GB2114288A - Method for the detection of malaria antigens in human blood - Google Patents

Abstract

A high sensitivity method for detecting the presence of malarial parasites of the order of 1 parasite or less per 10<6> infected human red blood cells, which consists in the inhibition of binding of Plasmodial antibodies, to enriched Plasmodium falciparum antigen preparation or Plasmodium berghei antigen crossreacting with Plasmodium falciparum, present on a solid phase adsorbent. The method is capable to detect at a high sensitivity the ring forms which characterize the parasites in the blood of patients. The detection can be performed by utilizing radioactive materials, fluoresence assay or standard ELISA procedure. A kit for carrying out the method of the invention is also disclosed, comprising a solid adsorbent treated with enriched sonicated red blood cells infected with Plasmodium falciparum, or Plasmodium berghei crossreacting with Plasmodium falciparum, buffer, labelled anti-immunoglobulin and standard positive and negative samples for calibration purposes.

Description

SPECIFICATION Method for the detection of malaria in human beings The present invention relates to a new method for detecting the presence of malarial parasites in infected human blood. More particulariy, the invention relates to an improved method for detecting the presence of malarial parasites in infected human blood, said method being characterized by its high sensitivity.

Malaria is a serious acute or chronic relapsing infection in man, affecting hundreds of millions of people mainly in tropical countries. Transmitted by mosquitoes, it occurs in the forested regions of Asia, Africa, and America. It is caused by various species of Protozoa that belong to a single genus known as Plasmodium. The main species of Plasmodium causing maleria in man are: Plasmodium falciparum, Plasmodium vivax, Plasmodium malaria and Plasmodium ovale.

Eradication of maleriais a difficult and costly job, requiring a prolonged effort to eliminate the insect vectors and to free the population from disease, by treatment with antimalarial drugs. An essential element in any antimalarial campaign is to get information regarding the incidence of infection before, during and at the end of such a campaign. This requires a diagnostic method suitable for large scale studies and possessing a high degree of sensitivity.

The only method used today for the determination of malaria in man, is the microscopic examination of blood. Obviously, this is a difficult, laborious and time-consuming procedure, totally unsuitable for epidemiological investigations, in which tens or hundreds of thousands of samples need to be examined.

In our previous Israeli Patent Application Number 59310, an assay was described for the determination of malarial antigens and antibodies in rats infected with Plasmodium berghei species. According to the assay described in said patent application, a tube is coated with antigen prepared by sonicating red cells of a rat infected with the parasite. To detect the presence of the parasite, red cells of the infected animal were sonicated and reacted with antibodies against Plasmodium berghei, after which the mixture was incubated with the coated tube. The amount of antibodies bound to the tubes was then determined with labeled Protein A.

A reduction in the amount of label bound to the tube, as compared to the amount bound when the antibodies were used without preincubation with the infected blood, was indicative of the presence of the parasite in the blood. A similar method could be used for the diagnosis of infection in man, using Plasmodium falxiparum instead of Plasmodium berghei antigen but the method would be of relatively low sensitivity and could not be applied to detect low grade parasitemia.

A serodiagnostic test for the detection of malaria was described by L. Mackey et. al. in a paper entitled "Diagnosis of Plasmodium falciparum infection using a solid-phase radioimmunoassay", Bulletin of the World Health Organization, 58(3): 439, 1 980. The method is based on the use of parasite antigens, from red blood cells infected in vitro with Plasmodium falciparum and malaria antibody, wherein parasites were detected in a solid-phase radioimmunoassay that measures inhibition of binding of antibodies. However, the method cannot be considered suitable for immunodiagnosis, in view of its low sensitivity. As stated by the authors in said paper, a blood sample was considered as positive when the inhibition was greater than the mean + 3 times the standard deviation of the controls, which means greater than 18%.

Using this figure, it can be estimated from their data that actually they detected by the radioimmunoassay only about half of the patients diagnosed as positive by microscopy. As specified in said paper (page 442): "The solid-phase radioimmunoassay used in these experiments is not proposed as a definitive immunodiagnostic method but serves to demonstrate the principle that the immunodiagnosis of malaria infection is practicable".

Another technique was recently described for the detection of malarial parasites in experimentally infected monkeys (British Medical Journal, 82, 1747, 1981) but this method was not tested or even suggested for man.

It is an object of the present invention to provide an improved assay for the determination of malaria antigens and antibodies in human blood. It is another object of the present invention to provide an improved assay for the determination of malaria antigens and antibodies in human blood, which is very sensitive and suitable to detect minute quantities of malaria antigens and antibodies. Thus, the invention consists in an improved method for the detection of malaria parasite antigens or antibodies in human blood, by utilizing an enriched Plasmodium falciparum antigen preparation on a solid phase adsorbent which enables the detection of parasitemia of the order of 1 parasite or less per 106 red blood cells.

The solid phase adsorbent may be in the form of beads, tubes or microtiter plates to which is attached the enriched Plasmodium falciparum preparation. The principle of the method is based on the fact that when sera of individuals with a history of malaria, (or antisera prepared in animals) are reacted with the solid adsorbent, antibodies remain bound to said adsorbent and can be quantified with radioisotope or enzyme labeled Protein A or an appropriate anti Immunoglobulin. The presence of Plasmodium falciparum antigen in the red blood cells is assayed by its ability to inhibit the binding of the antibodies.The sensitivity of the method was found to increase appreciably when the solid phase adsorbent was prepared with enriched Plasmodium falciparum antigen, such as obtained by preliminary separation of the cultured, infected red blood cells by the gelatin sedimentation technique, followed by sonication and ammonium sulfate precipitation, as described below.

Enriched Plasmodium falciparum infected red blood cells (60-85% parasitemia) can be obtained by a modification of the gelatin sedimentation technique as described by Jensen, J. B.

(Amer. J. Trop. Hyg. Med., 27: 1274, 1978) or other methods utilizing isopycnic centrifugation in appropriate gradients. Of course this method of enrichment is not related per se to the present invention as other methods which will produce the same result could be successfully applied in the assay according to the present invention. A short review of these methods is hereafter presented although there are other methods which might be conceived to be utlized for said enrichement: a. Fractionation of parasitized cells from non-infected ones on Percoll density gradients. Percoll (produced by Pharmacia Fine Chemicals, Sweden) is made of colloidal silica coated with polyvinyl-pyrrolidone. it has a low osmolality and low viscosity being virtually non-toxic.

Gradients were formed during cell separation. The procedure was as follows: Parasitized erythrocytes were washed once in RPMI and once in saline solution. A 50% erythrocyte suspension was prepared in 72% isotonic Percoll. An amount of 1 ml of the suspension was placed in a tube with 10 ml of 72% Percoll solution and centrifuged for 1 hour at 27,000 9.

The layer of the gradient above the bulk of the packed erthrocytes contained mainly the ring forms of the parasites and parasitaemia was about 5 times higher than in the unfractioned blood.

b. Fractionation of parasitized cells with Ficoll (J. E. Mrema et al., Bull. WHO 57, 133, 1979).

It is suggested to obtain high concentrations and high yields of infected cells by low g-force centrifugation through 5% (wt precent) solutions of Ficoll.

c. Fractionation of parasitized cells with aqueous solutions of 5% D sorbitol (Lambros and Vandenberg, J. Parasitol. 65, 418, 1979). Parasitized erythrocytes were suspended in a solution of 5% D sorbitol for ten minutes at room temperature. Immediately after the sorbitol treatment, the parasites consisted mainly of ring forms. Erythrocytes infected with more mature stages of the parasite were lysed. Of course any combination of the above enrichement methods could be also utilized and sometimes even preferred when a particular advantage could be achieved. Thus for instance, the supernatant of gelatin separation mixed with normal erythrocytes followed after 1 8 hours by a sorbitol treatment will induce synchronization of the cultures.

On further incubation these cultures yield red blood suspensions infected almost exclusively with ring forms.

According to the assay of the present invention, parasitemia of the order of 1/106 to 1/107 red blood cells could be detected in a reproducible manner by the tests, which represents an improvement in sensitivity of 10 to 100 fold over the results obtained with Plasmodium berghei in rats, as described in our previous patent application number 59310.

According to one embodiment, the invention relates to an assay for the detection and determination of antiplasmodial antibodies which comprises: a. preparing a solid adsorbent containing an enriched Plasmodium falciparum antigen preparation; b. incubating the serum to be tested for the presence of said antibodies on said solid adsorbent; c. incubating with radiolabeled Protein A or anti-immunoglobulin; and d. determining the radioactivity bound to the said solid adsorbent.

The present invention also relates to an assay for the detection and determination of antiplasmodial antibodies using an enzyme system.

Therefore the assay will comprise the following steps: a. Preparing a solid adsorbent containing an enriched Plasmodium falciparum antigen preparation; b. incubating the serum to be tested for the presence of said antibodies on said solid adsorbent; c. incubating with enzyme linked Protein A or anti-immunoglobulin against the immunoglobulin of the serum donor, and d. determining the enzyme bound on said solid adsorbent.

According to a further aspect of the present invention it is possible to assay for the detection and determination of Plasmodium falciparum antigens which comprises the same steps as described for the antibodies as mentioned above.

The presence of parasites in the red blood cells was determined by an inhibition test: an amount of 1 25 pl of immune serum sample was mixed with 1 25 yl of the solubilized red cells and the mixtures were incubated for 1 hour at 37"C and overnight at 4"C. Serum mixed with buffer or with solubilized non-infected red blood cells served as control. One hundred microliters of each mixture were added to the solid phase adsorbent containing the antigens and the residual antibody binding capacity was measured.The present inhibition was calculated from the formula: % inhibition =

Radioactivity bound by serum mixed with blood sample 1 by bound by by serum mixed with diluent A blood sample was considered as positive if it caused an inhibition of at least 15% or an increased binding, also known as enhancement, of at least 15% over the background binding obtained with antibody alone. Enhancement is a known phenomemon encounted in antibody binding-inhibiton tests, when the concentration of the inhibitor is just below that required to give marginal inhibition.The entire method is relatively simple, therefore large numbers of samples can be processed in one experiment.

The preparation of soluble Plasmodium falciparum antigen for use with the solid phase absorbent was as follows: a. Human red blood cells were infected with P. falciparum parasites "in vitro", as described in a previous publication (Avraham et al., Trans. Roy. Soc. Trop. Med. Hyg. 1981, 75: 451). This yielded a suspension with 10-20% parasitemia.

b. The infected red blood cells suspension was enriched in parasite content by a modified gelatin sedimentation technique (Jensen, J.B., mentioned above). The red blood cells suspension was washed once with RPMI-1640 (Roswell Park Memorial Institue), the tissue culture medium used to grow the parasite Plasmodium falciparum in human red blood cells, and mixed with 9 volumes of 0.7% by wt. gelatin in RPMI. The mixture was left for 30 minutes at 37"C to separate into an upper layer, consisting mainly of trophozoites and schizonts and a lower layer consisting largely of ring forms and non-infected red blood cells. The cells in the lower layer were washed with RPMI and refractionated with gelatin, to obtan a second fraction enriched in mature parasites.The upper layers obtained in the first and second fractionation were pooled, yielding a suspension containing about 60-85% parasitized red blood cells. The parasites thus obtained were found to be viable and metabolically active and consisted mainly of trophozoites and schizonts. Suspensions enriched in ring forms, which are charactristic to infected human red blood cells were obtained as follows: Parasitized red blood cells were separated with gelatin as described above. The upper layer containing trophozoites and schizonts was washed, mixed with 4 volumes of non-parasitized red blood cells to yield a 5% final cell suspension and returned to culture. After about 1 8 hours, the red blood cells were washed and resuspended in a 5% by wt.

sorbitol solution in distilled water for about fifteen minutes (as described by Lambros and Vandenberg, J. Parasitol., 65 41 8: 1 979). The resulting suspension contained non-infected red blood cells, red blood cells with ring forms and some lysed parasites of different stages. The cells were washed three times and used as the preparation of enriched ring forms.

c. Non enriched or parasite enriched, infected, red cells were sonicated and cleaned of debris as described by Avraham et al., (Trans. Roy. Soc. Trop. Med. Hyg. 1981, 75, 451). Both fractions (the enriched sonicated infected red blood cells E-SIRC and non-enriched) where diluted so as to contain the equivalent of 1 ml packed infected red blood cells per 100 ml solution.

Ammonium sulfate fractions of E-SIRC were prepared as follows: E-SIRC was brought to 50% saturation with saturated ammonium sulfate. The formed precipitate was washed with a 50% solution of saturated ammonium sulfate in phosphate buffered saline and subsequently dissolved in a solution of phosphate buffered saline (PBS) and reprecipitated as before. The second precipitate (P50) was further dissolved in water containing about 0. 1% by wt. of a detergent (e.g. Triton X-100 of NP-40 or Tween 20, etc.). The supernate (S50) and P50 were dialyzed against 1 5 mM of phosphate buffer at a pH of about 7.2 and assayed for activity and for protein content. The P60 fraction contained about 2 mg and the supernate (S50) about 6 mg protein.One ml of the P50 was spun in an ultracentrifuge at 100,000 g (Spinco L65 ultracentrifuge type). The separated sediment (sed P50) was redissolved in 1 ml of PBS with a solution of 0. 1% of detergent (e.g. Tween 20) and was tested for activity, together with the supernate fraction (Sup P50).

A description of the assay technique using Plasmodium falciparum will be hereafter presented for a better understanding of the invention. it should be understood that the invention is not limited only to the present description only. Thus, modifications envisaged by persons skilled in the art should also be considered to be included by this invention: Biological reagents were centrifuged for about 30 minutes at 12,000 g before use. The solid adsorbent utilized in the experiment, was the microtiter plate (of U type as produced by Nunc.

Denmark). The microtiter plate was coated with antigen by filling the wells with 0.1 ml of the antigen solution (undiluted sonicated infected red cells or enriched sonicated infected red cells, 50 yg/ml all others) and incubating 3 hours at 37"C and 15 hours at 4"C. The plates were then washed 3 times and used immediately. To test for binding of antibodies to the antigen coated plate, 0.1 ml volumes of the test serum, diluted in 10% (by volume) of Foetal Calf Serum (FCS), were added to the microplate wells and incubated for 4 hours at 37"C. The plates were then washed rapidly, two times with 1 % of Tween-20 (as detergent) in phosphate buffered salined and once with pure phosphate buffered saline.An amount of 0.1 ml radioiodinated Protein A was added to each well and the plates were left to stand for about 1 hour at room temperature.

The wells were subsquently washed quickly with a solution of 1 % (by volume) Tween 20 (as detergent) three times and 1 20 ul aliquots of 0.1 N sodium hydroxide solution were placed in each well. Finally, the contents of each well were transferred to plastic tubes and the radioactivity of the tubes was measured.

The activity of the various antigen preparations was tested by their capacity to inhibit binding of antibodies to the solid adsorbent and by their effectiveness as coating antigens. Coating effectiveness was assayed by two techniques: (a) the highest dilution of the immune serum giving detectable binding when reacted with wells coated with the antigen under test; and (b) the highest dilution of infected red blood cells giving detectable inhibition (about 15% or more) when reacted with microplates coated with the antigen under test.

The determinations of protein concentrations, were carried out in accordance with the Lowry method as described in J.Biol.Chem. 1951, 193: 265.

The results of tests of the efficiency of various Plasmodium falciparum preparations as coating antigens, as assayed by the antibody binding capacity, are presented in the following Table 1.

The purified antigens were used for coating at a constant protein concentration of 50 iug/ml.

Table 1.

Plasmodium falciparum antigens Antibody binding titer expressed used for coating as reciprocal of dilution Sonicated infected red blood cells 1 5,625.

Enriched sonicated infected red blood cells 78,125 The ammonium sulfate precipitate (P50) 78,125 The supernate of P50 78,125 The sediment of P50 625 The binding of antibodies to the coated plates was tested by treating the wells with fivefold dilutions of serum obtained from a patient recovered from Plasmodium falciparum infection. The binding titer of the antiserum was the highest dilution causing significant binding of radioactivity on subsequent treatment of the plates with radioiodinated Protein A. The results from the above Table 1, clearly show that the use of purified antigens for coating of the wells, caused a 4-fold increase in binding of antibodies.

The inhibitory activity of the various antigens is presented in Table 2 below. An appropriate dilution of the immune serum was incubated overnight with tenfold dilutions of the antigens, after which the residual binding activity was determined.

Table 2.

Antigen used for coating Inhibiting (end point) of antibody binding determined with: sonicated infected red P50 Sup. P50 blood cells (parasites/108 RBC) qtg/ml) (ug/ml) Sonicated infected red blood cells 102-105 10-1 10-2 The (NH4)2SO4 precipitate P50 10-4 10-4 The supernate of P50 10-4 10-4 As it appears, there was no substantial difference between the precipitate P50 and the supernate of P50, both antigens giving detectable inhibiton at concentrations of the order of 0.1 ng protein/ml.

As mentioned above, most of the parasites in the infected, cultured red cells are the mature forms trophozoites or schizonts. However, the parasites in the blood of patients are mostly ring forms. It was important, consequently, to determine whether ring forms are also detectable by our assay.

A preparation highly enriched in ring forms was solubilized and tested for inhibitory activity using microtiter plates coated with P50 fraction. The results obtained are presented in a graphic form in Fig. 3. As can be noticed, the ring forms could be detected at a concentration of about 1 ring/3 X 106 of red blood cells which is considered as being a very high sensitivity. It might be expected that further improvements could be obtained by purifying the major parasite antigens to a higher degree.

One of the drawbacks of utilising Plasmodium falciparum antigens is connected with the relative high costs and great lability of these antigens, which might hamper the development of clinically usefui imunodiagnostic reagents for malaria in according with the present invention.

This drawback can be obviated by using Plasmodium berghei antigens, crossreacting with Plasmodium falciparum.

The preparation of Plassmodium berghei is much simpler and less expensive than the preparation of Plasmodium falciparum, and therefore a net advantage would be expected to this embodiment in which Plasmodium berghei would be used as coating antigen in Radioimmunoassay tests for the detection of Plasmodium falciparum. It was found that microtiter plates coated with a Plasmodium berghei preparation could be useful to detect Plasmodium falciparum, in red blood cells samples with parasitemia of the order of 0.01% or higher. Plasmodium berghei parasites were freed from infected red blood following the technique of Zuckerman et al, (Bull, WHO, 37, p. 431, 1967). The parasite suspension thus recovered, which consisted largely of intact parasites, was used to coat microtiter plates as described by Kim et al (J.

Immun. 125, p. 2565, 1980). Red blood cells infected in vitro with Plasmodium falciparum were solublized with Brij-36. Serial ten-fold dilutions were then prepared using as diluent a sonicate of 10% normal human red blood cells. Aliquots of these dilutions were then tested for inhibitory activity by the standard procedure. As appears from Fig. 5 where the results obtained are graphically presented, although the inhibition curves were rather flat, parasitemia of the order of 10 to 100 infected red blood cells/105 non infected red blood cells could be detected.

The radioimmunoassay according to the present invention is based on the inhibition of binding of antiplasmodial antibodies by parasite antigens from the blood, wherein its sensitivity is inversely correlated to the amount of antibody used in the test. By decreasing the amount of antibody, less antigen will be required for inhibition. However, as the amount of antibody is reduced it will also reduce at the same time the base-line binding. Thus in order to obtain an acceptable level of binding while reducing the amount of antibody, it will be required to increase the efficiency of coating the solid adsorbent by antigen, which of course will be a function of the purity of the antigen.According to the present invention it was found that by utilizing an enriched Plasmodium antigen preparation in the range of 20% to 85% parasitemia it will be possible to obtain an acceptable level binding with an increased sensitivity of detection up to 100 times as much as in the method describd in our previous Israeli Patent Application Number 59310 for detection of malaria in rats. The effect of the purity of the antigen was studied by coating microtiter plates with various Plasmodium falciparum antigen preparations and performing inhibition tests, in which an appropriate serum dilution was reacted with sonicated infected red blood cells which are similar to infected red blood cells obtained from patients.The results obtained with plates coated with sonicated infected red blood cells, enriched sonicated infected red blood cells and the precipitate P50 are summarized in a graphic form in the Figs. 1 and 2.

Fig. 1 illustrates the inhibition of binding to microtiter plates coated with SIRC or enriched SIRC and Fig. 2 illustrates the inhibition of binding to microtiter plates coated with SIRC or ammonium sulfate precipitate (P50).

The results from the above graphs clearly show that always the sensitivity of the detection of the parasitized red blood cells is higher when enriched Plasmodium antigen preparations were utilized.

Although in the context of the present specification a 1125 labelled radioactive compound was mentioned it should be understood that any other suitable labelled radioactive compound could be utilized for its determination on the solid adsorbent in the assay.

According to another embodiment which obviates a major problem of loss of Plasmodium falcipararum antigen activity during fractionation and storage, it is suggested to utilize a stable immunoadsorbent. Furthermore, a stable antigen will be essential for use in clinical work. An immunoadsorbent was prepared by binding P50 to Ultrogel and it was found to be stable for at least 6 weeks and useful both in Radioimmunoassay and ELISA experiments. The Ultrogel was coated with Plasmodium falciparum antigens in according to the technique described by Guesdon et al (Guesdon, J.L. and Avrameas S., J.lmmun.Methods, 11, 129, 1976).Activated Ultrogel was incubated for 48 hours at 4"C with equal volume of Plasmodium falciparum antigen (1 mg protein/ml), after which the coated gel was washed and kept at 4"C as a 50% suspension in phosphate buffered saline. Before use, coated Ultogel was diluted + with noncoated Ultrogel. The resulting adsorbent was compared to P50 coated microtiter plates as regards efficiency of detection of Plasmodium falciparum, in "in vitro" infected red blood cells. The results are presented in the attached Fig. 4, from which it appears that the Ultrogel adsorbent was approximately 10 times less sensitive than the plates but the P50 coated Ultrogel retained most of its activity after 6 weeks storage in the refrigerator, which is an important advantage.

The approximate sensitivity of the various reagents expressed as number of infected/l06 noninfected red blood cells detectable in the test was as follows: --Radioimmunoassay (RIA) with microtiter plates 1:106-107.

-ELISA with microtiter plates . 1:106.

-RlA and ELISA with the Ultrogel adsorbent 10-100/108.

The method according to the present invention can also be performed in assays which do not employ radioactive materials or modified enzymes as the labelling substance. One approach envisaged will be the utlization of the fluorescence assay technique or the standard ELISA procedure.

It was found that with Ultrogel as adsorbent, the radioimmunoassay and ELISA tests had substantially the same sensitivity of about 1 infected/105 non-infected red blood cells detected.

However, preliminary tests showed that in the tests carried out with microtiter plates, the radioimmunoassay was about ten fold more sensitive than the ELISA test. As known ELISA testing has a net advantage over the radioimmunoassay, since it does not require radioactive reagents, a fact which is most desirable for many clinical laboratories. Two pools of human immune sera were tested. Five fold dilutions of the sera were incubated with the adsorbents, after which the bound antibodies were assayed with '251-SPA or by ELISA. The titer was the maximal dilution giving significant binding (2 x higher than control, normal human serum). In the following Table 3 are presented the results on the assays of Plasmodium falciparum antibodies in two different human immune sera (HITS1 and HIS2) carried out by radioimmunoassay (RIA) and by ELISA.

Table 3.

Assay of anti Plasmodium falciparum antibodies in human immune sera (HIS, and HIS2) by RIA and ELISA.

Coating Binding titer (reciprocal of dilution) Test Adsorbent Antigen HIS1 HIS2 RIA Microplates P50 1:78,125 1:15,625 E-SIRC 1:78,125 1:15,625 Ultrogel P50 1:15,625 1:3125 E-SIRC 1:15,625 1:3125 ELISA Microplates P50 1:78,125 1:15,625 E-SIRC 1:78,125 1:3125 Ultrogel P50 1:15,625 1:3125 E-SIRC 1:15,625 1:3125 The present invention further relates to a kit for carrying out an assay for antiplasmodial antibodies, comprising a solid adsorbent such as tubes, beads or microtiter plates treated with enriched sonicated red blood cells, infected with Plasmodium falciparum, an adequate buffer, and either labelled Pr A or labelled anti-immonoglobulin, and standard positive and negative samples for calibration purposes.In carrying out the subject assays, in order to obtain reproducible results, it is desirable that the critical reagents be provided in predetermined ratios, so as to optimize the sensitivity of the assay. The incorporation in the kit of the ancillary materials, such as buffer, so that dry powders or concentrates may be diluted to form assay solutions directly avoiding the necessity of weighing the various materials. Therefore all reagents will be provided in relative proportions so as to substantially optimize the sensitivity of the assay to the concentration range of interest.

While the invention has been described with specific embodiments thereof, it will be understood that it is capable of further modifications, and this patent is intended to cover any variation, uses or adaptations of the invention and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as fall within the scope of the invention. Thus although in the specification microtiters plates were examplified, any other solid adsorbent could also successfully be utilized. It is, therefore, desired that the embodiments described should be considered in all respects as illustrative and not restrictive, reference being made to the appended Claims, rather than to the foregoing description, in which it is intended to Claim all modifications coming within the scope and spirit of the invention.

Claims (21)

1. An improved method for the detection and determination of malaria antibodies and malaria antigens in human blood, which consists in the inhibition of binding of Plasmodial antibodies to enriched sonicated infected red blood cells located on a solid phase adsorbent, which enables the detection of parasitemia of the order of 1 parasite or less per 106 of red blood cells.

2. A method according to Claim 1, wherein said solid phase adsorbent is selected from beads, tubes or microtiter plates.

3. A method according to Claim 1 or 2, wherein said enriched infected red blood cells are in the range of 20% to 85% parasitemia.

4. A method according to Claim 3, wherein said enriched infected red blood cells are in the range of 40% to 65% parasitemia.

5. A method for the detection and determination of antiplasmodial antibodies which comprises: (a) preparing a solid adsorbent containing antigens derived from cells of blood enriched with Plasmodium falciparum parasite; (b) incubating the serum to be tested for the presence of said antibodies on said solid adsorbent; (c) incubating the solid adsorbent with a labelled protein compound which is suitable for the present system, and (d) determining the labelling compound on said solid adsorbent.

6. A method according to Claim 5, wherein the labelling of the protein is carried out by a radioactive compound.

7. A method according to Claim 6, wherein said radioactive compound is 1125.

8. A method according to Claim 5, utilizing the ELISA procedure.

9. A method for the detection and determination of antiplasmodial antibodies which comprises: (a) preparing a solid adsorbent containing antigens derived from cells of blood enriched with Plasmodium falciparum parasite; (b) incubating the serum to be tested for the presence of said antibodies on said solid adsorbent; (c) incubating with enzyme linked Protein A of antiimmunoglobulin against the immunoglobulin of the serum donor, and (d) determining the enzyme bound on said solid adsorbent.

10. A method for the detection and determination of malaria antigen which comprises: (a) preparing a solid adsorbent containing antigens derived from cells of blood enriched with Plasmodium falciparum parasite; (b) incubating a quantity of antibodies with the sample to be tested for the presence of malarial antigen then incubating the mixture with the solid adsorbent on the solid adsorbent; (c) incubating said solid adsobent with a labelled protein compound which is suitable for the present system, and (d) determining the labelling compound on said solid adsorbent.

11. A method according to Claim 10, wherein the labelling compound is radioactive.

1 2. A method according to Claim 11, wherein said radioactive compound is 1125.

1 3. A method for the detection and determination of malaria antigen which comprises: (a) preparing a solid adsorbent containing antigens derived from cells of blood enriched with Plasmodium faciparum parasite; (b) incubating a quantity of antibodies with the sample to be tested for the presence of malarial antigen then incubating the mixture with the solid adsorbent on the solid adsorbent; (c) incubating said solid adsorbent with an enzyme linked immunoglobulin, and (d) determining the enzyme bound on said solid adsorbent.

14. A method according to Claims 1 to 13, wherein said solid adsorbent is a stable immunoadsorbent obtained by binding P50 to Ultrogel.

1 5. A method according to Claims 1 to 14, wherein the enrichment of infected red blood cells is carried out by a method selected from: gelatin sedimentation, fractionation with Percoll, fractionation with Ficoll, fractionation with aqueus D sorbitol or combinations thereof.

1 6. A method according to Claims 1 to 8, wherein the species of Plasmodium to be assayed are selected from Falciparum, Berghei crossreacted with Vivax, Malariae and Ovale.

1 7. A kit for carrying out an assay for antiplasmodial antibodies according to Claims 1 to 16, comprising solid adsorbent with antigens from enriched infected sonicated red blood cells, a suitable buffer, either a labelled protein compound or enzyme labelled anti-immunoglobulin, and standard positive and negative samples for calibration purposes.

1 8. A kit for carrying out an assay for plasmodium antigen according to Claims 1 to 16, comprising solid adsorbent with antigens from enriched infected sonicated red blood cells, a suitable buffer, a standarized antibody preparation, either a labelled protein compound or enzyme labelled anti-immunoglobulin and standard positive and negative samples for calibration purposes.

19. A method for the detection and quantitative determination of antiplasmodial antibodies, substantially as herein before described.

20. A method for the detection and quantitaive determination of malarial antigen, substantially as hereinbefore described.

21. A kit for the detection and determination of malarial antigens and antibodies substantially as hereinbefore described.