Intended use

This  ELISA kit is intended for use in quantitating Carboxypeptidase B,  recombinant from Rattus norvegicus. The kit is for research use only and  is not intended for diagnostic use in human or animals.

2. Principle of the Procedure

This  Carboxypeptidase B assay is a two-site immuno enzymetric assay. The  recombinant Carboxypeptidase B from Rattus norvegicus, expressed in  E.Coli, from YAXINBIO, in the samples are reacted in micro titer wells  coated with a purified capture antibody. An anti-Carboxypeptidase B  monoclonal antibody conjugated with the enzyme horseradish peroxidase  (HRP) is reacted simultaneously forming a sandwich complex of solid  phase antibody-Carboxypeptidase B-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 Carboxypeptidase B present in the  samples. Accurate quantitation is achieved by comparing the signal of  recombinant Carboxypeptidase B 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 wavelength.

2. Pipettors – 50 μL and 100 μL

3. Repeating or multichannel pipettor – 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 ~141 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 ~250 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 (~2.0 ng/mL)  and high concentrations (~16.0 ng/mL).

Intra-assay

Inter-assay

3. Specificity/Cross-Reactivity

• This  ELISA kit is intended to detect Carboxypeptidase B, recombinant from  Rattus norvegicus, It is suggested that the compatibility of the kit  calibration be conducted, if it is used for other type of  Carboxypeptidase B.

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

7. Materials not cross reactive for recombinant Carboxypeptidase B

1. Limitations

Carboxypeptidase  B 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 Carboxypeptidase B  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 Carboxypeptidase B. The  readings of samples greater than 32 ng/mL (80 ng,100 ng,500 ug/mL) may  give absorbance less than 60 ng/mL standard. It is highly recommended  that samples should be assayed by diluted, when their readings were ≧32  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 cell culture samples can be used for Carboxypeptidase B determination.

3. It is recommended to test the sample matrix for interference, when it is necessary.

4. It  is recommended to dilute the samples (e.g., 1:20 to1:100) with sample  diluents, which Carboxypeptidase B concentration of approximately >32  ng/ml are expected in the samples