Benzo(a)pyrene Ab (Mouse) ELISA Kit

Catalog Number KA1490 96 assays Version: 02

Intended for research use only

www.abnova.com

Table of Contents

Introduction ...... 3

Intended Use ...... 3

Principle of the Assay ...... 3

General Information ...... 5

Materials Supplied ...... 5

Storage Instruction ...... 5

Materials Required but Not Supplied ...... 5

Precautions for Use ...... 6

Assay Protocol ...... 7

Reagent Preparation ...... 7

Assay Procedure ...... 7

Data Analysis...... 9

Calculation of Results ...... 9

Performance Characteristics ...... 9

Resources ...... 10

Plate Layout ...... 10

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Introduction

Intended Use

Benzo(a)pyrene Ab (Mouse) ELISA Kit is intended for qualitative or quantitative detection of anti-Benzo(a)pyrene IgG and IgM antibodies in mouse serum. The test does not differentiate the IgG and IgM type of antibodies.

Principle of the Assay

Benzo(a)pyrene Ab (Mouse) ELISA Kit is a solid-phase immunoanalytical test. Microtiter wells are coated with specific antigen. If there are antibodies to Benzo(a)Pyrene present in the serum samples, they will bind to the immobilised antigen. The bound antibodies react in the next step with animal anti- mouse IgG+IgM antibody labelled with horseradish peroxidase. The amount of the bound labeled antibodies is determined by enzymatic reaction with a chromogenic substrate. Negative sera do not react, a mild change in colour, if present, is attributed to the reaction background.

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Flow Chart Step 1. Prepare reagents and samples, dilute it ↓ Step 2. Add 100 μl/well of standards and serum samples ↓ Incubate 60 min at room temperature ↓ Wash 5 times (250 μL/well), aspirate ↓ Step 3. Add 100 μl/well of Px-conjugate ↓ Incubate 60 min at room temperature ↓ Wash 5 times (250 μL/well), aspirate ↓ Step 4. Add 100 μl/well TMB substrate ↓ Incubate 10 min at room temperature, dark ↓ Step 5. Add 100 μl/well of Stop solution

↓ Step 6. Read optical density for 450 nm (ref. 620-690 nm) till 10 minutes

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General Information

Materials Supplied

List of component

Component Amount 8-well double-strips coated with the antigen (colourless) within a plastic frame, STRIPS Ag 1 microplate Standard A (conc. Of anti-B(a)P=1 μg/ml), r.t.u.* 1.3 mL Standard B (conc. Of anti-B(a)P=0.5 μg/ml), r.t.u.* 1.3 mL Standard C (conc. Of anti-B(a)P=0.1 μg/ml), r.t.u.* 1.3 mL Standard D (conc. Of anti-B(a)P=0.05 μg/ml), r.t.u.* 1.3 mL Standard E (conc. Of anti-B(a)P=0.025 μg/ml), r.t.u.* 1.3 mL Standard F (conc. Of anti-B(a)P=0.01 μg/ml), r.t.u.* 1.3 mL Standard G (conc. Of anti-B(a)P=0.001 μg/ml), r.t.u.* 1.3 mL Px-conjugate concentrate (animal anti-mouse IgG+IgM antibodies labelled with 0.2 mL horseraddish peroxidase), 101x concentrated, CONJ 101x Wash buffer concentrate, 10x concentrated, WASH 10x 125 mL Dilution buffer, DIL, r.t.u.* 100 mL Chromogenic substrate , TMB, r.t.u.* 13 mL Stop solution, STOP, r.t.u.* 13 mL Sealable pouch for unused strips 1 piece * (ready to use)

Storage Instruction

 Store the kit reagents at +2 to +10°C, in a dry place and protected from the light. Avoid freezing. Expiration date is indicated at the ELISA kit label and at all reagent labels.  Store unused strips in the sealable pouch and keep the desiccant inside. Transport in thermo bags until 72 hours. Any damages of packaging of kit reagents advise to the producer without delay.  Do not store diluted samples and diluted Px conjugate. Always prepare fresh.

Materials Required but Not Supplied

 Distilled or deionised water for dilution of the Wash buffer concentrate.  Appropriate equipment for pipetting, liquid dispensing and washing.  Spectrophotometer/colorimeter (microplate reader – wavelength 450 nm).

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Precautions for Use

 Safety Precautions  All ingredients of the kit are intended for laboratory use only.  Mouse serum samples handle as with hazardous waste. However they should be regarded as contagious and handled and disposed of according to the appropriate regulations. Autoclave all reusable materials that were in contact with mouse serum samples for 1 hour at 121°C or decontaminate with 3% chloramines for 30 minutes.  Liquid wastes containing acid (Stop solution) should be neutralized in 4% sodium bicarbonate solution. Handle Stop solution with care. Avoid contact with skin or mucous membranes. In case of contact with skin, rinse immediately with plenty of water and seek medical advice.  Do not smoke, eat or drink during work. Do not pipette by mouth. Wear disposable gloves while handling reagents or samples and wash your hands thoroughly afterwards. Avoid spilling or producing aerosol.

 Handling Precautions  Avoid contamination of samples and kit reagents.  Avoid cross-contamination of reagents.  Standards, dilution buffer (DIL) and chromogenic substrate (TMB substrate) contain preservative ProClin 300® .  Avoid contact of the TMB substrate with oxidizing agents or metal surfaces.  Follow the assay procedure indicated in the Instruction manual.  Variations in test results are usually due to: Insufficient mixing of reagents and samples Inaccurate pipetting and inadequate incubation times Poor washing technique or spilling the rim of well with sample or Anti- mouse IgG+IgM Px-conjugate Use of identical pipette tip for different solutions

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Assay Protocol

Reagent Preparation

 Allow all kit components to reach room temperature.  Vortex serum samples and standards in order to ensure homogeneity and mix all solution well prior use. Dilute serum samples 1:101 in dilution buffer (DIL) – e.g. 5 μL of serum sample + 500 μL of dilution buffer (DIL). Do not dilute Standards – they are ready to use (r.t.u.).  Prepare Wash buffer by diluting the concentrate 10 times with an appropriate volume of distilled or deionised water (100 mL of the concentrated Wash buffer WASH 10x + 900 mL of distilled water). If there are crystals of salt present in the concentrated Wash buffer, warm up the vial to 32 to 37°C in a water bath. Diluted Wash buffer is stable for one week if stored at 2 to 10°C.  Dilute concentrated Anti-mouse IgG+IgM Px-conjugate CONJ 101x 1:101 with Dilution buffer (DIL) – e.g. 0.12 mL of Px-conjugate + 12 mL of Dilution buffer (DIL). (Note: For one microtitre plate you will need approx. 12 mL of diluted Px-conjugate.)  Do not dilute TMB substrate and Stop solution, they are ready to use.

Assay Procedure

1. Allow the antibody coated strips to reach room temperature before opening in order to prevent water condensation within the wells. Withdraw an adequate number of antibody coated strips (colourless). Put the remaining strips back in the aluminium pouches and seal them if possible, keep the desiccant inside. 2. Pipette 100 μL of standards and diluted serum samples to the wells according to the Plate Layout: Start with filling the first well with Dilution buffer (DIL) to estimate the reaction background, fill the next seven wells with Standards (ST1,ST2,...ST7). Fill the remaining wells with the diluted serum samples (S1, S2, S3,...). It is satisfactory to apply samples as singlets, however, if you want to minimise the laboratory error then apply the control serums and samples in doublets. 3. Incubate for 60 ( +/- 5) minutes at room temperature. 4. Aspirate the liquid from the wells into a collecting bottle containing appropriate disinfectant (see Safety Precautions). Wash and aspirate the wells five times with 250 μl/well of Wash buffer. Avoid cross-contamination between wells! If some liquid remains in the wells, invert the plate and tap it on an adsorbent paper to remove the last remaining drops. 5. Add 100 μL of the diluted Anti-mouse IgG+IgM Px-conjugate into each well. 6. Incubate for 60 ( +/- 5) minutes at room temperature. 7. Aspirate and wash the wells five times with 250 μl/well of Wash buffer as in step “4”. 8. Dispense 100 μL of the TMB substrate into each well. Pipette in a regular rhythm or use an appropriate dispensing instrument. 9. Incubate for 10 minutes (+/-5 seconds) at room temperature. The time measurement must be started at the beginning of TMB dispensing. Cover the strips with an aluminium foil or keep them in the dark during

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the incubation with TMB substrate. 10. Stop the reaction by adding 100 μL of Stop solution. Use the same pipetting rhythm as with the TMB substrate to ensure the same reaction time in all wells. Tap gently the microplate for a few times to ensure complete mixing of the reagents. 11. Read the absorbance at 450 nm with a microplate reader within 10 minutes. It is recommended to use reference reading at 620-690 nm.

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Data Analysis

Calculation of Results

 Calculate the concentration for each serum sample:  First subtract the absorbance of the background (absorbance of the DIL well) from the absorbancies of all other wells.  Construct the calibration curve by plotting the absorbance (Y) of Standards versus the known concentration (X) of standards.  Calculate the concentration of anti-B(a)P antibodies by equation of calibration curve. Use the value of serum sample absorbance (OD) instead of the value y. The values x represent the concentration of anti-B(a)P antibodies in serum samples. Note that you need to use the diluting factor in the calculation (i.e. if you dilute sample 100 times, you have to multiply the final concentration of anti-B(a)P antibodies by 100).

Performance Characteristics

 Validity of the test The test is valid if:  The mean value of Standard A (STA) absorbance is higher than 1.800.  The mean value of DIL absorbance is less than 0.050.  The mean values of absorbencies of Standards (STA,STB,...STG) can be lined up as follows: ST G < ST F < ST E < ST D < ST C < ST B < ST A

 Precision of the test The intraassay variability (within the test) and the interassay variability (between tests) evaluation was performed with samples of variable absorbance values.  Intraassay variability (N = number of parallels): N Absorbance SD CV 10 0.570 0.022 3.8% 10 1.433 0.062 4.3%  Interassay variability (N = number of paralells): N Absorbance SD Range (min – max) CV% 20 0.123 0.013 0.106-0.147 10.2 16 0.270 0.026 0.229-0.305 9.8 16 0.496 0.042 0.432-0.567 8.5

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Resources

Plate Layout

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1 2 3 …

ample ample ample ample

2 S S S S

IL

1 D A Standard B Standard C Standard D Standard E Standard F Standard G Standard

A B C D E F G H

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