1. Intended use

This  ELISA kit is intended for use in quantitating Trypsin, recombinant from  porcine pancreas. The kit is for research use only and is not intended  for diagnostic use in human or animals.

2. Principle of the Procedure

This  Trypsin assay is a two-site immunoenzymtric assay.  The recombinant  trypsin from porcine pancreas, expressed in E.Coli, from YAXINBIO, in  the samples are reacted in microtiter wells coated with a purified  capture antibody. An anti-trypsin monoclonal antibody conjugated with  the enzyme horseradish peroxidase (HRP) is reacted simultaneously  forming a sandwich complex of solid phase antibody-trypsin-HRP labeled  antibody. After a wash step to remove any unbound reactants, the wells  are then reacted with tetramethylbenzidine (TMB) substrate. The amount  of hydrolyzed substrate is read on a microtiter plate reader and will be  directly proportional to the concentration of recombinant trypsin  present in the samples. Accurate quantitation is achieved by comparing the signal of recombinant trypsin standards assayed at the same time.

3. Reagents & Materials Provided

4. Materials & Equipment Required But Not Provided

1. Microtiter  plate reader spectrophotometer with dual wave length capability at 450  & 650 nm. If your plate reader does not provide dual wave length  analysis, you may read at just the 450 nm wave length.

2. Pipettors – 50 μL and 100 μL

3. Repeating or multichannel pipette – 100 μL

4. Incubator, 37°C

5. Clean absorbent pad

5. Precautions

1. It is not intended for diagnostic use in human or animals.

2. Stop reagent is 0.5M H₂SO₄.  Avoid contact with eyes, skin and clothing. At the concentrations used  in this kit, none of the other reagents are believed to be harmful.

3. This kit should only be used by qualified technicians.

6. Performance Characteristics

YAXINBIO has validated the assay by conventional criteria as indicated below.

1. Sensitivity

The  lower limit of detection (LOD) is defined as that concentration  corresponding to a signal two standard deviations above the mean of the  zero standard. LOD is ~11 pg/mL in the recommended protocol. The lower  limit of quantitation (LOQ) is defined as the lowest concentration,  where concentration coefficients of variation (CVs) are <20%. The LOQ  is ~156 pg/mL.

2. Precision

Precision  is defined as the percent coefficient of variation (%CV). This is  calculated by dividing the standard deviation by the mean value for a  number of replicate determinations of two different control samples in  the low and high concentration range of the assay. Both intra-assay and  inter-assay precision were determined on 2 pools with low (~1.25 ng/mL)  and high concentrations (~10 ng/mL).

Intra-assay

Inter-assay

3. Specificity/Cross-Reactivity

• This  ELISA kit is intended to detect Trypsin, recombinant from porcine  pancreas. It is suggested that the compatibility of the kit calibration  be conducted, if it is used for other type of trypsin.

• The  following materials were tested for cross reactivity at the  concentrations indicated both in the absence of recombinant trypsin and  in the presence of 5 ng/mL recombinant trypsin.  None of these materials  were found to yield any statistically significant false increase or  decrease in apparent recombinant trypsin concentrations. It is  recommended that each user test known materials in their sample matrices  for cross reactivity in a similar experiment, when it is necessary.

7. Materials not cross reactive for recombinant trypsin

1. Limitations

Trypsin  from other suppliers other than YAXINBIO may show a different  reactivity with regard to sensitivity and accuracy. Therefore the  compatibility of the kit calibration to the individual trypsin product  must be verified.

2. Matrix interference

Although  the assay is designed to minimize matrix interference, certain sample  matrices may interfere in this assay. Materials, such as detergents and  salt in high concentration, extremes of pH (<6.0 and >8.5) or very  high protein concentrations may give erroneous results. It is  recommended to test the sample matrix for interference by diluting the  10 ng/mL standard to analyze the recovery.

3. Hook Capacity

High Dose Hook Effect may be observed in samples

with  very high concentrations of recombinant trypsin. The readings of  samples greater than 10 ng/mL (80ng,100ng,500ug,1mg/mL) may give  absorbencies less than the 40ng/mL standard. It is highly recommended  that samples should be assayed by diluted, when their readings were ≧10  ng/ml.

8. Quality Control

Precision  on duplicate samples should yield average coefficients of variation of  less than 10% for samples greater than 1 ng/mL. CVs for samples <1  ng/mL may be greater than 10%. It is recommended that each laboratory  assay appropriate quality control samples in each run to insure that all  reagents and procedures are correct.

9. Assay Time

Incubation time: 1 h 45 min to 2 hours

Total assay time: approximately 2 to 2.5 hours

10. Notes for samples

1. Samples  should be assayed within 7 days, when stored at 4°C, or divided and  stored at -20°C (≤1 month) or -80°C (≤3 months). Avoid repeated  freeze-thaw cycles.

2. The supernatants, centrifuged of