What does high avidity mean
1. Single determinations
Single determinations are used to clarify the following questions:
- Is there immunity?
- Is there an active infection?
- Has an infection occurred in the past (in the case of persistent pathogens)?
- Can the time of infection be narrowed down?
IgM antibodies can be detected with certainty after 10-14 days at the earliest, IgG antibodies after 14-21 days. Taking samples too early after infection is a common cause of negative results. In order to reliably rule out an infection, it is advisable to repeat the test 4-6 weeks after the first one. If an HIV infection is suspected, exclusion can take place 3 months after exposure at the earliest.
The course of an infection process can basically only with one Serum pair (Acute serum and convalescence serum) can be assessed which im 2-3 weeks apart is removed. Only the examination of both sera in parallel in the same experimental approach (so-called "parallel approach") allows an exact quantitative comparison. A four-fold increase in titer or a significant increase in the measured value indicates an acute infection. In the case of an antibody determination with quantitative information, even a single high reading can indicate an acute infection.
The Time requirement Depending on the complexity of the order, it takes 1-3 days for the complete processing of a serological test order. For technical and personnel reasons, not all serological tests can be carried out every day. Each determination is carried out at least twice a week (exception: determination of neutralizing antibodies against poliovirus 1 // 3).
The ones required for a serological order Material quantities vary depending on the number of examinations required. However, the minimum amount should not fall below 0.5 ml of serum. Smaller amounts of blood can be accepted for pediatric blood samples. The minimum volume required, however, depends on the number of examinations required. In case of doubt, please inquire directly in the laboratory.
The reproducibility of qualitative test results is ensured by the controls included in every test batch and the standards that are included periodically. For quality assurance purposes, the laboratory also participates in round robin tests by renowned European quality control organizations (NEQAS, Instant).
2. Measurement uncertainty in quantitative results
The methods for the determination of antibodies show fluctuations due to their biological nature, both with multiple determinations of a given serum in one run (intratest variation) and with repeated tests over time (test-to-test variation). The results of individual determinations of the same type that have been determined in different test series that are separated in time can consequently differ considerably from one another. Therefore, only the parallel investigation of two samples in the same test approach (parallel approach) allows a direct quantitative comparison and thus a correct assessment of the course of a parameter over time.
The measurement uncertainty for quantitative investigations can be asked in the laboratory.
3. Determination of IgG, IgM and IgA antibodies
The different antibody classes are determined using different methods depending on the pathogen.
In general, the detection of specific IgG antibodies is sufficient for the detection of a previous infection or the determination of the immunity.
A detection of specific IgM and IgA antibodies is an indication of a fresh or recent infection. If viruses of the herpes family are reactivated, IgM antibodies may (but need not necessarily) occur.
When interpreting positive IgM results, caution should be exercised as confounding factors can occur:
- With certain clinical pictures (e.g. lupus erythematosus) one finds positive IgM results, which are caused by cross-reactions of antinuclear antibodies and not of pathogen-specific IgM.
- In pregnant women, care should be taken when interpreting IgM results, as non-specific IgMs can occur in addition to confounding factors. In this case, it is advisable to additionally perform an IgM confirmation test (see 4.2: µ-capture ELISA) or a determination of the avidity of the IgG antibodies (see 4.3: IgG avidity) to confirm the diagnosis. Testing a second serum is also useful.
- Cross-reactions of IgM antibodies cannot be ruled out with the herpes (HSV, VZV, CMV, EBV) or flaviviruses (TBE, Zika virus, yellow fever, dengue, HCV).
4. Methods of serology
4.1 ELISA (Enzyme-Linked Immunosorbent Assay)
The ELISA method is the most widely used method today for the determination of antibodies and is also preferred in our institute.
The results are given as follows:
- IgG: qualitative: positive / negative / borderline
semi-quantitative: -in AU / ml (arbitrary units / ml) (CMV, Toxoplasma)
-as an index (ratio of measured value above cut-off; positive if> 1)
quantitative: in IU / ml or mIU / ml (international or milli-international units / ml serum)
(Rubella: ≥ 15 IU / ml = immunity, 10-14 IU / ml = borderline)
- IgM, IgA: qualitatively positive / negative / (borderline)
4.2 µ-capture ELISA (sandwich ELISA) for IgM
The clear clarification of a questionably positive IgM finding is of the greatest importance, especially for pregnant women. The µ-capture ELISA (sandwich type) is used as a confirmatory test in the case of questionable positive IgM results in the screening test for:
- CMV IgM
- Rubella IgM
- Toxoplasma IgM
The results are given purely qualitatively as IgM positive / negative. If the confirmatory test shows a different result than the screening test, then the confirmatory test is to be regarded as relevant.
4.3 ELISA for IgG avidity
The avidity of the IgG antibodies is used to estimate the time of infection:
- IgG antibodies after a fresh infection show a low avidity.
- IgG antibodies after an infection show a high avidity. The maximum avidity occurs approx. 4 months after infection.
The determination of IgG avidity is used as an additional parameter to clarify seroconversion during pregnancy and is possible with:
- Rubella (belongs to the non-accredited area)
- CMV (belongs to the non-accredited area)
The results are given as a ratio between 0 and 1 together with a corresponding interpretation.
4.4 Immunofluorescence (IF)
Indirect immunofluorescence is a reliable method for the detection of antibodies against complex pathogens and is used by us for the following antibody detection:
- Dengue Virus IgG and IgM
- Measles Virus IgM
- Mumps Virus IgM
- Zika Virus IgG and IgM
The indirect immunofluorescence method is somewhat less sensitive (sensitivity) in the weakly positive range, but more specific than the ELISA.
The results are given purely qualitatively as IgG or IgM positive / negative.
4.5 Neutralizing antibodies against polioviruses
Neutralizing antibodies against polio virus types 1 and 3 are determined by means of a neutralization test on cell cultures.
The test is very complex and is used to control seronegative, immunocompromised individuals who have to be passively immunized by periodic administration of immunoglobulins, but cannot be actively vaccinated (e.g. children with leukemia).
The test should NOT be used, or only in specially justified cases, to check the vaccination status of immunocompromised persons!
The determination of neutralizing antibodies against poliovirus type 1/3 requires at least 0.5 ml of serum.
Time required: The test is only performed when necessary. A waiting period of up to two weeks must be expected.
The results are given as the reciprocal number (1: X) of the serum dilution that still guarantees neutralization, together with an interpretation.
4.6 Characterization of individual antibodies (line blot immunoassay)
In the case of individual viruses (HIV, HTLV, HCV) so-called line blot immunoassays are carried out to specify the antibodies present. These tests are mainly used to check reactive results from screening ELISA tests and thus have a confirmation function.
Time required: The test is carried out 2-3 times a week if necessary
The results are shown semi-quantitatively with 0 / ± / + / ++.
5. CSF diagnostics:
Antibodies against neurotropic viruses, with the exception of enteroviruses, can also be detected in the CSF. For these examinations, however, a serum-CSF pair from the same collection date is required.
The cerebrospinal fluid is only examined if the desired antibodies have been detected in the blood. If there is negative antibody detection in the blood, no CSF analysis is carried out.
By means of comparative analyzes of blood and liquor samples taken at the same time, statements can be made about possible intrathecal antibody production. To do this, however, we need the information on the respective concentrations of albumin and total IgG from the blood and liquor in order to be able to calculate the specific antibody index. This scan is carried out for the following viruses:
The results are given as an index number together with an interpretation. A specific antibody index of> 1.5 is pathological. Specific, intrathecal antibody production can only be expected approx. 2 - 3 weeks after the onset of symptoms.
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