[ INTENDED USE ]

The Chemiluminescent Immunoassay kit is designed for the in vitro sensitive quantitative measurement of SAA in rabbit serum, plasma and other biological fluids.

[ REAGENTS AND MATERIALS PROVIDED ]

[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1. Luminometer capable of reading 96-well microplates with the following parameters:

  lag time 30.0secs; read time 1.0 sec/well .

2. Precision single or multi-channel pipettes and pipette tips with disposable tips.

3. Eppendorf Tubes for diluting samples.

4. Deionized or distilled water.

5. Absorbent paper for blotting the microtiter plate.

6. Container for Wash Solution

[ STORAGE OF THE KITS ]

1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20^oC upon receipt while the others should be at 4 ^oC.

2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.

Note:

It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit.

[ SAMPLE COLLECTION AND STORAGE ]

Serum - Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4^oC before centrifugation for 20 minutes at approximay 1000×g. Assay freshly prepared serum immediay or store samples in aliquot at -20^oC or -80^oC for later use. Avoid repeated freeze/thaw cycles.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8^oC within 30 minutes of collection. Remove plasma and assay immediay or store samples in aliquot at -20^oC or -80^oC for later use. Avoid repeated freeze/thaw cycles.

Other biological fluids - Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediay or store samples in aliquot at -20^oC or -80^oC for later use. Avoid repeated freeze/thaw cycles.

Note:

1.  Samples to be used within 5 days may be stored at 4^oC, otherwise samples must be stored at -20^oC （≤1 month） or -80^oC （≤2 months） to avoid loss of bioactivity and contamination.

2.  Sample hemolysis will influence the result, so hemolytic specimen can not be detected.

3.  When performing the assay, bring samples to room temperature.

[ TEST PRINCIPLE ]

The microtiter plate provided in this kit has been pre-coated with an antibody specific to SAA. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for SAA. Next, Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. Then the mixture of substrate A and B is added to generate glow light emission kinetics. Upon plate development, the intensity of the emitted light is proportional to the SAA level in the sample or standard.

[ ASSAY PROCEDURE SUMMARY ]

   1. Prepare all reagents, samples and standards;

   2. Add 100μL standard or sample to each well. Incubate 2 hours at 37^oC;

   3. Add 100μL prepared Detection Reagent A. Incubate 1 hour at 37^oC;

   4. Aspirate and wash 3 times;

   5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37^oC;

   6. Aspirate and wash 5 times;

   7. Add 100μL Substrate Solution. Incubate 5-10 minutes at 37^oC;

8. Read RLU value immediay.

[ CALCULATION OF RESULTS ]

Average the duplicate readings for each standard, control, and samples and subtract the average zero standard relative light unit （RLU）. Create a standard curve on log-log graph paper, with SAA concentration on the y-axis and the RLU value on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, such as curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

[ SENSITIVITY ]

The minimum detectable dose of rabbit SAA is typically less than 10. 2ng/mL.

The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined as the lowest protein concentration that could be differentiated from zero. It was determined the mean RLU value of 20 replicates of the zero standard added by their three standard deviations.

[ SPECIFICITY ]

This assay has high sensitivity and excellent specificity for detection of rabbit SAA.

No significant cross-reactivity or interference between rabbit SAA and analogues was observed.

Note:

Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between rabbit SAA and all the analogues, therefore, cross reaction may still exist.

[ RECOVERY ]

Matrices listed below were spiked with certain level of recombinant rabbit SAA and the recovery rates were calculated by comparing the measured value to the expected amount of SAA in samples.

[ LINEARITY ]

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of rabbit SAA and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

[ PRECISION ]

Intra-assay Precision （Precision within an assay）: 3 samples with low, middle and high level rabbit SAA were tested 20 times on one plate, respectively.

Inter-assay Precision （Precision between assays）: 3 samples with low, middle and high level rabbit SAA were tested on 3 different plates, 8 replicates in each plate.

CV（%） = SD/meanX100

Intra-Assay: CV<10%

Inter-Assay: CV<12%

[ STABILITY ]

The stability of CLIA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.

The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37^oC for 3 days, and compare RLU values of the kit kept at 37^oC with that of at recommended temperature. （referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For CLIA kit, 1 day storage at 37^oC can be considered as 2 months at 4^oC, which means 3 days at 37^oC equaling 6 months at 4^oC）.

Note:

To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

flb <h]s ??s > Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediay or store samples in aliquot at -20^oC or -80^oC for later use. Avoid repeated freeze/thaw cycles.

Note:

1.  Samples to be used within 5 days may be stored at 4^oC, otherwise samples must be stored at -20^oC （≤1 month） or -80^oC （≤2 months） to avoid loss of bioactivity and contamination.

2.  Sample hemolysis will influence the result, so hemolytic specimen can not be detected.

3.  When performing the assay, bring samples to room temperature.