Developed & Manufactured by Biomedica

Borrelia IgM ELISA | BI-21042

Cat. No.: BI-21042

1 kit / €  320,00Please Inquire

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Borrelia IgM ELISA highlights:
Therapeutic Areas
Assay Characteristics
  • Method

    Indirect ELISA, HRP/TMB, 12×8-well detachable strips

  • Sample type

    Serum, plasma and CSF

  • Sample volume

    10 µl / sample (serum and plasma), 100 µl / sample (CSF)

  • Assay time

    1 h / 30 min / 15 min

  • Standard range

    0 – 30 BBU/ml (Biomedica Borrelia Units)

  • Sensitivity

    100% (serum)

    82% (CSF)

  • Cross-reactivity

    The assay works with human samples only.

  • Specificity

    Endogenous and recombinant human anti-Borrelia IgM (B. burgdoreri ss, B. afzelii, B. garnii, B. bavariensis)

  • Use

    CE marked – for IVD use in the EU

  • Validation Data

    See validation data tab for: precision, specificity, sensitivity

Product details

Borrelia IgM ELISA Product Overview

The Borrelia assay is a 2 hour, 96-well indirect ELISA for the qualitative or quantitative determination of Borrelia IgM antibodies in human serum, plasma and CSF samples. The assay employs human serum-based standards to ensure the measurement of biologically reliable data.

The Borrelia test kit uses a recombinant antigen mix to improve the diagnostic specificity:

- p21 = OspC (outer surface protein C): B. afzellii (pKo), B. garinii (20047)

- p41/I = (inner part of flagellin): B. bavariensis (pBi)

- VIsE: fusion proteins of different Borrelia genospecies

Borrelia IgM ELISA Assay Principle

The Borrelia IgM ELISA kit is an indirect enzyme immunoassay for the qualitative or quantitative determination of Borrelia IgM antibodies in human serum, plasma and CSF samples.

The figure below explains the principle of the Borrelia ELISA:


Borrelia IgM ELISA kit Assay Principle

Capture antigen: recombinant Borrelia antigens (see Product Overview)
Detection antibody: HRP-conjugated rabbit anti-human IgM antibody
Target:  human anti-Borrelia IgM antibodies

In a first step, the standard/sample (STD/SAMPLE) is pipetted into the wells of the microtiter strips. IgM antibodies present in the STD/SAMPLE bind to the antigen mix that is pre-coated in the wells. After incubation, the plate is washed. This removes all non-specific unbound material. In a next step, the conjugate (HRP-conjugated rabbit anti-human IgM antibody) is pipetted into the wells and reacts with the anti-Borrelia IgM antibodies present in the sample. After another washing step, the substrate (Tetramethylbenzidine; TMB) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of IgG antibody present in the sample. This color change is detectable with a standard microtiter plate reader. The optical density (OD) of the cut-off STD represents 10 BBU/ml (Biomedica Borrelia units). This is the basis for the calculation of the results; see calculation of results in the instructions for use section for more information.

Borrelia IgM ELISA Kit Components

Contents

Description

Quantity

PLATE

Detachable microtiter strips, breakable, color coded, coated with recombinant antigen

12 x 8 tests

STD

Human serum-based standards: positive (pos), cut-off (c.o.), negative (neg), ready to use

Each 1 x 1.5 ml

CONJ

Conjuaget, HRP-conjugated rabbit anti-human IgM antibodies

1 x 14 ml

DIL

Sample diluent with green dye, ready to use

1 x 105 ml

SUB

Tetramethylbenzidine (TMB) substrate solution, ready to use

1 x 13  ml

WASHBUF

Wash buffer: 20 x concentrate

1 x 50 ml

STOP

Stop solution: sulfuric acid, ready to use

1 x 10 ml

Storage instructions: All reagents of the Borrelia IgM ELISA kit as supplied are stable at 4°C until the expiry date stated on the label of each reagent.

Instructions for use

Sample Collection & Storage                   

Serum, EDTA plasma, heparın plasma, citrate plasma, CPD plasma and CSF are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples and standards. The sample collection and storage conditions listed are intended as general guidelines.

Serum & Plasma

Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparın, citrate or CPD as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. The use of heat-inactivated, icteric, haemolytic, lipemic or turbid samples is not recommended. If the tests are not carried out immediately, the samples can be stored for up to two weeks at 2-8°C. Prolonged storage of the samples is possible at -25°C or below. Avoid more than three freeze-thaw cycles.

Reagent Preparation

Wash Buffer

1.

Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature.

2.

Dilute the WASHBUF concentrate 1:20, e.g. 50ml WASHBUF + 950ml distilled or deionized water. Only use diluted WASHBUF when performing the assay.

The diluted WASHBUF is stable up to one month at 4°C (2-8°C).

Standards for Serum and Plasma

Standards are ready-to-use and MUST NOT be diluted.

Sample Preparation

Sample Dilution

Bring samples to room temperature and mix samples gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.

Serum/plasma: dilute sample 1:101 (1 + 100) with sample diluent, e.g. 10 µl of sample + 1 ml of sample diluent, mix well.

CSF: dilute cerebrospinal fluid 1:2 (1 + 1) with sample diluent, e.g. 100 µl of sample + 100 µl of sample diluent, mix well.

For samples with results >30 BBU (Biomedica Borrelia Units)/ml the samples can be diluted with sample dilution buffer and retested. The obtained results need to be multiplied with their respective dilution factor to determine the final concentration.

Borrelia IgM ELISA Assay Protocol

Read the entire protocol before beginning the assay.

1.

 Bring samples and reagents to room temperature (18-26°C).

2.

Mark positions for BLANK/STD/SAMPLE (blank/standard/sample) on the protocol sheet. Take a minimum of one well as blank.

3.

Take the microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label.

4.

Pipette 100 µl STD/SAMPLE (standards, diluted sample) into the respective wells. Leave wells for blank measurement empty.

5.

Cover strips with the plastic film and incubate for 1 hour at 37°C in an incubator.

6.

Aspirate and wash wells 4 x with at least 250 µl diluted WASHBUF (wash buffer). Remove remaining WASHBUF by tapping the plate against paper towel after the last wash.

7.

Add 100 µl CONJ (HRP-conjugated anti-human IgM) into each well.

8.

Cover strips with plastic film and incubate for 30 min at room temperature (18-26°C).

9.

Aspirate and wash wells 4 x with at least 250 µl diluted WASHBUF (wash buffer). Remove remaining WASHBUF by tapping the plate against paper towel after the last wash.

10.

Add 100 µl SUB (TMB substrate) into each well.

11.

Incubate 15 min at room temperature (18-26°C) in the dark.

12.

Add 50 µl STOP (stop solution) into each well. Swirl gently.

13.

Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Calculation of Results

Subtract the optical density (OD) of the blank from the OD’s of the samples and standards.

Calculate the mean OD’s of all samples and standards.

The OD of the cutoff standard represents 10 BBU/ml (Biomedica Borrelia Units). This is the basis for calculation of the results.

The quality control protocol supplied with the kit shows the results of the final release QC for each kit. OD data obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as the following results are achieved:

Blank

The OD must be ≤ 0.250.

Negative standard

The OD must be ≤ 0.200 (blank subtracted).

Cut-off standard

The OD must be at least twice the negative standard (blank subtracted).

Positive standard

The OD must be at least twice the OD of the cut-off standard (blank subtracted).

Qualitative Calculation for Serum/Plasma

Positive result

Samples with an OD of more than cut-off +10% (≥ 11 BBU/ml)

Borderline result

Samples between 9 - 11 BBU/ml. These patients should be retested with a new sample.

Negative result

Samples with an OD of less than cut-off -10% (≤ 9 BBU/ml)

Quantitative Calculation for Serum/Plasma

Calculation of the BBU/ml is identical to the qualitative calculation.

The test is linear between 5 - 30 BBU/ml. Values outside this range are semi-quantitative.

Alternatively, samples with results >30 BBU/ml can be diluted with sample dilution buffer and retested. Obtained results need to be multiplied with respective dilution factor for final concentration.

Weak positive

Samples between 11 - 20 BBU/ml

High positive

Samples between 21 - 30 BBU/ml

Very high positive

Samples above 30 BBU/ml

Qualitative Calculation for Cerebrospinal Fluid

The calculation of the BBU/ml is identical to the qualitative calculation.

Normal values CSF

The mean of 30 CSF samples was 1.3 BBU/ml.

Neuroborreliosis negative

Samples with values ≤ 5 BBU/ml

Suspected for neuroborreliosis

Samples with values > 5 BBU/ml
Background & Therapeutic Areas

Lyme borreliosis is a bacterial infection caused by the spirochete Borrelia Burgdorferi, Borrelia Afzelii and Borrelia Garinii and is the most vector-transmitted human disease in Europe. The spirochetal bacteria were first described and isolated in 1981 by Willi Burgdorfer in the tick Ixodes dammini in Montana (USA). The infection with Borrelia is characterized by a variety of clinical symptoms and can be divided into 3 stages:

  • Stage 1, early dermatitis, appearing days or weeks after the infection. Clinical: erythema migrans.
  • Stage 2, early disseminated infection, appears weeks to months after infection. Clinical: lymphocytic meningoradiculitis (Bannwarth's syndrome), neuroborreliosis.
  • Stage 3, late disseminated infection, occurring up to years after infection. Clinical: chronic progressive encephalomyelitis, acrodermatitis chronica atrophicans (ACA), chronic arthritis.

Antibiotic therapy is indicated in all stages of the disease. It shortens disease duration significantly, when instituted in stage 1. Diagnosis of the bacterial infection relies on the detection of specific IgM antibodies against Borrelia.

To ensure diagnostic specificity, the Borrelia IgM ELISA microwell strips are coated with the following Borellia-specific recombinant antigens:

  • p21 = OspC (outer surface protein C): B. afzellii (pKo), B. garinii (20047)

  • p41/I = (inner part of flagellin): B. bavariensis (pBi)

  • VIsE: fusion proteins of different Borrelia genospecies

Many features of later infection are not specific to Lyme Borreliosis and occur in other conditions. The diagnosis of Lyme Borreliosis should be made only after careful evaluation of the patient´s clinical history, physical findings, laboratory evidence and exposure risk evaluation.

    Validation Data

    Borrelia IgM ELISA Sensitivity & Specificity

     

    Borrelia IgG
     

    Serum

    CSF

    Sensitivity

    100%

    91%

    Specificity

    96%

    82%

    The data for specificity and sensitivity are internal data from Biomedica. They were received from following serum panel:

     

    Clinical diagnosis

    Number of samples

    Lipaemic sera

    No Borreliosis

    9 sera

    Haemolytic sera

    No Borreliosis

    12 sera

    Early infection state

    Tick bite with Erythema Migrans

    5 sera

    Later stage infection

    Clinically defined positive sera

    30 sera

    The results were confirmed by Western Blot analysis using the product MG-4203, Borrelia IgM Western Blot from MIKROGEN GmbH, Martinsried, Germany.

    Borrelia IgM ELISA Crossreactivity

      n Crossreactive
    Rheumafactor-positive 15 1
    Syphilis-positive 7 0

    This ELISA uses recombinant antigens. Nevertheless, crossreactions to autoimmune-positive samples cannot be completely excluded.

    Borrelia IgM ELISA Precision

    The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different ELISA lots (in-between-run precision or reproducibility).

    Within-Run Precision

    Within-run precision was tested by measuring the same samples eight times within Borrelia IgG ELISA lot. 

    ID

    n

    Mean (BBU/ml)

    SD (BBU/ml)

    CV (%)

    Positive

    12

    24

    2

    6.7

    High positive

    12

    38

    3

    6.8

    In-Between-Run Precision

    In-between-run precision was assessed by measuring the same samples twelve times within multiple Borrelia IgG ELISA lots.

    ID

    n

    Mean (BBU/ml)

    SD (BBU/ml)

    CV (%)

    Positive

    17

    22

    2

    8.3

    High positive

    20

    36

    3

    8.1

    Comparison with other Assays

    A total of 32 samples has been tested with the Borrelia ELISA and a very good correlation to the results with the ELISA kits from Behring, Mikrogen and Virotech was found (anonymized Competitor ELISA A-D). The ELISA results were confirmed by Western Blots.

     

    Competitor ELISA

    Western Blot

     

    Biomedica

    A

    B

    C

    D

    Mikrogen

    Virotech

    Positive

    15

    9

    8

    15

    9

    15

    15

    Borderline

    2

    0

    1

    1

    0

    0

    7

    Negative

    11

    19

    6

    12

    19

    13

    5
    Citations

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    Cat. No.: BI-21042

    1 kit / €  320,00Please Inquire

    Contact distributor

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