Abcam EIA-6132 Bordetella Pertussis Toxin (PT) IgA ELISA Kit Instruction Manual

Abcam EIA-6132 Bordetella Pertussis Toxin (PT) IgA ELISA Kit

Product Information

Specifications

• Product Name: Bordetella pertussis Toxin (PT) IgA ELISA
• Product Code: EIA-6132
• Version: 2.0
• Date: 2023-11-06

Introduction

• Bordetella pertussis is a respiratory pathogen that causes pertussis, commonly known as whooping cough. It is a localized infection of the ciliated epithelium of the bronchial tree. Pertussis is characterized by a prolonged paroxysmal cough often accompanied by an inspiratory whoop.
• In addition to B. pertussis, three other Bordetella species can cause disease in humans: B. parapertussis, B. holmesii, and B. bronchiseptica. B. parapertussis causes a pertussis-like illness that is generally milder than pertussis because the bacteria do not produce pertussis toxin. Co-infection of B. pertussis and B. parapertussis is not unusual.
• B. pertussis is of worldwide prevalence, with 20 – 40 million cases occurring each year. The majority of cases (90%) are in developing countries, and there are up to 400,000 fatalities each year, mostly in young infants.
• Transmission of B. pertussis occurs primarily through close direct contact with an infected person or inhalation of airborne droplets. Symptoms develop following inhalation of the airborne pathogen. The organism is highly contagious, with up to 90% of household contacts developing the disease. Infected persons are most contagious in the catarrhal and paroxysmal stages.
• The incubation period for pertussis is usually seven to 10 days, with a range of 4 – 21 days. Typical pertussis symptoms occur in three different stages: catarrhal, paroxysmal, and convalescent. However, atypical presentations can occur due to various factors.

Intended Use
The Bordetella pertussis Toxin (PT) IgA ELISA is intended for the quantitative determination of IgA class antibodies against B. pertussis toxin (PT) antigens in human serum or plasma (citrate, heparin).

Product Usage Instructions

Materials

• 12 break apart 8-well snap-off strips coated with B. pertussis toxin (PT) antigens; in resealable aluminium foil.
• Standard A
• Standard B
• Standard C
• Standard D

FAQ

What is the purpose of the Bordetella pertussis Toxin (PT) IgA ELISA?
The purpose of the Bordetella pertussis Toxin (PT) IgA ELISA is to quantitatively determine IgA class antibodies against B. pertussis toxin (PT) antigens in human serum or plasma.

INTRODUCTION

• Bordetella pertussis is a respiratory pathogen that causes pertussis, commonly known as whooping cough, a localized infection of the ciliated epithelium of the bronchial tree. Pertussis is characterized by a prolonged paroxysmal cough often accompanied by an inspiratory whoop.
• The disease affects mainly children, but adults have also been increasingly reported to be affected. The pathogen produces toxins which cause local damage to the cilia of epithelial cells, which leads to prolonged illness and pertussis. Disease presentation varies with age and history of previous exposure or vaccination. Severe disease is infrequent in healthy, vaccinated persons. Infants, particularly those who have not received the primary vaccination series against pertussis, are at risk for complications and mortality.
• In addition to B. pertussis, three other Bordetella species can cause disease in humans: B. parapertussis, B. holmesii, and B. bronchiseptica. B. parapertussis causes a pertussis-like illness that is generally milder than pertussis because the bacteria do not produce pertussis toxin. Co-infection of B. pertussis and B. parapertussis is not unusual.
• B. pertussis is of worldwide prevalence. Globally, 20 – 40 million cases of pertussis occur each year, 90 % of which are in developing countries, and there are up to 400 000 fatalities each year, mostly in young infants.
• Transmission of B. pertussis occurs primarily via close direct contact with an infected person or inhalation of airborne droplets. Symptoms develop following inhalation of the airborne pathogen. The organism is highly contagious, with up to 90 % of household contacts developing the disease. Infected persons are most contagious in the catarrhal and the paroxysmal stages.
• The incubation period is usually seven to 10 days, with a range of 4 – 21 days.
• Typical pertussis symptoms occur in three different stages: catarrhal, paroxysmal, and convalescent.
• The catarrhal stage lasts for about 1 – 2 weeks, and is characterized by non-specific symptoms such as rhinorrhea, sneezing, low-grade fever and cough. The second stage is the paroxysmal stage, lasting for about 4 – 6 weeks, and is characterized by various pathognomonic symptoms of pertussis such as episodes of paroxysmal cough with a characteristic whooping sound. The final stage is the convalescent stage. During this stage, the respiratory symptoms gradually decrease although cough can continue for months.
• Many factors can alter the usual course of pertussis, causing an atypical presentation. Previously vaccinated adolescents and adults may have less severe paroxysmal symptoms.
• Infection or presence of pathogen may be identified by
  • Microscopy
  • PCR
  • Serology: e. g. by ELISA

INTENDED USE

The Bordetella pertussis Toxin (PT) IgA ELISA is intended for the quantitative determination of IgA class antibodies against B. pertussis toxin (PT) antigens in human serum or plasma (citrate, heparin).

PRINCIPLE OF THE ASSAY

• The quantitative immunoenzymatic determination of specific antibodies is based on the ELISA (Enzyme-linked Immunosorbent Assay) technique.
• Microtiterplates are coated with specific antigens to bind corresponding antibodies of the sample. After washing the wells to remove all unbound sample material a horseradish peroxidase (HRP) labelled conjugate is added. This conjugate binds to the captured antibodies. In a second washing step unbound conjugate is removed. The immune complex formed by the bound conjugate is visualized by adding
• Tetramethylbenzidine (TMB) substrate which gives a blue reaction product.
• The intensity of this product is proportional to the amount of specific antibodies in the sample. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450/620 nm is read using an ELISA Microtiterplate reader.

MATERIALS

Reagents supplied

• Microtiterplate:
  12 break apart 8-well snap-off strips coated with B. pertussis toxin (PT) antigens; in resealable aluminium foil.

• IgA Sample Dilution Buffer:
  1 bottle containing 100 mL of phosphate buffer (10 mM) for sample dilution; pH 7.2 ± 0.2; coloured yellow; ready to use; white cap, ; ≤ 0.0015% (v/v) CMIT/ MIT (3:1).Stop Solution:
  1 bottle containing 15 mL sulphuric acid, 0.2 mol/L; ready to use; red cap.

• Washing Buffer (20x conc.):
  1 bottle containing 50 mL of a 20-fold concentrated phosphate buffer (0.2 M), pH 7.2 ± 0.2, for washing the wells; white cap.

• Conjugate:
  1 bottle containing 20 mL of peroxidase labelled antibody to human IgA in phosphate buffer (10 mM); coloured violet; ready to use; black cap.

• TMB Substrate Solution:
  1 bottle containing 15 mL 3,3′,5,5′-tetramethylbenzidine (TMB), < 0.1 %; ready to use; yellow cap

• Standards: 4 vials, each containing 2 mL standard; coloured yellow; ready to use; yellow cap. ≤ 0.02% (v/v) MIT.

  • Standard A : 0 IU/mL
  • Standard B: 10 IU/mL
  • Standard C: 25 IU/mL
  • Standard D: 50 IU/mL
    The standards are calibrated in accordance with the ”1st WHO International Standard Pertussis antiserum (human)”, Code 06/140, of the National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK.

• Control Low: 1 vial containing 2 mL control; coloured yellow; ready to use; blue cap. Concentration in IU/mL is indicated on the label; ≤ 0.02% (v/v) MIT.

• Control High: 1 vial containing 2 mL control; coloured yellow; ready to use; red cap. Concentration in IU/mL is indicated on the label ; ≤ 0.02% (v/v) MIT.

  • For hazard and precautionary statements see 12.1
  • For potential hazardous substances please check the safety data sheet.

Materials supplied

• 1 Cover foil
• 1 Instruction for use (IFU)
• 1 Plate layout

Materials and Equipment needed

• ELISA Microtiterplate plate reader, equipped for the measurement of absorbance at 450/620 nm
• Incubator 37 °C
• Manual or automatic equipment for rinsing Microtiterplates
• Pipettes to deliver volumes between 10 μL and 1000 μL
• Vortex tube mixer
• Distilled water
• Disposable tubes

STABILITY AND STORAGE

• Store the kit at 2 °C – 8 °C.
• The opened reagents are stable up to the expiry date stated on the label when stored at 2 °C – 8 °C.

REAGENT PREPARATION

It is very important to bring all reagents and samples to room temperature (20 °C – 25 °C) and mix them before starting the test run!

Microtiterplate

• The break-apart snap-off strips are coated with B. pertussis toxin (PT) antigen.
• Immediately after removal of the strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2 °C – 8 °C.

Washing Buffer (20x conc.)

• Dilute Washing Buffer 1 + 19; e. g. 10 mL Washing Buffer + 190 mL distilled water.
• The diluted buffer is stable for 5 days at room temperature (20 °C – 25 °C). In case crystals appear in the concentrate, warm up the solution to 37 °C e.g. in a water bath. Mix well before dilution.

TMB Substrate Solution

• The reagent is ready to use and has to be stored at 2 °C – 8 °C, away from the light.
• The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away.

SAMPLE COLLECTION AND PREPARATION

• Use human serum or plasma (citrate, heparin) samples with this assay. If the assay is performed within 5 days after sample collection, the samples should be kept at 2 °C – 8 °C; otherwise, they should be aliquoted and stored deep-frozen (-70 °C to -20 °C). If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing.
• Heat inactivation of samples is not recommended.

Sample Dilution

• Before assaying, all samples should be diluted 1+100 with IgA Sample Dilution Buffer.
• Dispense 10 μL sample and 1 mL IgA Sample Dilution Buffer into tubes to obtain a 1+100 dilution and thoroughly mix with a Vortex.

ASSAY PROCEDURE

• Please read the instruction for use carefully before performing the assay. Result reliability depends on strict adherence to the instruction for use as described. The following test procedure is only validated for manual procedure. If performing the test on ELISA automatic systems we recommend increasing the washing steps from three up to five and the volume of Washing Buffer from 300 μL to 350 μL to avoid washing effects. Pay attention to chapter 12. Prior to commencing the assay, the distribution and identification plan for all samples and standards/controls (duplicates recommended) should be carefully established on the plate layout supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder.
• Perform all assay steps in the order given and without any delays.
• A clean, disposable tip should be used for dispensing each standard/control and sample.
• Adjust the incubator to 37 °C ± 1 °C.
  1. Dispense 100 μL standards/controls and diluted samples into their respective wells. Leave well A1 for the Substrate Blank.
  2. Cover wells with the foil supplied in the kit.
  3. Incubate for 1 hour ± 5 min at 37 °C ± 1 °C.
  4. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 300 μL of Washing Buffer. Avoid overflows from the reaction wells. The interval between washing and aspiration should be > 5 sec. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! Note: Washing is important! Insufficient washing results in poor precision and false results.
  5. Dispense 100 μL Conjugate into all wells except for the Substrate Blank well A1.
  6. Incubate for 30 min at room temperature (20 °C – 25 °C). Do not expose to direct sunlight.
  7. Repeat step 4.
  8. Dispense 100 μL TMB Substrate Solution into all wells.
  9. Incubate for exactly 15 min at room temperature (20 °C – 25 °C) in the dark. A blue colour occurs due to an enzymatic reaction.
  10. Dispense 100 μL Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution, thereby a colour change from blue to yellow occurs.
  11. Measure the absorbance at 450/620 nm within 30 min after addition of the Stop Solution.

Measurement

• Adjust the ELISA Microtiterplate reader to zero using the Substrate Blank.
• If – due to technical reasons – the ELISA Microtiterplate reader cannot be adjusted to zero using the Substrate Blank, subtract its absorbance value from all other absorbance values measured in order to obtain reliable results!
• Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard/control and sample in the plate layout.
• Bichromatic measurement using a reference wavelength of 620 nm is recommended.
• Where applicable calculate the mean absorbance values of all duplicates.

RESULTS

Run Validation Criteria
In order for an assay run to be considered valid, these Instructions for Use have to be strictly followed and the following criteria must be met:

• Substrate Blank: Absorbance value < 0.100
• Standard A: Absorbance value < 0.200
• Standard B Absorbance value > Standard A
• Standard C Absorbance value > Standard B
• Standard D : Absorbance value > 1.000
• Control Low: Result in IU/mL within range indicated on the label
• Control High: Result in IU/mL within range indicated on the label
  Standard A < Standard B < Standard C < Standard D
  If these criteria are not met, the test is not valid and must be repeated.

Calculation of Results

• In order to obtain quantitative results in IU/mL plot the (mean) absorbance values of the 4 Standards A – D on (linear/Linear) graph paper in a system of coordinates against their corresponding concentrations (0, 10, 25 and 50 IU/mL) and draw a standard curve (absorbance values on the y-axis, concentrations on the x-axis).
• Read results from this calibration curve employing the (mean) absorbance values of each patient sample and control.
• For the calculation of the standard-curve mathematical Point to Point function should be used.

Typical Standard Curve

Interpretation of Results
According to recent literature and recommendations from reference laboratories the following interpretation of results is recommended:

1. Antibody Isotypes and State of Infection  

SPECIFIC PERFORMANCE CHARACTERISTICS

The results refer to the groups of samples investigated; these are not guaranteed specifications.
For further information about the specific performance characteristics please contact DRG.

Precision

Diagnostic Specificity

• The diagnostic specificity is defined as the probability of the assay of scoring negative in the absence of the specific analyte.
• It is 90.91% (95% confidence interval: 75.67% – 98.08%).

Diagnostic Sensitivity

• The diagnostic sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte.
• It is 100.0% (95% confidence interval: 69.15% – 100.0%).

Analytical Sensitivity
The analytical sensitivity (according to CLSI EP17-A) is defined as the apparent concentration of the analyte that can be distinguished from the zero calibrator. It is 1.56 IU/mL.

Interferences
Interferences with hemolytic, lipemic or icteric samples are not observed up to a concentration of 10 mg/mL hemoglobin, 5 mg/mL triglycerides and 0.5 mg/mL bilirubin.

Cross Reactivity
Investigation of a sample panel with antibody activities to potentially cross- reacting parameters (including antibodies to several respiratory pathogens) did not reveal evidence of false-positive results due to cross-reactions.

Measurement range
The measurement range is 1.56 IU/mL – 50 IU/mL.

LIMITATIONS OF THE PROCEDURE

Bacterial contamination or repeated freeze-thaw cycles of the sample may affect the absorbance values.

PRECAUTIONS AND WARNINGS

• The test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the test kits with analyzers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples.
• Only for in-vitro diagnostic use.
• All materials of human or animal origin should be regarded and handled as potentially infectious.
• All components of human origin used for the production of these reagents have been tested for anti-HIV antibodies, anti-HCV antibodies and HBsAg and have been found to be non-reactive.
• Do not interchange reagents or Microtiterplates of different production lots.
• No reagents of other manufacturers should be used along with reagents of this test kit.
• Do not use reagents after expiry date stated on the label.
• Use only clean pipette tips, dispensers, and lab ware.
• Do not interchange screw caps of reagent vials to avoid cross-contamination.
• Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination.
• After first opening and subsequent storage check conjugate and standard/control vials for microbial contamination prior to further use.
• To avoid cross-contamination and falsely elevated results pipette patient samples and dispense reagents without splashing accurately into the wells.
• The ELISA is only designed for qualified personnel following standards of good laboratory practice(GLP).
• For further internal quality control each laboratory should additionally use known samples.

Safety note for reagents containing hazardous substances
Reagents may contain CMIT/MIT (3:1) or MIT (refer to 4.1)
Therefore, the following hazard and precautionary statements apply.
Warning

• H317 May cause an allergic skin reaction.
• P261
  • Avoid breathing spray
  • Wear protective gloves/ protective clothing.
• P302+P352 IF ON SKIN: Wash with plenty of soap and water.
• P333+P313 If skin irritation or rash occurs: Get medical advice/ attention.
• P362+P364 Take off contaminated and Wash it before reuse.

Disposal Considerations
Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste is regulated through national and regional laws and regulations. Contact your local authorities or waste management companies which will give advice on how to dispose hazardous waste.

SCHEME OF THE ASSAY

Test Preparation

• Prepare reagents and samples as described.
• Establish the distribution and identification plan for all samples and standards/controls on the plate layout supplied in the kit.
• Select the required number of microtiter strips or wells and insert them into the holder.

**Assay Procedure

**

SYMBOLS USED

•  European Conformity
•  Consult instructions for use *
•  In vitro diagnostic medical device *
•  Catalogue number *
•  Batch code *
•  Contains sufficient for tests *
•  Temperature limit *
•  Use-by date *
•  Manufacturer *
•  Distributor *
•  Date of manufacture *
•  Biological risks *
•  Caution *
•  Unique device Identifier *
•  For research use only
•  Content
•  Volume / No.
•  Microplate
• Conjugate
•  Negative Control
•  Positive Control
•  Cut off control
•  Standard or Calibrator
•  IgA Sample Diluent
•  IgG Sample Diluent
•  IgM Sample Diluent
•  Stop solution
•  TMB Substrate solution
•  Washing Buffer 20x concentrated

References

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