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.

1. 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.

1. Reagents & Materials Provided

1. Materials & Equipment Required But Not Provided
2. 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.
3. Pipettors – 50 μL and 100 μL
4. Repeating or multichannel pipettor – 100 μL
5. Incubator, 37°C
6. Clean absorbent pad

1. Precautions
2. It is not intended for diagnostic use in human or animals.
3. 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.
4. This kit should only be used by qualified technicians.

1. 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.

1. 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

1. Specificity/Cross-Reactivity
2. 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.
3. 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. 

1. 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.

1. 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.

1. 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.

1. 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.

1. Assay Time

Incubation time: 1 h 45 min to 2 hours

Total assay time: approximately 2 to 2.5 hours

1. Notes for samples
2. 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.
3. The supernatants, centrifuged of cell culture samples can be used for Carboxypeptidase B determination.
4. It is recommended to test the sample matrix for interference, when it is necessary.
5. 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
6. Avoid the assay of samples containing sodium azide (NaN₃) , which will destroy the HRP activity of the conjugate and could result in lower readings.

1. Procedural Notes
2. All standards, controls and samples should be assayed in duplicate. Maintain a repetitive timing sequence from well to well for all assay steps to insure that all incubation times are the same for each well.
3. Completely washing of the plates to remove excess unreacted reagents is essential to good assay reproducibility and sensitivity. The manual wash procedure described below generally provides lower backgrounds, higher specific absorbance, and better precision. If duplicate CVs are poor, or if the absorbance of the 0 ng/mL standard is greater than 0.200, evaluate plate washing procedure for proper performance.
4. If the substrate has a distinct blue color prior to assay, it may have been contaminated. If the absorbance of 100 μL of substrate plus 100 μL of stop solution against a water blank is greater than 0.1, it may be necessary to obtain new substrate or the sensitivity of the assay may be compromised. Strips should be read within 30 minutes after adding stop solution, since the color will fade over time.
5. Do not mix or use components from other lots (except for washing buffer and stop solutions).

1. Preparation of Reagents
   Bring all reagents to room temperature.
2. Working Diluents for Standard, Samples & HRP-Ab (P1)

With deionized or distilled water in a ratio of 1:25, bring Diluents Concentrate (P1, 25×) for Standard, Samples & HRP-Ab to working Diluents.

1. Wash Buffer (P2)

Dilute Wash Concentrate (P2, 25×) with deionized or distilled water in the proportion of 1:25 to make working Wash Buffer. If crystals have formed in the concentrate, warm it in 40°C water bath and mix it gently, until the crystals have completely dissolved.

1. Working solution for standard curve.
2. Briefly centrifuge (1,000×g for 1 min) the Carboxypeptidase B standard lyophilizates and carefully reconstituted it with 1 mL standard & sample diluents. The final concentration of the reconstitution is 32 ng/mL.
3. Doubling dilution
4. Liquate Standard & Sample Diluents to seven EP tubes, 500 μL of each.
5. Pipette 500 μL of the 32 ng/mL solution to the first tube and mix up to produce a 16 ng/mL working solution. Pipette 500 μL solutions from former tube to the latter one in order, accordingly to make serial concentrations (32,16,8,4,2,1,0.5 and 0 ng/ml) as working solution for the standard curve. Be aware that do not pipette solution from the former tube to the last tube, as it is regarded as blank control.
6. Working solution for Antibody-HRP Conjugate (A2)

Dilute the Concentrated HRP Conjugate (A2,125×) in a proportion of 1:125 with P1. Based on the required amount (100 μL/well), extra amount of 100~200 μL is recommended.

1. TMB Substrate (A3)

Dilute TMB substrate (A3, 20×) with P3 in a ratio of 1:20 to make a working solution (A3).

1. Assay Protocol
2. Pipette 100 μL of standards, controls and samples into the bottom of wells and avoid touching the inside wall and foaming as possible. Cover microplate with plate sealer. Then incubate at 37°C for 60 minutes.
3. Aspirate or decant the solution from each well. Fill wells generously to overflowing with diluted wash solution using a squirt bottle or by pipetting in~350 μL. Soak for 1~2 minutes and aspirate or decant the solution and pat it.

Dry against clean absorbent paper. Repeat this wash step 3 times.

Note:

• A microplate washer can be used in this step and other wash steps.
• Blot and gently but firmly tap over absorbent paper to remove most of the residual liquid. It is not necessary in an attempt to remove all residual liquid and may cause variable dissociation of antibody bound material resulting in lower ODs and worse precision.
• Pipette 100 μL of anti-rCarboxypeptidase B-HRP into each well. Cover & incubate 37°C for 30 minutes.
• Dump contents of wells. Repeat for a total of 5 washes with Wash buffer. Wipe off any liquid from the bottom outside of the micro titer wells, as any residue can interfere in the reading step. Do not allow wells to dry before adding substrate.
• Pipette 100 μL of TMB substrate.
• Incubate at room temperature for 10-30 minutes. DO NOT SHAKE.
• Pipette 100 μL of Stop Solution.
• Read absorbance at 450/650 nm.

1. Calculation of Results
2. Average the duplicate readings for each standard and samples, then subtract the average zero standard optical density.
3. The standards may be used to construct a standard curve with values reported in ng/mL. This data reduction may be performed through computer methods using curve-fitting routines, such as 4-parameter logistic fit or 5-parameter logistic fit. Do not use linear regression analysis to interpolate values for samples as this may lead to significant inaccuracies!
4. Absorbances of samples are then interpolated from the standard curve. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. If the OD of the sample surpasses the upper limit of the standard curve, you should re-test it, after appropriate dilution.
5. Typical data As the OD values of the standard curve may vary, according to the conditions of actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish standard curve for each test.
6. Typical standard curve and data below is provided for reference only.

Experimental Data