were measured in
duplicates, interpolated from the AST standard curves and corrected for
sample dilution. Undiluted samples are as follows: HepG2 cell culture
supernatant 50%. The interpolated dilution factor corrected values are
plotted (mean +/- SD, n=2). The mean AST concentration was determined to
be 13 ng/mL in HepG2 cell culture supernatant

ab263881 Human AST SimpleStep ELISA Kit 24

Figure 6. Interpolated concentrations of native AST in human liver extract
and HepG2 cell extract samples based on 10 and 75 μg/mL extract loads,
respectively. The concentrations of AST were measured in duplicate and
interpolated from the AST standard curve and corrected for sample dilution.
The interpolated dilution factor corrected values are plotted (mean +/- SD,
n=2). The mean AST concentration was determined to be 14 ng/mL in
human liver extract and 14 ng/mL in HepG2 cell extract.

ab263881 Human AST SimpleStep ELISA Kit 25

 200

Human AST (ng/mL) 150

100

50

0
1 2 3 4 5 6 7 8 9 10
Donor #

Figure 7. Serum from ten individual healthy human male donors was
measured in duplicate. Interpolated dilution factor corrected values are
plotted (mean +/- SD, n=2). The mean AST concentration was determined to
be 58 ng/mL with a range of 27 – 136 ng/mL.

ab263881 Human AST SimpleStep ELISA Kit 26

17. Assay Specificity

This kit recognizes both native and recombinant human AST protein
in serum, plasma, cell culture supernatant, cell extract and tissue
extract samples only.

18. Species Reactivity

This kit recognizes human AST protein.

Please contact our Technical Support team for more information.

ab263881 Human AST SimpleStep ELISA Kit 27

19. Troubleshooting

Problem Reason Solution

Difficulty Prepare 1X Cell Extraction Buffer
Genomic DNA
pipetting lysate; PTR (without enhancer). Add
solubilized
viscous lysate. enhancer to lysate after extraction.
Inaccurate
Check pipettes
Pipetting
Poor standard Prior to opening, briefly spin the
curve Improper standard stock standard tube and dissolve
dilution the powder thoroughly by gentle
mixing
Ensure sufficient incubation times;
Incubation times
increase to 2 or 3 hour
too brief
standard/sample incubation
Inadequate
Check pipettes and ensure correct
Low Signal reagent volumes or
preparation
improper dilution
Ensure sufficient incubation time
Incubation times
until blue color develops prior
with TMB too brief
addition of Stop solution
Review manual for proper wash
Plate is insufficiently
technique. If using a plate washer,
washed
Large CV check all ports for obstructions.
Contaminated
Prepare fresh wash buffer
wash buffer
Store your reconstituted standards
at -80°C, all other assay
Improper storage of
Low sensitivity components 4°C. Keep TMB
the ELISA kit
Development Solution protected
from light.
Precipitation and/or
Precipitate in coagulation of Precipitate can be removed by
Diluent components within gently warming the Diluent to 37ºC.
the Diluent.

ab263881 Human AST SimpleStep ELISA Kit 28

20. Notes

ab263881 Human AST SimpleStep ELISA Kit 29

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