ENZYME

### ENZYME-LINKED-IMMUNO-SORBENT-ASSAY (ELISA)

>     INTRODUCTION
>     PREPARATION OF THE ANTIGEN FOR THE ELISA
>     COATING OF THE ELISA PLATES
>     MATERIALS (SOLUTIONS)
>     PERFORMANCE OF THE TEST
>     READING OF THE TEST RESULTS
>     SETTING UP ELISA PROCEDURES
>     STANDARDIZATION OF THE ELISA
>     RESULTS
>     ROLE OF BOVINE SERUM ALBUMIN AND TWEEN 20
>     DEFINITION OF ELISA TITRE
>     ADVANTAGES OF ELISA
>     REFERENCES

INTRODUCTION

If one looks at the literature it is clear that the ELISA is often used as an additional test or alternative to the MAT. Other serological methods such as counter-current-immuno-electrophoresis, or an immuno fluorescence test for the detection of antibodies also have shortcomings that prevent their effective competition with the ELISA. The IFT giving largely similar results in the ELISA comes near to the ELISA.

The success of the ELISA is probably that the method provides highly useful information on clas-specificity of the antibodies which is of clinical improtance.

IgM-antibodies are compatible with current or recent disease. ELISAs come in a large variety. Their main advantage in comparison with the MAT is probably the stability of antigenic preparations and their so-called genus-specificity meaning that as far as we know with a single antigenic prepration all types of leptospirosis irrespective of the causative serovars can be diagnosed.

As the nature of the serogroup and serovar antigens are better understood the value of this test becomes more appreciated. Based on antigen selection it can either show broad group specificity or the other extreme considerable serovar specificitiy.

From the very beginning we aimed in our laboratory at an ELISA that could be applied in tropical countries. Such an ELISA should conform to important criteria such as a relative simplicity, avoidance of the need for sophisticated, vulnerable and expensive apparatus and stability of reagents (Terpstra at al., 1980, 1985)

We started with the examination of various antigenic preparations that could be coated to wells in polysterene microwell plates that are generaly used for ELISAs. We obtained acceptable results when we tested the following antigenic preparations that were put into the wells in a microtitre plate to adherte to the polysterene:

1. A suspension of liptospires that were discrupted by sonification. This preparation could be stored for only a few weks in the refrigerator.
2. Whole leptospires that were coated to the polysterene by the use of poly-L-lysine and glutaraldehyde. This preparation could also be stored for only a few weeks at 4°C.
3. An ethanol extract from leptospires. This antigenic extract of leptospires remains stable for a long term but we found that the yield of antigen was low.
4. The supernatant of a heated culture of leptospires .We considered this antigenic preparation useful and economic as large amounts could be obtained from cultures in a relatively easy way. Last but not least the antigen is very stable. We found that the antigen coated to polystyrene in microtitre plates was still fully reactive after storage for over a decade at room temperature in a dry place.

In ELISA procedures the test results can be read as the intensity of the colour reaction to gauge the antibody level. This procedure demands the use of apparatus to determine optical densites (OD). We decided that we preferred the detgermination of an antibody titre using twofold serial dilutions of the serumsample to be examined, to be applied into subsequent wells in microtitre plates. This procedure allwos the reading of the results with the naked eye.

It should be mentioned that this procedure requires the use of a chromogen that can be clearly discerned by the human eye. We chose 5-amino-salicylic acid.

The titre can be determined by observed a gradual shift in the staining intensity from dark reddish-brown via light reddish-brown to uncouloured in a row of wells containing the serially diluted serum specimenn. This procedure has the advantage that it largely avoids inconsistency in test results that may occur easilyin a procedure using a single serum dilution due to variability between wells in their capacity to bind antigen.

We observed that specific anti-leptospira antibodies of the IgM class appeared approximately six weeks and decreased subsequently over the months.

Specific IgG antibodies were observed somewhat later than IgM antibodies. IgG antib odies peaked also after a few weeks and dropped off to low levels.

PREPARATION OF THE ANTIGEN FOR THE ELISA

1. Leptospira copenhageni strain Wijnberg is grown in EMJH medium for 10-12 days at 30°C in a shaking incubator. Abundant growth is necessary to produce a good antigen.
2. If necessary add 5 ml of sterile diluted Tween 80 (1:10) to 500 ml culture under sterile conditions 4-5 days after inoculation.
3. Incubate for another 5-6 days.
4. Before harvesting check for abundant growth and purity. Also check for the absence of Tween. This can be done by killing a sample from the culture with formalin (final concentration 0.5%) centrifuging at 10,000 r.p.m. for 30 minutes and heating the supernatant in a glass tube in a boiling waterbath. If the supernatan becomes turbid not all the Tween has been cosumed. The turbidity disappears quickly upon cooling. If there is turbidity, leave the cultures a few days longer in the shaking incubator initil all of the Tween has been used up.
5. After checking, the leptospires are killed with formalin (0.5% final concentation).
6. After 1 hour the killed leptospira culture is heated in a boiling waterbath for 30 minutes, shaking every 5 minutes.
7. After cooling to roomtemperature the culture is centrifuged for 30 minutes at 10,000 r.p.m. Centrifutation at lesser rotations per minute is also possible. The supernatant is used as antigen.
8. In the liquid form the antigen is stable for years if stored at 4°C.

COATING OF THE ELISA PLATES

1. 100 ul of antigen are pipetted into every well of the microtitre plate.
2. The plates are left at roomtemperature until complete evaporation of the fluid has taken place (1-3 days, depending on temperature and humidity); avoid exposure to sunlight.
3. The coated lates are kept in a closed box, or in sealed plastic bags, at romtemperature. The antigen is stable for years.

MATERIALS (SOLUTIONS)

PBS pH 7.2

- Dissolve per litre distilled water:
  8.5 g NaCl
  0.85 g Na2HPO4.2H2O
  0.25 g KH2PO4
  Adjust to pH 7.2 with 1N NaOH or 1N HCl

PBS/Tween

- PBS pH 7.2 + 9.05% Tween 20

PBS/Tween/BSA

- PBS/Tween + 0.5% BSA

Substrate

- Dissolve 80 mg 5-amino-2-salicylic-aid (5 AS) in 100 ml distilled water at 75°C. This solution is stable for 1 month if kept at 4°C. Before using bring pH to 6.0 with 1N NaOH. Add to 9 parts 5-AS 1 part of 0.05% H2O2 just before use (1.5 ml 1% H2O2 to 28.5 ml distilled water)

PERFORMANCE OF THE TEST

1. Rinse the coated plates 4 times with PBS/Tween.
2. During the last two washings leave the fluid in the wells for 1 minute.
3. Dry the plates by tapping them on a cloth of filter paper.
4. Make doubling dilutions of the patient's serum and control era in PBS/Tween/BSA. Fill the second column of the wells with 180 ul. Use mixing throughly, 100 ul to the 3rd well, etc. Discard the final 100 ul. Use the first column as blaknk.
5. Cover the plate with another microtitre plate and incubate 1 hour at 30°C.
6. After 1 hour wash the plate as under 1.
7. Add to all wells 100 ul conjugate, diluted in PBS/Tween/BSA. For each of the conjugates an optimal dilution should be used (i.e. block titration).
8. Incubate 1 hour at 30°C as before.
9. Discard the contents and wash four times as above.
10. Add to all wells 100 ul substrate. Mix well.
11. Leave at room temperature for 2 hours.

READING OF THE TEST RESULTS

By scanner:

Read the optical densities using a multiscanner with a 492 nm filter. Use column 1 for measuring "blaknk". The end-point (titre) is the last well (dilution) in which you find a colour giving an OD equal to or greater than half the value of the darkest well of the positive control.

By eye:

See above. The substrate gives a reddish brown colour which makes it easy to read by eye.

SETTING UP ELISA PROCEDURES

Choice of antigen and choice of plates

The choice of the antigen determines the nature of the plates to be used. In general:

- protein binds well to polystyrene or pvc
- lipids bind to pvc
- polysaccharides do not bind well so a covalent cross-linkery may be used (glutarldehyde, poly-L-lysine) with polysterne or pvc

We suppose that heat-stable antigen is mainly of a polysaccharide nature and bound to polysterene during the evaporation procedure.

If crude preparation of antigen is used and its composition is not well defined, start with a soluble fraction of the preparation and try different types of plates. Many bacteria and unicelluar organisms can be coated whole onto a plate which may facilitate recognition of surface antigens.

Choice of conjugate

This is determined by:

- primary antibody to be detected (species; class, IgM for early detection)
- stability: peroxidase conjugates are usually the most stable
- availability and cost: will vary with cointry or supplier
- substrate desired: when a colour detectable by the naked eye is needed then substrates must be selected that are clearly discrenable and the enzyme of the conjugate must be able to produce it.

Choice of substrate

This is determined by:

Sensitivity of the substrate: for example, peroxidase substrates in the order of sensitivity are:

- 5AS 5-amino-calicylic acid (low)
- ABTS Azinobis-3-ethylbenzthiazoline-sulfonic-acid (moderate)
- OPD ortho-phenylenediamine (high)
- TMB Tetra-methylbenzidine (high)

> In this case high sensitivity means that a higher Optical Density (OD) is obtained with the same amount of enzyme, which makes the reading easier.

Stability of the substrate itself, or of the reagents in which it is dissolved. 5 AS is most stable, since a solution can be stored for several weeks. OPD/TMB are less stable, a fresh solution (containing methanol) has to be prepared shortly before use.

Availability of an automatic reader: if reading is to be done by the naked eye, 5 AS is best because of the clear distinciton between the purple colour of positive wells and the colourless negative and blank wells.

Health hazards: TMB is mutagenic and OPD is carcinogenic, so both of these must be handled with great care under safe conditions.

Type of standardization used in the test:

If the end-point titres are read, 5 AS can be used without stopping the reaction. 5AS inactivates itself during the enzyme reaction and once that reaction is complete, the colours will remain stable, even for days.

STANDARDISATION OF THE ELISA

Antigen concentration

Too high concentrations of antigen will result in overcoating, so that the antigens are not only attached to the platge but also to each other, by relatively weak hydrophobic interactionis. These interactions can be broken during later steps in the washing and incubation so that antibodies bound to the top layer of antigen will be lost from the plate during washing. This will give a false estimation of the amount of antibodies bound and thus an understimation of positive reactions. Overcoating may also result in insufficient exyposure of the antigenic determinants which are most important or of most interest; this will also give an underestimation of positive reactions.

If lower concentrations of antigen are used, not all of the active binding sites on the plate are blocked.

Antibodies and other serum proteins will bind to these sites and the test will have a high background (high readings for blanks and negative controls).

Conjugate dilution

In general, use the dilution advised by the manufacturer. If there are practical reasons (eg. to reduce costs) you can try several dilutions of the conjugate.

The concentration is determined by performing a checkerboard titration between antigen (different concentrations) and a known positive and a known negative serum sample.

Example of conjugate titration:

Dilution of conjugate | Control sera | Titres
1:1000 | positive | 1:10,240
1:1000 | negative | 1:40
1:2000 | positive | 1:5,120
1:2000 | negative | neg/1:10
1:3000 | positive | 1:2,560
1:3000 | negative | negative
Colour in wells is lighter than 1:1000 and 1:2000
1:4000 | positive | 1:2,560
1:4000 | negative | negative
Colour in wells is lighter than 1:1000, 1:2000 and 1:3000

Conclusion: A conjugate dilution of 1:2000 is chosen.

Substrate

In all of the standardisation procedures, you must use known positive and negative serum samples, i.e. serum samplsfrom healthy individuals and from individuals known to have leptospirosis by other detection methods (bacteriological/clinical).

The high degree of cross-reacticity in many endemic area amongst the organisms present can give problems.

RESULTS

ELISA results can be interpreted using a cut-off point, which is separately determined for each test.

After following all of these steps, one will have a standard ELISA protocol providing reproducible results. The cut-off point depends on the presence of persisting antibodies of past infections in the community. the cut-off point must be determined in one's own laboratory, using local positive and negative controls.

ROLE OF BOVINE SERUM ALBUMIN AND TWEEN 20

For the preparation of antigens as well as in performance of ELISAs one often uses Tween and/or Bovine Serum Albumin (BSA). What can be said about the funciton of these reagents in the coating of the plates as wells as for the washings?

Tween is a detergent used as a locking agent for non-specific binding. Tweens are also components of the medium in which leptospires are grown, but can block the coating of the antigen to the plates. So, it is very important that all the Tween has been consumed by the leptospires before the antigen is propared.

BSA as a protein source has a high affinity to plastic of the ELISA plates. BSA is a constituent of the growing medium.

The leptospira ELISA antigen will be bound to the plate very easily.

DEFINITION OF ELISA TITRE

The titre is the last dilution (well) giving an absorbance of more than half of the highest absorbance (darkest colour) of the positive control.

ADVANTAGES OF ELISA

The advantage of ELISA using serial dilutioins is that the influence of possible initer-well variation on the test results is reduced.

The major advantage of the heat extracted antigen is its stability, as it can be stored for a long time both in the liquid state as well as coated to polyssterene.

Furhermore, the test is genus specific, no expensive tools (ELISA reader) are required, can be read by naked eye, and IgM and IgG antibodies can be detected.

The ELISA can be prformed using a single antigenic preparation, while for the MAT often a panel of serovars for antigen is needed.

The use of a single antigenic preparation can be of value when an acute cliical condition suspected to be leptospirosis needs to be assessed and the type of the causative strain is of no immediate relevance to the patient in need of undelayed treatment.

REFERENCES

Terpstra WJ, Ligthart GS and Schoone GJ. Serodiagnosis of human leptospirosis by ELISA. Zbl.Bakt.I.Abt. Orig. 1980; A 247; 400-405.

Terpstra WJ, Ligthart GS and Schoone GJ. ELISA for the detection of specific IgM and IgG in human leptospirosis. J. Gen. Microbiol. 1985; 131: 377-385.