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Immunology basics

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PowerPoint Slideshow about 'Immunology basics' - meryl


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slide1

Immunology basics

Any normal person is ALWAYS making antibodies against anything that enters the body. The molecules that elicit antibody responses are called antigens. Antigens can be of any origin: animal, vegetal or even mineral. Any normal individual will have antibodies against antigens commonly encountered in the environment, such as pollen, food, and also against microorganisms dwelling in the skin and digestive tract (the symbiotic flora we ALL possess). During an acute infection with a pathogen antibodies are made, but they VERY RARELY exceed 1% of the TOTAL antibodies found in the blood. The HIV (or any serological) test works by detecting antibodies that recognize HIV antigens. If those antibodies are present (HIV antibody positive), then the person is assumed to be infected with HIV.

Mind that the same reasoning applies to finding antibodies against ANY antigen: positivity is indicative of an encounter with the source of that antigen (a current or past infection).

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What the "HIV test" measures

So, the HIV test is actually detecting a VERY small amount of serum antibodies capable of recognizing the HIV antigens used in the test, which probably amount to MUCH LESS than 1%.

Tuberculosis (TB) the result of infection with the bacterium Mycobacterium tuberculosis (Mt) has been taken to be one of the MOST representative of the AIDS dieseases, basically because there is "strong evidence" to support that immune dysfunction can allow TB to progress to overt disease and also because "several studies show" that TB and HIV seropositivity are often present at the same time in the same individual.

The possibilities for this frequent coincidence are two:

1- HIV-AIDS makes people more prone to develop TB as the "defenses are down" and although TB patients often have anti Mt antibodies, the HIV test truly detects those antibodies directed against HIV.

2- Mt, in a proportion of individuals, is responsible for generating antibodies that recognize HIV components (antigens) of the HIV test.

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The key points

1- At any given time, humans have antibodies against an enormous amount of different antigens from all sources (not only pathogens).

2- VERY rarely the antibodies directed against ONE antigen source (pathogen or not) exceed a small fraction of the total antibodies circulating.

3- If you want to ascertain the existence of ANY antibody capable of recognizing any particular antigen source (say HIV, Mt, etc.), you must make sure you can pick that small subset from a huge amount of "irrelevant" antibodies. This is what determines the specificity of the test.

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So how do you go about detecting these antibodies?

First thing, you need a source of the antigen the antibodies you wish to detect can bind.

Then you immobilize the antigens on a surface (very much like a coat hook on the wall) and you incubate some blood (or serum) on this surface which is covered with the antigen. The antibodies which can bind to the antigens will do so and become, in their turn, immobilized, such that you can remove the rest of the blood (or serum), which contains many things, among them that >99% of antibodies that do not bind the antigens.

Finally, there are ways to measure the amount on antibodies that stayed bound to the antigen-coated surface, the more there are, the more likely that the source of that antigen elicited an antibody response (say HIV or Mt).

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If the amount of antibodies that remained bound to the immobilized HIV antigens used in the HIV test is above a certain value (the "cut-off" value) then the sample in question is positive, and conversely, if the amount is below the cut-off, the sample is negative.

It is important to note that reproducibility (precision) is essential, if this is not so, a sample can score positive in the test and negative upon re-testing, or viceversa, and which one is the "true" value remains to be determined.

The cut-off value is determined by testing a sample known to be negative, and calculating the standard deviation of multiple determinations (generally three) and then adding the equivalent of three times the SD of the mean. In practice, the cut-off is the average of the negative control values + 0.1.

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Blood of a true HIV+ antibody subject

Addition of serum to the immobilized HIV antigens

Antibodies capable of binding the HIV antigens

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Blood of a true HIV+ antibody subject

Incubation to allow antibody binding to antigens

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Blood of a true HIV+ antibody subject

Wash and remove irrelevant (unbound) antibodies

Value above the cut-off.

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So, what happens if we remove antibodies which recognize antigens other than HIV's before incubating on the HIV antigen coated plates? [Preadsorption]

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Antibodies (if there are any) that bind the non-HIV antigens will be immobilized and removed from the sample, but the outcome of the HIV antibody test should not change

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Then we transfer the rest (unbound antibodies) to a plate for the measurement of remaining anti-HIV antibodies.

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In principle, if the antibodies against HIV antigens are SPECIFIC, they should not be adsorbed by other antigens, so the subject should remain HIV+.

Value should remain above the cut-off.

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Evaluation of cross-reactivity between anti-Mycobacterium tuberculosis antibodies and HIV antigens in South Africa.

Study design:

- Serum samples from clinically diagnosed tuberculosis patients were tested. Of these, 99 were of known to be positives in the HIV antibody test and 63 were of unknown HIV serological status. All were diagnosed and in treatment for TB.

- The idea was to test if antibodies responsible for positivity in the "HIV test" could be removed from serum by incubation against M. tuberculosis antigens before running the HIV antibody test.

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- The original study proposal called for a stratified group of samples. most of which would be clear but not off-the-scale positives. However, due to operating constraints, the study was done on an uncharacterized population of samples which consisted of mostly very high positives (values above 2 comprise about 90% of the samples).

- Additionally, an unexpected but very significant variation between duplicate samples was observed, which could be the result of the preadsorption of the sera OR to inherent variability in the HIV test.

The latter turned out to be the case, as will be seen. This variability is particularly dangerous for those that score low positive values or even negative values.

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High positives

Mid-positives

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From the previous graph, no significant differences can be observed between those samples that were preadsorbed against Mycobacterial antigens (of strains H37Rv and CSU93), those preadsorbed by an inert protein control (Gelatin) and those that were not treated.

However, a significant variation in those samples done in duplicate (CSU93 and gelatin) was observed.

The results can be interpreted as follows:

- Most of the samples are so high that removal of cross- reacting antibodies (if they exist) diminishes the signal insignificantly.

- Variability in the test gives values that are not-reliable for the quantitative assessment of decrease in signal.

slide17

Variability in duplicates preadsorbed with CSU93 or gelatin and comparison to untreated control. Values are those of chosen samples with values low to mid range.

Note the high standard deviations in many samples. At this stage we did not know if originating in the preadsorption step or in the test itself.

slide18

So, although we had not anticipated that the test could be so variable, we undertook to test if the results of the HIV antibody test were reliable by testing untreated (that is, unmanipulated) samples in triplicate and see how precise the test outcome was.

The results were surprising, in that we never expected this level of variability of samples tested 3 times on the same assay plate, as the following graph shows.

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*

*

The graph on the left are the indivdual test results, on the right is the average  SD.

Intra-assay variability

What happens if you test the same sample 3 times?

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For a number of samples, the HIV antibody status was not known, upon testing, 14 of 58 were negative ( 24%, prevalence of 76%???)

slide21

Conclusions:

The HIV antibody test used in this study was that of Biomérieux, and was used strictly according to the manufacturer's instructions and under the same conditions used in MEDUNSA's Department of Virology, where it serves as a "confirmatory" test for HIV seropositivity.

1- The test is highly variable (imprecise) at low to mid-range values.2- Most of the samples that turn out positive (by the test's criteria) are far up in the upper limits of detection.

3- In the upper limits of detection, cross-reactivity cannot be assessed, as we are far from the linear detection range of the instrumentation to detect and quantify drops in readings.

4- In the low to mid-range limits of detection, we could not assess the effects of preadsorption as the test is NOT acceptably reproducible.

slide22

This pilot study has illuminated several aspects of the HIV antibody test that were not known to us, mostly because IF they are known, they are not in the test's instruction manual. The most relevant is the lack of reproducibility, and it warrants further study, not only in the test we used in this occasion but on as many as possible of those which are in use in South Africa.

The preadsorption experiments must be carried out as originally planned, with more antigens and more samples, and the optimal conditions for preadsorption must be sought and established in the experimental procedures.

A critical aspect is to find samples which give positive reactivity within the linear range of the assay, which will have to be determined for each assay to be evaluated. Complementary to this, is to do a "normalization" of readings by diluting strong positive samples down to values within the linear range of the test.

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To the best of our knowledge, and besides the preadsorption experiments to be carried out, a stringent evaluation of the HIV antibody test's precision has not yet been conducted. Basically, we rely on what the manufacturer says about its test.

Our preliminary results indicate that it must be done, not only to enable the interpretation of the preadsorption data but also because it seems likely that an unacceptable number of samples of low to mid-range reactivity may turn positive or negative upon re-testing, limiting the confidence with which an HIV seropositivity result can be assigned.

Considering that an HIV+ result certainly ruins the life of anyone receiving it, the level of uncertainty has to be established to know whether it is acceptable or not, independently of the manufacturer's claims, which are usually based on optimal testing conditions more or less removed from the real working conditions encountered in the diagnostic laboratories in South Africa or anywhere else.