IT9022473A1 - COMPETITIVE IMMUNOENZYMATIC METHOD FOR THE DETERMINATION OF PLASMODIAL ANTIGENS IN HUMAN BLOOD - Google Patents

Description

DESCRIPTION

The present invention relates to a competitive immunoenzymatic method for the determination of plasmodial antigens in a human blood sample.

The invention also relates to a diagnostic kit for carrying out this method.

The causative agent of malaria is a protozoan of the genus Plasmodium which is transmitted to the vertebrate host (eg humans) through the bite of the Anopheles mosquito. Of the four human infectious species, Plasmodium malariae (P.malariae), Plasmodium_ ovale (P.ovale), Plasmodium falciparum (P. falciparum), and Plasmodium vivax (P.vivax), the latter two cause most of the morbidity and mortality associated with the disease.

Malarial parasites develop according to a multi-stage cycle presenting the host with a very large number of antigenic components and each form of development of the parasite contains antigens which are different and stage-specific.

Malaria is one of the major health problems worldwide. In fact, every year this disease affects more than 250 million individuals causing, in early childhood, a mortality that can reach 50% of cases.

Alongside the need to develop an effective antimalaria vaccine, there is in the art the need for a diagnostic method capable of detecting plasmodial antigens in a blood sample.

In fact, even in the presence of classic clinical symptoms such as fever, nausea, muscle pain, dizziness, chills of cold and heat, the clinical diagnosis is necessary both to confirm and to deny this disease.

The current situation regarding diagnostics in the field of malaria has been described in the report of the World Health Organization (WHO / MAL- / 88.1045) where, among other things, it is hoped that simple and economic methods for the determination parasite in human blood (clinical diagnosis).

Among the various methods developed in the art for the determination of plasmodial antigens both in blood and in mosquitoes, we can distinguish those of the IRMA (Immunoradiometric Assay) type, which, despite being very sensitive, have drawbacks (high costs and safety) deriving from the use of radioisotopes, and those of the ELISA type (Enzyme-Linked Immunosorbent Assay).

In particular, among the ELISA assays, competitive immunoenzymatic methods have been developed in which: i) the antigen (Ag) present in the sample and the antigen adsorbed on the solid phase (S-Ag) compete in binding with a limited amount of antibody labeled (Ab-E) specific for this antigen according to the following reaction:

The inhibition of the active-complex bound to the support (S-Ab-Ag-E) results in a reduction of the final color compared to the negative control (analysis carried out in the absence of the sample);

ii) the antigen (Ag) present in the sample and the labeled antigen (Ag-E) compete in binding with the specific antibody adsorbed on a solid support (S-Ab) according to the following reaction:

The inhibition of the active complex (S-Ab-Ag-E) results in a reduction of the final color compared to the negative control (absence of the sample);

The main limitations of these methods derive from the fact that:

Ag and Ab are present on the support in little controllable quantities and in heterogeneous phase (slow reaction); steric and mobility reasons disadvantage the antigen as a parasite with respect to the Ab-E or Ag-E conjugate in binding to supported Ag or Ab;

the presence in the biological sample of specific free antibodies or fragments of incompletely metabolized antigen can give rise to false negatives or false positives.

For all these reasons these methods show a reduced sensitivity.

Currently, therefore, the most suitable method for diagnosing malaria is to search for the parasite in the blood by observing an appropriately colored blood smear under an optical microscope.

Microscopic analysis allows to reveal a minimum of 4 parasites per 1 μl of blood and to distinguish the different Plasmodium species. However, this method requires an efficient microscope, highly skilled personnel and is not suitable for analyzing large numbers of samples.

An assay suitable for the clinical diagnosis of malaria must be characterized by high sensitivity, i.e. ability to detect low levels of extraneous antigens (parasites) in the blood, specificity and reproducibility.

Furthermore, such an assay must be simple (not requiring numerous steps or reagents), quick and cost-effective.

The present invention makes available a competitive immunoenzymatic method for the determination of a plasmodial antigen in a blood sample which allows to overcome the problems of the known art.

The immunoenzymatic method according to the present invention is basically based on the fact that the competition reaction between the labeled antigen (Ag-E) and the free antigen (Ag), possibly present in the sample, in the binding with the specific antibody for the formation of the antibody-antigen complexes (Ab-Ag and Ab-Ag-E) it is carried out in the liquid phase and the resulting complexes are bound to Protein A adsorbed on a solid support.

In particular, the immunoenzymatic method according to the present invention comprises:

a) reacting a sample containing the plasmodial antigen to be determined with the specific antibody for said antigen and with the labeled antigen so as to obtain the antibody-antigen complexes Ab-Ag and Ab-Ag-E;

b) binding the complexes formed in step a) to Protein A adsorbed on a solid support; and finally c) determining the labeled antibody-antigen complex bound to the solid support by detecting the label.

Plasmodial antigens which can be determined by the method of the present invention include those of the erythrocyte stage of Plasmodium.

Solid supports useful for carrying out the method of the present invention can be selected from those commonly used in an ELISA assay as long as they do not give non-specific background interference. For example, commercially available polystyrene plates can be used.

The quantity of Protein A adsorbed on a solid support, for example the wells of a microplate, must be such as to bind all the antibody used in the competition reaction.

Generally, an excess amount of Protein A will be used with respect to the antibody.

Thus, for example, using plates in which 100 ng of protein A were used in the adsorption (coating), each well was found to be capable of adsorbing 6 ng of monoclonal antibody (MAb).

It was also observed that doubling the amount of Protein A (200 ng) in the coating the results of the inhibition tests remained unchanged.

The adsorption of Protein A on such supports can be carried out according to conventional techniques.

For carrying out the method of the present invention, polyclonal or, preferably, monoclonal antibodies capable of reacting specifically with the plasmodial antigen to be determined can be used.

Amounts of antibody ranging from 1 to 6 ng per well, preferably from 1 to 2 ng per well, are generally used.

Antigens used for the preparation of the antigen-labeled conjugate (Ag-E) are generally synthetic peptides having the sequence corresponding to that of an epitope of a Plasmodium surface antigen, preferably antigens. of the erythrocyte stage.

Marking substances can be chosen from the group of enzymes capable of developing, in the presence of a specific substrate, a colorimetric reaction such as, for example, peroxidase, alkaline phosphatase and β-galactosidase, or biotin.

The preparation of the antigen-labeling conjugate can be carried out according to conventional techniques The quantity of Ag-E conjugate used in the method according to the present invention is chosen as the minimum quantity that favors the inhibitory activity of the free antigen present in the sample (sensitivity of the method) and, at the same time, allow sufficient development of the final color in the negative control against which the inhibition given by the positive samples is calculated.

Amounts of conjugate ranging from 0.1 to 2 ng / well, preferably from 0.2 to 0.5 are generally used.

The stoichiometric ratio between antibody (Ab) and Ag-E conjugate is chosen so that Ab is <Ag-E.

In step c) of the method according to the present invention, after suitable washing to eliminate the material in solution, the quantity of the Ab-Ag-E complex bound to the support is determined by adding a specific substrate for the marker used. The reduction in color, which is observed when the antigen is present in the sample, compared to that of the negative control (in the absence of sample or with negative sample) is comparable to the quantity of antigen present in the sample based on the standard inhibition curve of Ag.

The method according to the present invention shows the following advantages:

- the simple and reproducible coating of the plate requires a low consumption of Protein A;

- the dried plate is stable over time;

- there is no need for plate post-coating, i.e. saturation of the residual binding sites with an inert protein. In fact, there was no non-specific background absorption even when human erythrocyte lysate is used;

- the possibility of using reduced quantities of labeled antigen and antibody compared to those used in competitive ELISA methods in which Ag or Ab are bound directly to a support; and finally - the possibility of using automatic equipment available in any immunodiagnostic laboratory.

A diagnostic kit suitable for conducting the method according to the present invention will comprise a solid support, preferably a polystyrene ELISA plate, adsorbed with Protein A, the specific antibody for the antigen to be determined, the labeled antigen, the enzymatic substrate specific for the marker and swabs.

The experimental examples which follow are illustrative and not limitative for the invention.

Example 1

Determination of the optimal amounts of antibody and labeled antiqene in the competition assay.

Purified Protein A (Pharmacia) is dissolved in TBS buffer (25 mM tris-HCl, 0.15 M NaCl, pH 7.8) at a final concentration of 1 ug / ml. Aliquots (0.1 ml) of this solution are introduced into each well of a 96-well Dynatech M129A plate. Then the plate is incubated at room temperature (20-25 ° C), in a humid chamber, for one night and then washed twice with TBS containing 0.05% Triton X-100 (TBS-TX).

If the plate is to be preserved, after incubation it is washed twice with TBS, dried, at 37 ° C for 1 hour (dried plate) and then stored at 4 ° C. Before being used, the plate is washed twice with TBS-TX.

The peptide (NANP) 20 (70 mg) and the enzyme peroxidase (Calbiochem) (10 mg) are conjugated by operating as described in the Italian patent application N.

22817-A / 86.

In each well of the microplate adsorbed with Protein A, 0.1 ml of the following solutions in TBS-TX buffer are dispensed in duplicate:

a) (NANP) 20-peroxidase (5 and 10 ng / mL), MAb 2A10 antisporozoite antibody (10, 20, 30, 50 and 60 ng / mL) (negative controls) and

b) (NANP) 20-peroxidase (5 and 10 ng / mL), anti-sporozoite MAb 2A10 antibody (10, 20, 30, 50 and 60 ng / mL) (NANP) 20 (10 ng / mL) (positive controls ).

After 1 hour at room temperature the plate is washed with 200 μl of TBS-TX (4 times) to remove the dissolved components.

In each well, 0.1 ml of peroxidase substrate solution prepared at the time of use is added by adding 1 volume of 0.1 M citric acid containing 3,3 ', 5,5'-tetramethylbenzidine (TMB) (1 mg / ml ) to 9 volumes of 0.1 M acetate buffer (pH 5.0) containing H-2O2-1.3 mM.

After 1 hour at room temperature, the enzymatic reaction is stopped by adding in each well 50 μl of 1 M sulfuric acid. The color that develops is determined by evaluating the absorbance (A) at 450 nm with an automatic plate reader "Lambda Automatic Microtiter Reader "from Perkin-Elmer.

The inhibition values are calculated as follows:

The results in Figure 1 show that the minimum quantities of MAb and (NANP) 20-peroxidase used, respectively 1 ng and 0.5 ng, correspond to the maximum inhibition (about 60%) and a sufficiently high negative control value. Therefore these quantities were used in the subsequent examples.

Example 2

Determination of the inhibition curve with synthetic sporozoite antigen (NANP) 20

In each well of the microplate adsorbed with Protein A, 0.1 ml of the following solutions in TBS-TX are introduced in triplicate:

a) (NANP) 20-peroxidase (5 ng / ml), antibody

anti-sporozoite MAb 2A10 (10 ng / ml) (negative control)

b) (NANP) 20-peroxidase (5 ng / ml), anti-sporozoite MAb 2A10 antibody (10 ng / ml) and (NANP) 20 (from 0, la 80 ng / ml) (positive controls).

We operate as reported in example 1 above.

Figure 2-A shows the inhibition curve obtained.

The sensitivity of the system, indicated by the high inhibition value (about 50%) produced by 1 ng of (NANP) 20, and the regular profile of the inhibition curve confirm the applicability of the method to the determination of P. falciparum sporozoites in a biological sample.

Example 3

Determination of the inhibition curve with synthetic sporozoite antigen (NANP) 20 in the presence of human erythrocyte lysate.

In order to investigate the applicability of the method in the detection of malarial antigens present in human erythrocytes, the effect of erythrocyte lysate was examined.

a) Preparation of erythrocyte lysate

Whole blood samples, added with EDTA as an anticoagulant, are stored at 4 ° C and used within three days of collection.

In each of 18 1 ml Eppendorf tubes, 20 μl of blood and 1 ml of TBS buffer containing 0.1% Tween-20 (TBS-T) are introduced and, after mixing, centrifuge at 2,000 rpm for 4 minutes (Eppendorf Centrifuge) . Then the supernatant is removed by aspiration and the sedimented erythrocytes are washed 3 times with 1 ml of TBS-T, centrifuging each time as above.

Finally the combined final sediments are taken up with 4.5 ml of TBS-TX haemolysing buffer (TBS, pH 7.8 containing 0.05% Triton X-100) and lysed at room temperature for 1-2 minutes,

b) Competitive enzyme immunoassay.

0.05 ml of lysate (equivalent to 4 μl of blood) are introduced, in duplicate, into each well of the microplate together with 0.05 ml of the following solutions in TBS-TX buffer:

a) (NANP) 20-peroxidase (10 ng / ml), antisporozoite antibody MAb 2A10 (20 ng / ml) (negative control) and b) (NANP) 20-psroxidase (10 ng / ml), antisporozoite antibody MAb 2A10 (20 ng / mL) and (NANP) 20 (0.2 to 160 ng / mL) (positive controls).

After 1 hour at room temperature the plate is washed 4 times with TBS-TX to remove the components in solution.

Then in each well 0.1 ml of the solution containing the enzymatic substrate 3,31,5,5 '-tetramethylbenzidine (TMB) (0.1 mg / ml), H202 1.3 mM, in acetate / citrate buffer pH are introduced. 4.7. After incubation at room temperature for 1 hour, the enzymatic reaction is stopped by adding 50 μΐ of 1 M sulfuric acid to each well. The color is determined by evaluating the absorbance at 450 nm with Perkin's "Lambda Automatic Microtiter Reader". -Elmer.

The inhibition curve shown in figure 2-B, very similar to that shown in fig. 2-A, shows the absence of background interference from the US.

The inhibition values in the presence of lysate are slightly higher than those obtained in the absence of lysate (curve A). These results are due to the slight reductive effect of the lysate on NANP 20-peroxidase which leads to an increase in competition (inhibition) by NANP20.

Example 4

Determination of P. falciparum sporozoitic antigens.

P. falciparum sporozoites extracted from Anopheles stephensi mosquitoes are suspended in TBS-TX buffer at a concentration of 250,000 sporozoites / ml.

Then dilute solutions (from 200 to 40,000 sporozoites / ml) of said suspension in TBS-TX are prepared. In each well of an ELISA microplate adsorbed with Protein A, 0.05 ml of these solutions and 0.05 ml of the solution containing (NANP) 20-peroxidase (10 ng / ml) and anti-sporozoite antibody are introduced in duplicate. MAb 2A10 (20 ng / ml).

The negative control, examined in parallel, is prepared by introducing into each well 0.1 ml of a solution in TBS-TX buffer containing (NANP) 20-peroxidase (5 ng / ml) and anti-sporozoite MAb 2A10 antibody (10 ng / ml).

The inhibition values, shown in figure 3, show a detection limit of about 125 sporozoites, corresponding to 20% of inhibition.

Example 5

Determination of P. falciparum sporozoitic antigens.

One operates as in example 4 using biotinylated MAP4- (NANP) 10 compound (prepared as reported in Italian patent application No.19800 A / 90) as the labeled antigen [Seyer et al., (1989), Int. J. Peptide Protein Res., 24: 235) 0.2 ng / well, 1 ng / well of MAb 2A10 antibody and up to a maximum of 2000 sporozoites per well.

After incubation, 0.1 ml of a diluted solution (100 times) of Streptavidin-alkaline phosphatase (Boehringer) in TBS-TX buffer is introduced into each well.

After about 1 hour at room temperature, 0.1 ml of p-nitrophenylphostat (NPP) enzyme substrate in diethanolamine buffer pH 9.8 (BIORAD) is added to each well.

The enzymatic reaction is stopped by introducing 50 μl of 1.5 N NaOH into each well and the absorbance is read at 405 nm.

From the inhibition curve (figure 4) a detection limit of about 500 sporozoites is observed. The inhibition value of the positive control (1 ng MAP4- (NANP) 10) is shown in the same figure.

Claims (4)

CLAIMS 1. Competitive immunoenzymatic method for the determination of plasmodial antigens in a human blood sample which includes: a) reacting a sample containing the antigen (Ag) to be determined with the specific antibody (Ab) for said antigen and with the labeled antigen (Ag-E) so as to obtain the antibody-antigen complexes Ab-Ag and Ab-Ag-E; b) binding the complexes formed in step a) to Protein A adsorbed on a solid support; and finally c) determining the labeled antibody-antigen complex bound to Protein A adsorbed on the solid support by detecting the label. 2. The immunoenzymatic method of claim 1, wherein plasmodial antigens are selected from those of the erythrocyte stage. The immunoenzymatic method of claim 1, wherein the antibody is a polyclonal or monoclonal antibody capable of reacting specifically with the plasmodial antigen to be determined. 4. The immunoenzymatic method of claim 1, wherein the marker is an enzyme selected from peroxidase, alkaline phosphatase and β-galactosidase or biotin. The immunoenzymatic method of claim 1, wherein the labeled antigen is a synthetic peptide.