• Leucine aminopeptidase (LAP) is an enzyme used to distinguish catalase-negative, gram-positive cocci of Aerococcus and Leuconostoc (LAP test negative) from other organisms in the group, such as Streptococcus, Enterococcus, Lactococcus, and Pediococcus (almost always positive).
• The LAP test is mostly used for the preliminary characterisation of organisms with Streptococcus-like characteristics.
• For identifying catalase-negative, Gram-positive cocci, the LAP test is simply one of several helpful tests. The streptococcal identification panel (API Rapid Strep) includes an LAP test, for instance.
• Streptococcus pneumoniae and Streptococcus pyogenes, Pediococcus, Lactococcus, and Enterococcus species are LAP positive in general, whereas other beta-hemolytic Streptococci, Aerococcus, and Leuconostoc species are LAP negative.
• The discs are impregnated with leucine-ß-naphthylamide, which is degraded by LAP-positive organisms’ leucine aminopeptidase.
• This enzymatic activity releases ß-naphthylamine, which, when combined with pdimethylaminocinnamaldehyde reagent (Dalynn RP95), forms a very visible red Schiff base.

Purpose

• To distinguish the catalase-negative, gram-positive cocci of Aerococcus and Leuconostoc (negative LAP test) from other organisms in the same genus, such as Streptococcus, Enterococcus, Lactococcus, and Pediococcus (almost always positive).

Principle

On a paper disc inoculated with Streptococcus, Enterococcus, Lactococcus, and Pediococcus, hydrolysis of the leucine-pnaphthylamide substrate by the enzyme LAP liberates leucine and free b-naphthylamide. The combination of bnaphthylamide with cinnamaldehyde reagent produces a pink to cherry-red hue.

Quality control

Perform QC on each new lot or shipment of LAP discs and colour reagent prior to use. Since the test is conducted infrequently and the vast majority of Gram-positive cocci are LAP-positive, potency must be confirmed by testing the positive control each time a negative test result is obtained using test organisms.

• Enterococcus faecalis ATCC 29212—LAP positive (Red color)
• Aerococcus viridans ATCC 11563—LAP negative (No color change)

Procedure

1. Place the disc in a petri dish or on a slide.
2. Moisten (do not saturate) the disc with distilled water in a loop.
3. Using a sterile stick or loop, spread the isolate under suspicion.
4. Incubate at room temperature for 5 minutes.
5. Add a drop of cinnamaldehyde reagent and notice the colour for two minutes.

Requirement

• Test organisms – Fresh growth of catalase-negative, gram-positive cocci less than 24 hours old. A culture that has been incubated for up to 72 hours may be employed if growth is inadequate.
• LAP Disk 
• 0.01% p-Dimethylaminocinnamaldehyde (the same reagent as used in PYR test)
• Loop or stick Petri dish or slide sterile

Interpretation of Results

• Positive: After the addition of cinnamaldehyde reagent, a deep crimson to reddish-purple hue develops.
• Negative: No change or a slight yellow color 

Microorganisms	LAP test Result
Streptococcus pneumoniae	Variable
S. agalactiae	–
Other β Streptococcus	–
S. pyogenes	+
Enterococcus spp.	+
Alloiococcus otitis	+
G. morbillorum	+
A. viridans	–
A. urinae	+
G. hemolysans	Variable
Globicatella sanguis	–
Helcococcus kunzii	–
Lactococcus spp.	+
Leuconostoc spp.	–
A. adiacens	+
A. defectiva	+
Pediococcus spp.	–
Tetragenacoccus spp.	–
Vagacoccus spp.	+

Uses

• In conjunction with PYR and other biochemical assays, the LAP test is often used to distinguish catalase-positive, gram-positive cocci.
• Leucine aminopeptidase (LAP) is an enzyme used to differentiate catalase-negative, gram-positive cocci of Aerococcus and Leuconostoc (negative LAP test) from other organisms in the group, including Streptococcus, Enterococcus, Lactococcus, and Pediococcus (almost always positive).
• The LAP test is typically employed for the preliminary characterization of organisms exhibiting Streptococcus-like features.

Limitation

• Confirm that the test organism is a grampositive coccus and is catalase negative before performing the LAP test
• Ensure that adequate inoculum is used or false negatives may occur
• Streptococci should be tested prior to 48-hour incubation or subcultured prior to testing  

References

• https://www.ucsfhealth.org/medical-tests/leucine-aminopeptidase-blood-test
• https://www.abcam.com/leucine-aminopeptidase-lap-activity-assay-kit-fluorometric-ab234627.html
• https://www.dalynn.com/dyn/ck_assets/files/tech/DL10.pdf
• https://rml.rush.edu/Lists/Lab%20Test/MainDispForm.aspx?ID=921&ContentTypeId=0x0100795D141F45C16548A0C0634AC557B6C4
• https://www.clinmicronow.org/doi/abs/10.1128/9781683670438.CMPH.ch3.17-26
• https://test-guide-en.srl.info/hachioji_en/test/detail/000270102
• https://5minuteconsult.com/collectioncontent/2-152381/lab-tests/leucine-aminopeptidase
• https://universe84a.com/leucine-aminopeptidase-lap/
• Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
• Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition