Instruction for use

A solid-phase enzyme immunoassay kit

for the qualitative detection of IgG antibodies
to Borelia burgdorferi sensu lato
in human serum or plasma

Borelia burgdorferi IgG EIA

Catalogue number REF K118G

For 96 determinations
96

In vitro diagnostic medical device

ХЕМА LLC
Akademika Yefremova St. 23 Authorized Representative in EU:
03179, Kyiv, Ukraine Polmed.de Beata Rozwadowska
tel.:+38 044 422-62-16 Fichtenstr. 12A, 90763 Fuerth, Germany
tel.:+38 044 294-69-78 tel.:+ 49 911 931 639 67
E-mail: [email protected] E-mail: [email protected]
www.xema.com.ua www.polmed.de
 ASSAY PROCEDURE

EIA Buffer Dispensing of control sera and test samples Incubation 1

test
samples

DIL
SPE
10 µL

90 µL

Washing Incubation 2 Conjugate Solution

5 times Washing
5 times

CONJ
HRP

100 µL

Substrate Incubation 3 Stop Solution OD measuring, calculation

Solution of results

SUBS 15
TMB

450 /620-680 nm
100 µL
100 µL

During performing several independent series of tests, Positive and Negative Control Serum should be used each time.
K118G
 ХЕМА

CONTENT
1. INTENDED USE  2
2. GENERAL INFORMATION  2
3. TEST PRINCIPLE  3
4. KIT COMPONENTS  4
5. EQUIPMENT AND MATERIAL REQUIRED BUT NOT PROVIDED  5
6. WARNING AND PRECAUTIONS  5
7. SPECIMEN COLLECTION, TRANSPORTATION AND STORAGE OF SAMPLES  6
8. TRANSPORTATION AND STORAGE TERMS OF KIT, WASTE DISPOSAL  6
9. REAGENTS PREPARATION  7
10. ASSAY PROCEDURE  7
11. TEST VALIDITY AND CALCULATION OF RESULTS  8
12. INTERPRETATION OF THE RESULTS  8
13. PERFORMANCE CHARACTERISTICS  9
14. LIMITATIONS  9
15. REFERENCES  10
SAMPLES IDENTIFICATION PLAN 11

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K118GIE

Instruction for use

A solid-phase enzyme immunoassay kit
for the qualitative detection of IgG antibodies
to Borelia burgdorferi sensu lato
in human serum or plasma
Borelia burgdorferi IgG EIA
1. INTENDED USE
ELISA reagent kit Borelia burgdorferi IgG EIA is a solid-phase enzyme immunoassay for
the qualitative detection of IgG antibodies to Borelia burgdorferi sensu lato in human serum or
plasma.
The field of application is clinical laboratory diagnostics.
2. GENERAL INFORMATION
Borrelia burgdorferi sensu lato - is a group of borreliosis or Lyme disease pathogens, a
common infection, the main host and vector of which is the ixodid tick. The disease is transmitted
only through a tick bite.
In the early stages of borreliosis, fatigue, chills and headaches may be observed, and later
more serious symptoms may occur, such as joint pain, meningitis, numbness in the extremities,
facial nerve paralysis, memory disorders, and eye and heart damage. After the spirochete
penetrates the skin, a creeping erythema occurs, and after several days or weeks, it reaches
many organs by haematogenous or lymphatic means. In general, the incubation period is from
3 to 45 days.
Early diagnosis of the disease is based on clinical and epidemiological data. The diagnosis is
confirmed by laboratory, usually by serological methods - the detection of specific antibodies to
Borrelia burgdorferi in the blood.
IgM antibodies appear in the blood first, a few days after infection, but can be detected by
laboratory tests in 2-3 weeks. After about 6 weeks, the concentration of antibodies reaches a
maximum and then gradually decreases. IgG antibodies begin to be detected 4-6 weeks after
infection and the maximum amount of IgG antibodies is synthesised 2-3 months after the onset
of early symptoms of the disease. Then their number gradually decreases, but they remain in
the body for several years.

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3. TEST PRINCIPLE
The detection of IgG antibodies to Borrelia burgdorferi is based on the indirect enzyme
immunoassay principle. On the inner surface of the microplate wells are immobilized recombinant
Borrelia burgdorferi antigen. The analysis procedure includes three stages of incubation:
- during the first stage specific to Borrelia burgdorferi antibodies from the specimen are
bound onto the microwell surface;
- during the second stage horseradish peroxidase-conjugated specific monoclonal
anti-IgG antibodies bind to the antigen-antibody complexes, fixed in the formed at the previous
stage complexes;
- during the third stage, the complexes formed due to the reaction with the chromogen
3,3’,5,5’-tetramethylbenzidine are visualized.
After stopping the reaction with a stop solution, the intensity of the color of the microwells
is measured. The optical density (OD) in the microwell is directly related to the concentration of
the measured IgG antibodies to Borrelia burgdorferi in test specimen.

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 4. KIT COMPONENTS

Code of Symbol Name Volume Qty, Description

K118GIE

component pcs.
96-well polystyrene strip microplate coated with

Document: K118GIE
P118GZ SORB MTP Microplate - 1 recombinant antigen of Borrelia burgdorferi,
ready to use

Solution based on human serum, free of IgG antibodies to

CN118GZ CONTROL - Negative Control 0.5 mL 1 Borrelia burgdorferi, with preservative,
Serum К-
ready to use (yellow liquid)

Solution based on human serum, containing of IgG

Positive Control
CP118GZ CONTROL + 0.2 mL 1 antibodies to Borrelia burgdorferi,
Serum К+
with preservative, ready to use (red liquid)

4
Solution of monocnoclonal antibodies to IgG conjugated to
Conjugate
T118GZ CONJ HRP 12 mL 1 the horseradish peroxidase,
Solution
ready to use (red liquid)
Buffer solution with detergent and preservative,
SP118GZ DIL SPE EIA Buffer 12 mL 1
ready to use (purple liquid)

Substrate Tetramethylbenzidine (TMB) substrate solution,

R055Z SUBS TMB 12 mL 1
Solution ready to use (colourless liquid)

26х Concentrate Buffer solution with detergent, 26x concentrate

BUF WASH
S008Z Washing 30 mL 1
26X (colourless liquid)
Solution
5.0% solution of sulphuric acid, ready to use
R050Z STOP Stop Solution 12 mL 1
(colourless liquid)

The kit also includes instruction for use, quality control data sheet and plate sealing tape (2 pcs).

Instruction version/date: 2024.04

 ХЕМА

5. EQUIPMENT AND MATERIAL REQUIRED BUT NOT PROVIDED

–– microplate photometer with 450\620-680 nm wavelength;
–– dry thermostat for +37°С±1°C;
–– automatic plate washer (optional);
–– micropipettes with variable volume, range volume 5-1000 µL;
–– graduated cylinder of 1000 mL capacity;
–– distilled or deionized water;
–– timer;
–– vortex mixer;
–– disposable gloves;
–– absorbent paper.
6. WARNING AND PRECAUTIONS
In order to prevent incorrect results, strictly follow the recommended order and duration of
the analysis procedure.
6.1. The kit is for in vitro diagnostic use only. For professional laboratory use.
6.2. Follow the rules mentioned below during the kit using:
- do not use kit beyond expire date;
- do not use the kit if its packaging is damaged;
- in order to avoid contamination, use new tips to pipette samples and reagents;
- use only verified equipment;
- close each vial with its own cap, after using the reagent;
- do not use components of other kits or reagents of other manufacturers;
- do not let wells dry after completing the rinsing step; immediately proceed to the next
stage;
- avoid bubbles when adding reagents.
ATTENTION! The TMB substrate solution is light sensitive. Avoid prolonged exposure
of the component to light.
6.3. Some kit components, such as stop solution, substrate solution, and washing solution,
may cause toxic or irritant effects. If they get on the skin or mucosa, the affected area should
be washed with plenty of running water.
6.4. All human products, including patient samples, should be considered potentially
infectious. Handling and disposal should be in accordance with the procedures defined by an
appropriate national biohazard safety guidelines or regulations.
6.5. The positive and negative control serum included in the kit are negative for antibodies
to HIV 1,2, hepatitis C virus and HBsAg, but the reagents should be considered as potentially
infectious material and handled carefully.
6.6. Specimens must not contain any azide compounds, as they inhibit activity of peroxidase.
6.7. Wear protective gloves, protective clothing, eye protection, face protection.
6.8. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents
are handled.
6.9. Safety Data Sheet for this product is available upon request directly from XEMA LLC.
6.10. Serious incidents related to the kit must be reported to the manufacturer, Authorized
Representative, and to the Competent Authority of the EU member state(s) where the incident
has occurred.

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7. SPECIMEN COLLECTION, TRANSPORTATION AND STORAGE OF SAMPLES

7.1. Blood sampling should be carried out from the cubital vein with a disposable needle
using a vacuum blood sampling system. Serum or plasma specimens should be clearly labeled
and identified. Serum must be separated from the clot as early as possible to avoid hemolysis
of red blood cells. If there are any visible particles in the sample, they should be removed by
centrifugation at 3000-5000 rpm for 20 minutes at room temperature or by filtration.
Don’t use samples with high lipidemia, hemolysis as they may give false test results.
7.2. Specimen should be stored at +2…+8°C up to 3 days. Specimen held for a longer time,
should be placed in a freezer at -15°C or below, avoid no more than three cycles of thawing-
freezing samples.
7.3. For the transportation of samples, it is recommended to use triple packaging. The primary
package is the labeled tube containing the sample. Secondary packaging is a polyethylene bag
that is hermetically closed with a zip-lock. The outer packaging is a heat-insulating container,
while the secondary packaging is placed in the outer packaging for transportation in the center
of the thermal container. Frozen refrigerants are placed on the bottom, along the side walls of
the thermal container, and cover the samples with them.
8. TRANSPORTATION AND STORAGE TERMS OF KIT, WASTE DISPOSAL
Information about the singularity storage conditions, transportation of the kit, and disposal
of waste should be taken into account by all persons who participate in these processes.
8.1. Transportation
The Borrelia burgdorferi IgG EIA kit should be transported in the manufacturer’s packaging
at +2...+8°С. Single transportation at the temperature up to 25ºС for 5 days is acceptable.
8.2. Storage
The Borrelia burgdorferi IgG EIA kit should be stored in the manufacturer’s packaging at
+2...+8°С. Do not freeze.
The kit contains reagents sufficient for 96 determinations including Positive and Negative
Control Serum.
Once opened test-kit is stable for 2 months when stored properly as intended by manufacturer
at 2-8°C.
In case of partial use of the kit, the components should be stored in the following way:
–– the remaining strips should be immediately resealed in the bag along with the silica gel,
closed with the zip-lock, and stored at +2...+8°C within 2 months;
–– EIA Buffer, Substrate Solution, Stop Solution, and Washing Solution concentrate after opening
the vial, can be stored tightly closed at +2...+8°С until the kit’s shelf life;
–– Conjugate Solution, Positive and Negative Control Serum after opening the vial, can be
stored tightly closed at +2...+8°С within 2 months;
–– diluted Washing Solution can be stored at room temperature (+18…+25°С) for up to 5 days or
at +2…+8°С for up to 14 days.
Kits that were stored in violation of the storage condition cannot be used.
8.3. Disposal
Expired kit components, used reagents and materials, as well as residual samples must be
inactivated and disposed of in accordance with legal requirements.

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9. REAGENTS PREPARATION
9.1. All reagents (including microstrips) and test samples should be allowed to reach room
temperature (+18…+25 °C) for at least 30 minutes before use.
9.2. Microplate preparation
Open the package with the microplate and install the required number of strips into the
frame. The remaining strips should be immediately resealed in the bag along with the silica gel
and closed with the zip-lock to prevent moisture from affecting the plate’s strips.
9.3. Washing solution preparation
Add the contents of the 30 mL washing solution concentrate vial to 750 mL of distilled or
deionized water and mix thoroughly. In case of partial use of the kit, take the necessary amount of
washing solution concentrate and dilute it 26 times with distilled or deionized water.
The spending of the components in case of partial use of the kit is given in the table:
Quantity of strips 1 2 3 4 5 6 7 8 9 10 11 12
Volume of the washing
solution concentrate, 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30
mL
Volume of water, mL 62.5 125 187.5 250 312.5 375 437.5 500 562.5 625 687.5 750

10. ASSAY PROCEDURE

10.1. Put the desired number of strips into the frame based on the number of test samples
and 4 wells for Positive and Negative Control Serum (1 well for Positive Control (CP)
and 3 wells for Negative Control Serum (CN)).
10.2. Dispense 90 µL of EIA Buffer to all wells.
10.3. Dispense 10 µL of Positive and Negative Control Serum as well as 10 µL of
test serum/plasma samples (SAMP) to the wells of the microplate according to
the scheme below. The introduction of Positive and Negative Control Serum and test
samples should be carried out within 5 minutes to ensure equal incubation time for
the first and last samples.

NOTE: during performing several independent series of tests,

Positive and Negative Control Serum should be used each time.

Scheme of introduction of samples

1 2 3 4 5 6 7 8 9 10 11 12
A CP SAMP5 SAMP13 SAMP21

B CN SAMP6 SAMP14 SAMP22

C CN SAMP7 SAMP15 SAMP23

D CN SAMP8 SAMP16

E SAMP1 SAMP9 SAMP17

F SAMP2 SAMP10 SAMP18

G SAMP3 SAMP11 SAMP19

H SAMP4 SAMP12 SAMP20

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10.4. Carefully mix the contents of the microplate in a circular motion on a horizontal
surface, cover strips with a plate sealing tape and incubate for 30 minutes at
+37°С.
10.5. At the end of the incubation period, remove and discard the plate cover. Aspirate
and wash each well 5 times using an automatic washer or an 8-channel dispenser.
For each washing, add 300 µL of Washing Solution (see 9.3) to all wells, then
remove the liquid by aspiration or decantation. The residual volume of the Washing
Solution after each aspiration or decantation should be no more than 5 µL. After
washing, carefully remove the remaining liquid from the wells on the absorbent
paper. For the automatic washer/analyzer, the Washing Solution volume can be
increased to 350 µL.
10.6. Add 100 µL of Conjugate Solution to all wells.
10.7. Cover strips with a plate sealing tape and incubate for 30 minutes at +37°С.
10.8. At the end of the incubation period, aspirate and wash each well 5 times as
described in 10.5.
10.9. Add 100 µL of Substrate Solution to all wells. The introduction of the Substrate
Solution into the wells must be carried out within 2-3 minutes. Incubate the
microplate in the dark at room temperature (+18...+25°C) for 15 minutes.
10.10. Add 100 µL of Stop Solution to all wells in the same order as the Substrate
Solution. After adding the Stop Solution, the contents of the wells turn yellow.
10.11. Read the optical density (OD) of the wells at 450 nm and reference light filters
620–680 nm using a microplate photometer within 5 minutes of adding the stop
solution. Set photometer blank on air.

11. TEST VALIDITY AND CALCULATION OF RESULTS

11.1. The test results are valid only if Positive and Negative Control Serum are within the
specified ranges and if all other test parameters are also within the given assay specifications,
namely:
- OD of CONTROL- < 0.15;
- OD of CONTROL+ > 1.5;
- ОD(CN) x 0,5 < ОD(CN) < ОD(CN) x 2.
11.2. Calculate the mean OD value of the Negative Control Serum:
meanОD(CN) = (ОD1(CN) + ОD2(CN) + ОD3(CN))/3
11.3. Calculate the Cut Off value by adding to the mean OD value of the Negative Control
Serum the coeficient 0.3.
Cut off = meanОD(CN) + 0.3
11.4. Calculate Positivity Index (PI) for each sample by dividing the OD of the sample by
Cut off value:
PI = ОDsample/Cut off

12. INTERPRETATION OF THE RESULTS

If PI value > 1.1 the result is POSITIVE,
If PI value is between 0.9 and 1.1 the result is EQUIVOCAL,
If PI value < 0.9 the result is NEGATIVE.

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If equivocal results are obtained, it is recommended to conduct a reexamination of the

sample in several replicates. If the result is equivocal again, a new sample should be obtained
within 5-7 days and retested. If the result remains equivocal, the sample should be considered
negative.
13. PERFORMANCE CHARACTERISTICS
13.1. Analytical performance characteristics
13.1.1. Precision of Measurement
Reproducibility (Intra assay repeatability) was determined by evaluation the coefficient of
variation (CV) for 2 different samples, with different levels of IgG antibodies to the Borrelia
burgdorferi sensu lato antigen, during 1 day in 43 replicates on one series of ELISA kit.

№ serum mean OD mean PI CV PI, %

1 0.31 1.02 8.29
2 1.08 3.59 6.65

Reproducibility (Inter assay reproducibility) was determined by evaluating the coefficients

of variation (CV) for 2 samples of each serum for 4 days in 8-replicate determinations.

№ serum mean OD mean PI CV PI, %

1 0.27 0.9 8.1
2 1.1 3.66 6.8

13.1.2. Analytical specificity

For the analysis result is not affected by the presence in the sample of bilirubin in a
concentration of up to 0.21 mg/mL, hemoglobin in a concentration of up to 10 mg/mL and
triglycerides in a concentration of up to 10 mg/mL.
13.2. Diagnostic performance characteristics
The clinical sensitivity and specificity of the assay were evaluated using a serum panel with
8 positive and 8 negative clinical serum samples and were 100%. The relative sensitivity and
specificity of the assay were investigated in a sample of 96 donor sera characterised for the
content of IgG antibodies to Borrelia burgdorferi antigen in commercical Kits, and the ressults
were 99.7% and 97.5%, respectively.
14. LIMITATIONS
A positive result is evidence of the presence of IgG antibodies to Borrelia burgdorferi sensu
lato antigen. The diagnosis cannot be based on the results of an IgG antibody test to Borrelia
burgdorferi alone and requires confirmation, including an assessment of the patient’s clinical
presentation and history, the detection of IgM antibodies to Borrelia burgdorferi and conducting
an immunoblot test.
A negative result indicates the absence of IgG antibodies to Borrelia burgdorferi sensu lato
or antibody levels below the limit of sensitivity of the kit.
The results of serum tests in patients with immunosuppression and immunological disorders
should be interpreted with caution.

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15. REFERENCES
1. Lyme Borreliosis (Lyme disease). In: International travel and health. Geneva: World Health
Organization; 2014.
2. M Cinco 1, R Murgia, M Ruscio, B Andriolo. IgM and IgG significant reactivity to Borrelia
burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by
Lyme arthritis or neuroborreliosis. FEMS Immunol Med Microbiol . 1996 Jun;14(2-3):159-66.
doi: 10.1111/j.1574-695X.1996.tb00283.x.
3. Наказ МОЗ України №325 від 08.06.2015 «Про затвердження Державних санітарно-
протиепідемічних правил і норм щодо поводження з медичними відходами».
4. Постанова КМУ від 02 жовтня 2013р. №754 «Про затвердження технічного регламенту
щодо медичних виробів для діагностики in vitro».
5. НПАОП 85.14-1.09-81. Правила облаштування, техніки безпеки, виробничої санітарії,
протиепідемічного режиму і особистої гігієни при роботі в лабораторіях (відділеннях,
відділах) санітарноепідеміологічних установ системи Міністерства охорони здоров`я СРСР
(НАОП 9.1.50-1.09-81).

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ХЕМА

Instruction version/date: 2024.04

 SAMPLES IDENTIFICATION PLAN
K118GIE

1 2 3 4 5 6 7 8 9 10 11 12

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Instruction version/date: 2024.04

 Manufacturer

In vitro diagnistic medical device

Catalogue number

YYYY-MM Use-by date

Batch code

Temperature limit

Contains sufficient for <n> tests

Caution

Consult instructions for use

Conformity Marking with technical regulations in

Ukraine
Authorized representative in the European Com-
munity/European Union

CE Conformity Marking
For any issues related to operation of the kit and technical support,
please contact by telefon number
+38 044 294-69-78
or write to:
[email protected]

ХЕМА LLC
Akademika Yefremova St. 23
03179, Kyiv, Ukraine
tel.:+38 044 422-62-16
tel.:+38 044 294-69-78
E-mail: [email protected]
www.xema.com.ua