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INFECTIOUS MONONUCLEOSIS KIT

A latex agglutination screening test for the detection of infectious mononucleosis heterophile antibody in serum and in plasma.

Infectious mononucleosis (glandular fever) is an acute infectious disease caused by the Epstein-Barr virus and primarily affects lymphoid tissue. It is characterised by the appearance of enlarged and often tender lymph nodes, enlarged spleen, and abnormal lymphocytes in blood. Patients usually, but not always, develop a transient heterophile antibody response.
The detection of heterophile antibodies to infectious mononucleosis by the agglutination of sheep red blood cells was first reported by Paul and Bunnel¹. Subsequent work by Davidsohn^2,3 Lee³, and Beer⁴ showed the need for differential absorption of sera to remove non-infectious mononucleosis heterophile
antibodies. Fetcher and Woolfolk⁵ showed that antigens obtained from the membranes of bovine erythocytes were more effective in combining with the infectious mononucleosis heterophile antibodies than those antigens obtained from either sheep or horse erythocytes.
The Oxoid Infectious Mononucleosis Kit is a simple, two minute latex agglutination test for the detection
of the specific heterophile antibody associated with infectious mononucleosis in serum and plasma. The purified specific heterophile antigen from bovine red
cell membranes is used to coat uniform latex particles. The purity and potency of the antigen used in the Oxoid Infectious Mononucleosis kit lead to
improved sensitivity, and specificity, and eliminate the need to perform differential absorptions on test samples.
When a drop of serum or plasma containing the heterophile antibody associated with infectious mononucleosis is mixed with a drop of the latex,
visible agglutination of the latex occurs within two minutes. When no such antibody is present, agglutination will not occur.
If required, the latex reagent may be used in a semiquantitative assay for the antibody.
An accurate diagnosis of infectious mononucelosis however, should only be made when clinical and haematological findings as well as the results from the Oxoid Infectious Mononucleosis Test have been taken into consideration.

Components of the Kit
DR0681M Test Latex:
Consists of latex particles sensitised with purified bovine antigen. Each kit contains sufficient reagent for 50 tests.
DR0682M Positive Control Serum Control:
Consists of rabbit antiserum containing specific antibody reactive with the test reagent. Sufficient for 15 tests.
DR0683M Negative Control Serum:
Consists of rabbit antiserum tested for the absence of heterophile antibodies to infectious mononucleosis. Sufficient for 15 tests.
DR0500G Disposable Reaction Cards:
There are 15 reaction cards provided in the pack. Each card may be used for testing six sera. If fewer sera are to be tested, the card may be cut prior to use
with scissors and the unused rings saved for later use.
DR0699M Paddle Pastettes:
A minimum of 55 paddie pastettes are provided. These may be used to apply a drop of the serum to the test card and to mix the serum and latex together.
Instruction leaflet.
Materials required but not provided
Timer
Sodium hypochlorite solution (>1.3% w/w).
Additional items required for the optional semiquantitative assay:
Test tube (12 x 75 mm)
Pipettes (for delivery of 0.5 ml)
0.85% Nacl (w/v) solution

Precautions
This product is for in vitro diagnostic use only
The reagents contain sodium azide as a preservative. Sodium azide may react with lead or copper plumbing to produce metal azides which are explosive by contact detonation. To prevent azide accumulation in plumbing, flush with copious amounts of water immediately after waste disposal.
The appropriate precautions should be taken when handling human serum or plasma. Pipettes, test cards, etc. should be disposed of into hypochlorite disinfectant (>1.3% w/w).

Storage
The reagents must be stored at 2-8°C.
Do Not Freeze. Under these conditions the reagents will retain their activity until the date shown on the bottle.

Control Procedures
The positive and negative control sera provided should be used to check the correct working of the latex reagent each day before routine tests are performed.
The positive control serum must cause visible agglutination with the latex reagent within two minutes.
The negative control serum should cause no agglutination within two minutes.
Do not use the test if reactions with the control sera are incorrect.

Important procedure notes
Do not allow the test latex to become contaminated by letting the dropper tip touch the samples.
Ensure that the caps are secure for storage.
Do not touch the reaction areas on the reaction cards as this may introduce a greasy surface on the card and affect the reaction.
After use, return the kit to the refrigerator ensuring that the bottle is stored in the upright position.

Specimen collection and preparation of test material
Both serum and plasma may be used for the test. Blood collected by venipuncture in a clean tube without anticoagulant may be used to prepare
serum. For preparation of plasma, the anticoagulant EDTA is suitable. Centrifuge to obtain the clear plasma (Do not use specimens which are
contaminated or grossly haemolysed. Serum or plasma samples must be clear and particle free).
Specimens which are not tested on the same day as collection should be stored between 2-8°C for up to three days. For longer storage, freeze at -20°C. It
should be noted that specimens which are frozen and thawed more than once may result in an altered antibody level.

Qualitative assay
Method
1. The latex reagent and the serum or plasma must be brought to room temperature before performing the test. Make sure that the latex is in homogeneous suspension by shaking the bottle and expelling any latex from the dropper pipette for complete mixing.
2. Dispense one free-falling drop of the latex reagent onto a ring of the reaction slide.
3. Using a paddle pastette, add one drop of patient’s serum or plasma to the same ring.
4. Mix the latex and serum together and spread to cover the entire area of the ring.
N.B. The blade end of the paddle pastette can be used for mixing. It is advisable, for safety reasons to expel any excess sample from the pastette before mixing the sample and latex together. This will reduce the risk of accidental contact of the operator with human serum.
5. Gently rock the card in a circular motion for up to 2 minutes and observe for agglutination. Do not rock the card for longer than 2 minutes and do not use a magnifying glass to aid reading the result.
A POSITIVE sample will normally cause agglutination within 1 minute.
6. The card and paddle pastette should be disposed of after immersion in sodium hypochlorite solution.
7. Recap the bottle and return to the refrigerator.

Results
Agglutination of the latex reagent within two minutes is a positive result and indicates the presence of the heterophile antibody associated with infectious
mononucleosis: Disregard any reactions occurring after this time. If required, a semi quantitative assay may be performed (refer to the section below).
If no agglutination occurs, this indicates the absence or very low level of heterophile antibody and therefore constitutes a negative result.

Semi-quantitative assay
It may sometimes be useful to determine the heterophile antibody titre, particularly in the acute and convalescent stages of the disease, to establish if there is a declining titre and thus aid in monitoring the disease prognosis. This may be accomplished by performing the slide agglutination test on serial dilutions of the serum.

Method
1. Add 0.5 ml of isotonic saline to each of the six small test-tubes in a rack.
2. To the fist test tube, add 0.5 ml of the serum (test sample) then transfer 0.5 ml to the second tube. Continue the dilutions to the sixth tube to give dilutions of 1/2, 1/4, 1/8, 1/16, 1/32 and 1/64. Do not discard the excess 0.5 ml from the last tube.
3. Perform the slide agglutination test on each of the serum dilutions as described for the qualitative assay.
4. The heterophile antibody litre of the sample is taken as the reciprocal of the last sample dilution which produces a positive result.
5. If the sixth tube gives positive agglutination the sample should be further diluted in the same way in four more tubes to give 1/128, 1/256, 1/512 and 1/1024 and tested by the slide agglutination test.
6. The test tubes, pipette tips, cards should be immersed in sodium hypochlorite solution and then disposed of.

Performance characteristics
The performance of the Oxoid Infectious Mononucleosis kit has been validated against differential red cell absorption tests. Trials were conducted at an independent external centres, on a total of 300 paired serum/plasma samples. These studies demonstrated the sensitivity and specificity BM of the Oxoid Infectious Mononucleosis kit for the detection of heterophile antibodies specific for infectious mononucleosis.

The results of the trial are summarised below⁶:

* Throughout the evaluation, a small number of samples were found to have negative I.M. screens by both methods, but which apparently have morphology
suggestive of I.M. There are a number of possible explanations for this.
1. The patient is in the early stage of the disease and heterophile antibody is not yet demonstrable by agglutination methods.
2. Lymphocytosis and atypical mononuclears are not necessarily diagnostic of I.M. Similar changes can be found in other viral infections.
3. The differentiation of atypical mononuclears from reactive lymphocytes on a blood film is rather subjective. Reactive lymphocytes alone
are not usually indicative of I.M.

Limitations of the procedure
A negative result in a patient with obvious clinical signs of infectious mononucleosis may indicate that insufficient antibody has developed (in rare cases antibody may not be detectable for up to three months). If this situation occurs, further samples should be tested at a later date. Paediatric patients may fail to produce any detectable antibody at all.
With a very high antibody level, the phenomenon of prozone may occur and give a false negative result. Should this be suspected make a 1 in 20 dilution of
the serum (0.1 ml of serum added to 1.9 ml of saline) and test the diluted sample with the latex reagent.
The presence of heterophile antibody has been demonstrated in other disease states such as leukaemia, Burkitts lymphoma, rheumatoid arthritis, viral hepatitis and cytomegalovirus infections⁷.
As heterophile antibody may persist for several months after recovery, a positive result should not be regarded as indicative of acute infectious mononucleosis in isolation of the clinical and haematological information.
When performing the semi-quantitative assay, the demonstration of an antibody within individual patient is useful to gauge the prognosis of disease. The demonstration of an antibody titre on a single sample is not indicative of the severity of the disease.
The result obtained with OXOID Infectious Mononucleosis Kit must be considered with both the haematological findings and the clinical symptoms of
the patient before a diagnosis of Infectious Mononucleosis is made.
WARNING: This product contains sodium azide. Harmful if swallowed.

References
1. Paul J. R. and Bunnell W. N. The presence of heterophile antibodies in infectious mononucleosis. Am. J. Med. Sci. 1932; 183. 90-104.
2. Davidsohn I. Serologic diagnosis of infectious mononucleosis. JAMA 1937: 108. 289-295.
3. Davidsohn I. and Slaby R. Horse agglutinins in infectious mononucleosis. Am. J. Clin. Path. 1968: 49. 3-11.
4. Beer P. The heterophile antibodies in infectious mononucleosis and after injection of serum. J. Clin. Invest. 1935: 15. 591-599.
5. Fletcher M. A and Woolfolk B. J. Immunochemical studies of infectious mononucleosis. Isolation and Characterisation of heterophile antigens from hemoglobin-free stroma. J. Immunol. 1971: 107. 842-853.
6. Data on file at Oxoid Limited.
7. Henle G. E Horwitz C. A Hum. Pathol. 1974: 5. 551-565.

Paddle Pastettes is a registered trademark of Alpha Laboratories.