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| Porcine Toxo IgG Antibody Kit |
Brief
Porcine Toxoplasmosis IgG Antibody Test Kit is used for the detection the Toxoplasmosis antibodies in the serum; the test kit is employed to control Toxoplasmosis. |
Principle
Controlling this disease is based on method of serological diagnosis, test kit is enzyme immunassy for the detection Toxoplasmosis antibodies in swine serum. Toxoplasmosis antigens is coated on 96-well plate, put sample into well, antibodies react with antigens, subsequent to a wash step, enzyme conjugate solution is added, unreacted conjugate is removed by washing, and a substrate is added to generated a bule color. |
Specifications
Reagents
1 Antigen coated microplate 2 pieces (96 wells)
2 Negative control serum 2ml/tube
Positive control serum 2ml/tube
3 Enzyme conjugate(yellow lid) 1 bottle (22ml/bottle)
4 20×concentrated washing liquid(white lid) 1 bottle (30ml/bottle)
5 Substrate A (red lid)/B (black lid) 1 bottle each (12ml/bottle)
6 Stop solution (blue lid) 1 bottle (12ml/bottle)
7 Sample diluent 1 bottle (50ml/bottle) |
Instructions for use
Toxoplasma IgG ELISA Kit
Qualitative/semi-quantitative assay for anti- Toxoplasma gondii IgG antibodies
Product code GD80
96 tests
071103
1. Intended use
The Toxoplasma IgG kit is a rapid ELISA designed for the
qualitative/semi-quantitative detection of IgG antibodies to
Toxoplasma gondii in human serum. The assay is intended to be
used to evaluate serologic evidence of previous infection with
T.gondii and is for in vitro use. Plasma samples may also be
used.
2. Introduction
Toxoplasma gondii is an intracellular protozoan parasite with a
world-wide distribution. Although cats are the definitive host, the
parasite can infect almost all mammals and birds. Human
infection results from ingestion of contaminated soil, careless
handling of cat litter, ingestion of raw or undercooked meat or
transmission from mother to foetus through the placenta.
Serological data indicates that approximately 30% of the
population of most industrialised nations is chronically infected
with the organism.
Infection with T.gondii is asymptomatic in the majority of cases.
The most common clinical symptoms of acute toxoplasmosis in
the adult are asymptomatic lymphadenopathy, which may be
accompanied by fever and malaise, and atypical lymphocytosis
symptoms resembling infectious mononucleosis. While serious
complications, such as encephalitis, myocarditis and pneumonitis,
are rarely seen in the normal host, infection in an
immunocompromised host is often fatal.
When a seronegative woman becomes infected with T. gondii
during pregnancy, the organism is often transmitted to the foetus.
Infection during the first trimester may lead to spontaneous
abortion, stillbirth, or overt disease in the neonate. Infection
acquired later during pregnancy is usually asymptomatic in the
neonate, and may not be recognised. Approximately 75% of
congenitally infected newborns are asymptomatic. However,
nearly all children born with subclinical disease will develop
chorioretinitis and some may suffer blindness and mental
retardation.
3. Principle of the test
Diluted serum or plasma specimens (1:100) are incubated for 20
minutes to allow specific antibodies to T. gondii to bind to the
antigen-coated wells. After washing away unbound antibodies
and other serum constituents, T. gondii specific IgG is detected
using rabbit anti-human IgG conjugated to horseradish
peroxidase. After 20 minutes incubation, unbound conjugate is
removed by washing, and TMB enzyme substrate is added for 10
minutes. A blue colour develops if antibodies to T. gondii are
present. Addition of stop solution gives a yellow colour and the
optical densities of controls, standard(s) and samples are
measured using a microplate reader.
4. Materials included in the Kit
? Microplate 96 wells in 12 X 8 break-apart strips, pre-coated
with T.gondii purified membrane antigen
? Reagent 1: Sample diluent concentrate x15, 15ml, (blue),
dilute before use
? Reagent 2: Wash buffer concentrate (X 15), 75 ml, dilute
before use
? Reagent 3: Conjugate (peroxidase conjugated rabbit anti -
human IgG), 12 ml, (red), ready to use
? Reagent 4: TMB Substrate, 12 ml, ready to use
? Reagent 5: Stop solution, 12 ml, ready to use
? Standards1: (for semi-quantitative assays) 50 IU/ml; 150 IU/ml
(blue), ready to use
? Standard1: (for qualitative assa ys) 15 IU/ml, (yellow), ready to
use
? Positive control1: 100 IU/ml (red), 1ml, ready to use
? Negative control: 1 ml (green), 1ml, ready to use
? Instructions for use
1 Calibrated against the 3rd International Standard for Anti-
Toxoplasma serum, code TOXM – NIBSC, Potters Bar, UK
5. Other equipment required
10mm X 60mm tubes for dilution, pipettes 10 ? l, 100? l, 1000? l;
repeating dispenser 100 ? l, microplate reader with 450nm filter,
microplate washing device. Distilled or de-ionised water, general
laboratory apparatus.
6. Storage and precautions
On arrival, store the kit at 2 - 8ºC. Once opened the kit is stable
for three months (or until its expiry date if less than three months).
It is important to protect the unused wells from excess moisture.
Do not use kits beyond their expiry date.
The assay standards and controls are manufactured from dilute
non-infectious human serum. Normal clinical laboratory safety
procedures should be maintained at all times. Operators should
wear gloves and protective clothing when handling any patient
sera or serum based products.
The stop solution contains 0.24M sulphuric acid and is noncorrosive.
7. Samples
Only freshly drawn and properly refrigerated sera or plasma should
be used in this assay. Avoid haemolysed, lipemic or bacterial
contaminated sera. Sera should be stored at 2-8ºC for no longer
than 5 days. If delay in testing is anticipated, store test sera at –
20ºC. Avoid multiple freeze-thaw cycles.
8. Method
Ensure that all materials are at room temperature before
beginning the procedure. We recommend that the standards and
the controls are always run in duplicate. Samples may be run
singly or in duplicate.
1. Assemble the number of strips required for the assay.
2. The sample diluent X15 concentrate contains 0.09%
sodium azide as preservative. Prepare sufficient
working strength diluent for the assay run. However, if
the working strength diluent is to be stored for more than
1 week, add sodium azide (0.9g/L). Store unused
sample diluent concentrate and dilute sample diluent
at 2 – 8 oC. Prepare working strength sample diluent
(Reagent 1) by diluting it 1 in 15 in distilled/de-ionised
water.
3. Dilute patient samples 1:100 (e.g. 5 ? l serum plus 0.5 ml
diluent). It is important to dispense all samples and
controls into the wells without delay. Therefore ensure
that all samples are ready to dispense.
4. For qualitative determinations, dispense 100 ? l of the
negative control, the 15 IU/ml standard, the positive
control and the diluted patient sample into the wells.
For semi-quantitative determinations, use sample
diluent as 0 IU/ml and additionally dispense the 50
IU/ml and 150 IU/ml standards.
5. Place the strips into the incubation bag provided and
incubate for 20 minutes at room temperature. During
all incubations, avoid direct sunlight and close
proximity to any heat sources.
6. Dilute the wash buffer ( Reagent 2), 1 to 15 in
distilled/de-ionised water to make sufficient buffer for
the assay run. The diluted wash buffer is stable for two
months at 2 - 8ºC.
7. After 20 minutes, decant or aspirate the well contents
and wash the wells 3 times using an automatic plate
washer or the manual wash procedure (see below).
Careful washing is the key to good results. Blot the wells
on absorbent paper before proceeding. Do not allow
the wells to dry out.
Manual Wash Procedure:
Empty the wells by inversion. Using a multi-channel
pipette or wash bottle, fill the wells with wash buffer.
Empty by inversion and blot the wells on absorbent
paper. Repeat this wash process two more times.
8. Dispense 100 ? l of Conjugate (Reagent 3) into each well.
This reagent is colour coded red. Keep all pipettes and
other equipment used for Conjugate completely separate
from the TMB Substrate reagent! Incubate the wells for
20 minutes in the incubation bag at room temperature.
9. After 20 minutes, discard the well contents and carefully
wash the wells four times with wash buffer. Ensure that
the wells are completely washed. Blot the microplate on
absorbent paper to remove final drops of wash fluid. Do
not allow the wells to dry out.
10. Using a repeating dispenser, rapidly dispense 100 ? l of
TMB Substrate ( Reagent 4) into each well. Incubate the
plate for 10 minutes.
11. Add 100 ? l of Stop Solution (Reagent 5) to each well. To
allow equal reaction times, the stop solution should be
added to the wells in the same order as the TMB
Substrate.
12. Read the optical density in a microplate reader within 10
minutes.
9. Quality control
Quality control data is supplied on the lot-specific QC certificate
included in the kit. .
10. Interpretation
Qualitative determinations
Negative samples: OD < 15 IU/ml OD
Positive samples: OD >/= 15 IU/ml OD
Semi-quantitative determinations
Plot the optical densities of the standards against their respective
concentrations. Draw a line to join the points. Read the
concentrations of unknowns from this graph. Concentrations below
15 IU/ml are considered negative; concentrations above 15 IU/ml
are considered positive for anti -toxoplasma IgG.
A negative result indicates no current or previous infection with T.
gondii . Such individuals are presumed to be susceptible to
primary infection. However see Limitations below.
A positive result indicates a current or previous infection with T.
gondii.
11. Limitations
1. The antibody titre of a single serum specimen cannot
be used to determine recent infection. Paired samples
(acute and convalescent) should be collected and
tested concurrently to demonstrate seroconversion.
2. Test results for demonstration of seroconversion should
be interpreted in conjunction with the clinical
evaluation and the results of other diagnostic
procedures.
3. Samples collected too early in the course of the
infection may not have detectable levels of IgG. In
such cases, a second sample may be collected after 2-7
weeks and tested concurrently with the original
specimen to look for seroconversion or an IgM specific
assay should be performed.
4. A positive Toxoplasma IgG test in neonates should be
interpreted with caution since passively acquired
maternal antibody can persist for up to 6 months.
However, a negative test for IgG antibody in the
neonate may help exclude congenital infection.
12. Performance characteristics
Comparative study:
The Genesis Diagnostics Toxoplasma IgG kit was compared with
another commercially available ELISA procedure for the
detection of IgG antibodies to T. gondii. The Genesis kit showed
100% agreement with the other ELISA. The results are
summarised below.
Comparative Study
(n=83)
Reference Toxoplasm a IgG
ELISA kit
+ -
Genesis Diagnostics +
Toxoplasma IgG kit -
39 0
0 44
13. Assay characteristics
Within Assay Imprecision < 12%
Between Assay Imprecision < 12%
Method Summary
? Dilute sera 1:100 with sample diluent (Reagent 1)
? For qualitative assays, dispense 100? l of the 15 IU/ml
standard, the controls and diluted sample into the
microplate wells. For semi-quantitative determinations,
additionally run the 50 & 150 IU/ml standards.
? Incubate for 20 minutes at room temperature.
? Wash the wells three times
? Dispense 100? l of Conjugate (Reagent 3) into each well
? Incubate at room temperature for 20 minutes
? Wash the wells four times
? Add 100? l of TMB Substrate (Reagent 4) to each well
? Incubate at room temperature for 10 minutes
? Add 100? l Stop Solution (Reagent 5) to each well
? Read the optical density at 450nm
Further Reading
Krick JA, and Remington JS: Toxoplasmosis in the adult: An
overview. New Engl J Med 298: 550-553, 1978
Welch PC et al: Serologic diagnosis of acute lymphadenopathic
toxoplasmosis. J Infect Dis 142:256-264, 1980
Ruskin J, and Remington JS: Toxoplasmosis in the compromised
host. Ann Intern Med 84: 193-199, 1976
Highes HPA: Toxoplasmosis: The need for improved diagnostic
techniques and accurate risk assessment. Contem Topics Micro
Immunol 120: 10005-139, 1985 |