1

Human Serum Amyloid A (SAA) ELISA
Cat. No.: BA1006
Enzyme Immunoassay for the quantitative determination of Serum amyloid
A (SAA) in human serum, plasma, and other body fluids.
Serum amyloid A (SAA) is a highly sensitive acute phase reactant that has
been linked to inflammatory diseases, both infectious and non-infectious
origin. SAA is an apolipoprotein that interacts with HDL, promotes theaccumulation of leukocytes at the site of inflammation and the adhesion ofplatelets, and participates in the removal of damaged cell membranes. The
acute phase SAAs (SAA1, SAA2) are transcriptionally regulated inhepatocytes by a variety of inflammatory cytokines, and they can transiently
increase > 1000-fold. The combination of SAA, PCT, and CRP can improvethe differential diagnosis of early bacterial and viral infections. Several
studies indicated that SAA allows monitoring of disease activity in various
inflammatory rheumatic diseases and autoinflammatory diseases, including
SLE, amyloidosis, or rheumatoid arthritis. SAA can be also used to risk
prediction of coronary heart disease, the prognosis of tumour patients, andobservation of transplant rejection.

Kit Contents
Item
Antibody Coated Microtiter Plate
Biotin-labelled Antibody 100x conc.
Ab-bt diluent
Streptavidin-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer 2x conc.
Wash Buffer 15x conc.
Substrate Solution
STOP Solution
MATERIAL REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.

PRINCIPLE OF SAA ELISA
The microtiter plate is coated with the antibody specifically binding theSerum amyloid A (SAA1 and SAA2). The human serum or plasma is
incubated in the plate with the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith biotin-labelled detection antibody. Following another washing step,
Streptavidin-HRP conjugate is added into the well.
Unbound reagent is then washed out. Horseradish peroxidase (HRP) boundin the complex reacts with the chromogenic substrate (TMB) creating theblue colour. The reaction is stopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of SAA in the specimen. The
concentration of SAA in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Qty.
96 wells
140 µL
13 mL
13 mL
1 vial1 vial1 vial2x13 mL
50 mL
13 mL
13 mL

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS

Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human SAA Master Standard in Dilution Buffer, forthe volume information see the Certificate of Analysis. Let it rehydrate for
15 min. The concentration of human SAA in Master Standard is
200 ng/mL.
Prepare set of Standard solution as follows:
Use the Master Standard to produce a dilution series (as below). Mix eachtube thoroughly before the next transfer. The Dilution Buffer serves asBlank.
Volume of Standard

v_01

Std1

Standard 200 ng/mL (lyophilised)

Dilution
BufferSee CofA

Std2300 µL of Std1300 µL

100 ng/mL

Std3300 µL of Std2300 µL

50 ng/mL

Std4
Std5300 µL of Std3300 µL of Std4300 µL
300 µL

25 ng/mL
12.5 ng/mL

Std6Blank

300 µL of Std5300 µL
300 µL

6.25 ng/mL
0 ng/mL-Concentration
200 ng/mL

2Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls indeionized/distilled, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:50 in Dilution
Buffer, prior to use, see Preparation of samples.
Preparation of Wash Buffer 1x
Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at
4oC for two weeks or at -20oC for long term storage.

1.

Sensitivity

The limit of detection, defined as a concentration of human SAA giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 1.56 ng/mL of sample.
2.

Precision

Intra-assay
Sample12

Mean (µg/ml)
15.0
0.9SD
1.4
0.1

CV (%)
910SD
0.2
0.04

CV (%)
43Preparation of Dilution Buffer 1x
Prepare a working solution of Dilution Buffer by mixing 13 mL (26 mL) ofDilution Buffer 2x conc. and 13 mL (26 mL) of deionized/ distilled water(dH2O). Prepare only the amount for immediate consumption. Mix well.
Store at 4oC for two weeks.

Inter-assay (Run – to – run)
Sample12

Mean (µg/ml)
4.0
1.2

Preparation of Biotin-Labelled Antibody 1x
Prepare a working solution of Biotin-labelled Antibody by mixing 130 µL ofBiotin-labelled Antibody 100x conc. and 12.870 mL of Ab-bt Diluent in thespecial tube. Mix well. Store at 4oC for two weeks.

3.

Accuracy

Dilution linearity
SampleDilution

Measuredconcentration(µg/ml)
4.6
2.4
1.4
0.7
10.8
5.3
2.8
1.6

Expected
concentration(µg/ml)
2.3
1.1
0.6
5.4
2.7
1.4

Yield(%)

Measuredconcentration(µg/ml)
1.1
3.2
2.2
1.7

Expectedconcentration(µg/ml)
3.6
2.3
1.7

Yield(%)

Preparation of samples
Human serum or plasma may be used with this assay. For long-term storage
the samples should be frozen at minimum -70oC. Lipemic or haemolytic
samples may cause false results.

1

Recommended dilution of healthy individual samples is 1:50, i.e., 5 µL ofsample + 245 µL of Dilution Buffer, for duplicates and for singlets.

2

Recommended dilution of samples is 1: 2000 for individuals in condition inwhich is expected higher level of SAA5. It is recommended to use the twostep dilution.
Dilution A (40x) for both singlets and duplicates: 5 µL of samples + 195 µL ofDilution Buffer.
Dilution B (50x): 5 µL of Dilution A + 245 µL of Dilution Buffer, for singlets;
15 µL of Dilution A + 285 µL of Dilution Buffer, for duplicates.
Recommended dilution of urine is 1:10, i.e., 20 of samples + 180 µL ofDilution Buffer, for singlets, 30 of samples + 270 µL of Dilution Buffer, forduplicates.
Do not store the diluted samples.
ASSAY PROCEDURE
1.
2.

Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate for
1 hour at 25oC ± 2oC, shaking at 300 rpm.
3. Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
4. Pipette 100 µL of Biotin-labelled Antibody into each well.
Incubate for 1 hour at 25oC ± 2oC, shaking at 300 rpm.
5. Wash the wells as described in point 3.
6. Pipette 100 µL of Streptavidin-HRP into each well. Incubate for
30 min at 25oC ± 2oC, shaking at 300 rpm.
7. Wash the wells as described in point 3.
8. Pipette 100 µL Substrate solution, incubate for 15 min,at 25oC ± 2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450/630 nm within 15 min.

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:50 as recommended and assayed.
The results are multiplied by dilution factor.
v_01

2x4x8x2x4x8x103
119
117
98
104
120

Spiking RecoverySample

Spike(ng /mL)

12.5
1.3
0.6

8994102

Typical standard curve
The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

3

Human SAA Standard Curve
43.5

Absorbance (450/630 nm)

32.5
21.5
10.5
06.25

12.5

25

50

100

200

Concentration (ng/mL)

RESOURCES
1Levin M, Franklin EC, Frangione B, Pras M. The amino acid sequence of a major

nonimmunoglobulin component of some amyloid fibrils. J Clin Invest. 1972;51:2773–
6. https://doi.org/10.1172/ JCI107098. 3. Linke RP, Sipe JD, Pollock PS, Ignaczak TF,Glenner GG. Isolation of a low-molecular-weight serum component antigenicallyrelated to an amyloid fibril protein of unknown origin. Proc Natl Acad Sci U S A.
1975;72:1473–6. https://doi.org/10.1073/pnas.72. 4.1473.
2Johnson BD, Kip KE, Marroquin OC, Ridker PM, Kelsey SF, Shaw LJ, et al. Serum amyloid
A as a predictor of coronary artery disease and cardiovascular outcome in women: theNational Heart, Lung, and Blood Institute-Sponsored Women’s Ischemia Syndrome
Evaluation(WISE).
Circulation.
2004;109:726–32.
https://doi.org/10.1161/01.CIR.0000115516.54550.B1. 19. Kosuge M, Ebina T,
Ishikawa T, Hibi K, Tsukahara K, Okuda J, et al. Serum amyloid A is a better predictor ofclinical outcomes than C-reactive protein in non-ST-segment elevation acute coronarysyndromes. Circ J. 2007;71:186–90. https://doi.org/10.1253/circj. 71.186. 20. Deguchi
H, Elias DJ, Navarro S, Espana F, Griffin JH. Elevated serum amyloid A is associated withvenous
thromboembolism.
Thromb
Haemost.
2013;109:358–9.
https://doi.org/10.1160/ TH12-10-0722.
3Saiki O, Uda H. Ratio of serum amyloid A to C-reactive protein is constant in the same
patients but differs greatly between patients with inflammatory diseases. Scand JImmunol. 2022 Feb;95(2):e13121. doi: 10.1111/sji.13121. Epub 2021 Nov 24. PMID:34796986.

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