Field evaluation of an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum sporozoite detection in anopheline mosquitoes from Kenya

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes a repetitive epitope on the circumsporozoite protein of Plasmodium falciparum was used in Kenya to assess malaria infections in Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparum. The ELISA infection rate of 18.6% (n = 736) for individually tested mosquitoes for both species was significantly higher than the 10.4% (n = 537) salivary gland sporozoite rate determined by dissection. This difference was due to ELISA detection of medium and large sized oocysts on the midguts of infected mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax, ELISA tests on "head" and "body" portions showed that 29.5% of 95 positive mosquitoes contained circumsporozoite antigen in the body portion in the absence of salivary gland infections. This field evaluation demonstrates that the ELISA can most accurately be used to estimate sporozoite rates by cutting mosquitoes at the thorax and testing anterior portions.     

Identification of Plasmodium vivax sporozoites in mosquitoes using an enzyme-linked immunosorbent assay

A sandwich enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium vivax sporozoites in mosquitoes is described. Monoclonal antibodies produced against Thailand P. vivax sporozoites were used in an ELISA to detect and identify homologous sporozoites of Southeast Asian, Mexican and North Korean origin in extracts of frozen or dried infected mosquitoes. The assay was sensitive enough to detect 1 infected mosquito in a pool of 20 insects or 125-250 sporozoites per 30 microliter of mosquito extract. The use of a nonionic detergent and a single freeze-thaw to disrupt the circumsporozoite antigen significantly increased the sensitivity of the method.     

Identification of Plasmodium falciparum-infected mosquitoes by a double antibody enzyme-linked immunosorbent assay

A double antibody micro enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium falciparum sporozoites in mosquitoes is described. Using monoclonal antibodies made against South American P. falciparum sporozoites, the ELISA was able to detect and identify sporozoite antigens of South American and Asian origins in extracts of dried infected mosquitoes.     

The enzyme-linked immunosorbent assay (ELISA) for malaria. I. The use of in vitro-cultured Plasmodium falciparum as antigen.

Using the Panama II strain of Plasmodium falciparum obtained from continuous in vitro culture as antigen, the micro enzyme-linked immunosorbent assay (ELISA) was used to test serum samples from 50 persons from the southeastern United States and serum specimens collected weekly from four non-immune and nine semi-immune patients infected with P. falciparum. None of the 50 sera from the United States had ELISA antibody titers greater than 1:80. The nine semi-immune patients had rapid ELISA antibody responses (titers greater than 1:2560) following patent parasitemia. ELISA titers remained elevated despite disappearance of patent parasitemia, and declined gradually following curative antimalarial therapy. The ELISA responses observed in the four non-immune patients were more variable, though positive titers appeared rapidly with patent parasitemia. Maximum titers were lower than those observed in semi-immune patients. These results demonstrate that P. falciparum obtained from continuous in vitro culture is an excellent antigen for the micro-ELISA test for malaria. However, further assessments of the ELISA are needed to identify the conditions associated with positive responses.     

Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay

A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P. falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant GLURP489-1271 was expressed as a chimeric protein, fused with E. coli beta-galactosidase. However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase. Antigen from in vitro P. falciparum cultures of isolates from Tanzania (F32), Papua New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP.     

Development of immunoradiometric assay for detection of Plasmodium falciparum antigen in blood using monoclonal antibody

Specific monoclonal antibody (MAB) F2W22C1) produced by hybridoma technology against blood stage common antigens shared by almost all isolates of P. falciparum was selected. The 125I-labelled prepared from this MAB (125I-MIgG) was used as probe for detection of low grade P. falciparum infection. Recently, a two-site monoclonal antibody sandwich immunoradiometric assay (Mab-IRMA) has been developed. The assay showed good correlation with parasitaemia with the ability to detect as few as 2.4 parasites/10(8) erythrocytes. Two surveys were made in people living in a malaria endemic area during transmission and non-transmission season to detect the antigen of Plasmodium falciparum by using the Mab-IRMA. In the first survey involving 101 people, the IRMA positive rate was 56.4% and then significantly declined to 16.5% during the second survey involving 79 people of the same group. The parasitological positive rates were likewise decreased from 11.9% to 1.3% (p = 0.015) during these two seasons. IRMA positive rates were significantly higher than the corresponding parasitological positive rates (p less than 0.0001 and less than 0.002 for the first and the second surveys respectively). Regression analysis showed that IRMA activities were linearly correlated with the parasite counts by microscopic examinations (r = 0.629, p = 0.022). IRMA was specific for P. falciparum since all 30 healthy controls and six of seven vivax malaria cases were negative. IRMA has potential for use to monitor the malaria control and to assess the efficacy of the future field trial of malaria vaccine.     

A sensitive malaria immunoperoxidase assay for the detection of Plasmodium falciparum antibody

A new and rapid malaria immunoperoxidase assay using the enzyme horseradish peroxidase in place of fluorescein isothiocyanate was developed to allow the serological measurement of antimalarial antibody by light microscopy. Acetone-fixed thin blood films prepared from cultured Plasmodium falciparum were used as the source of antigen. This malaria immunoperoxidase assay is as sensitive as, and occasionally more sensitive than, the indirect fluorescent antibody assay. It is easy to perform and the antigen used does not show cross-reactivity with sera from nonmalarial diseases.     

An enzyme-linked immunosorbent assay for the measurement of thyroglobulin in human serum using mouse monoclonal antibodies

A robust, rapid, sensitive and specific enzyme-linked immunosorbent assay (ELISA), using an in-house immunoglobulin-A-sub-class mouse monoclonal human-thyroglobulin antibody (WNSM2), with a sensitivity of 1.51 pmol/l has been established for the measurement of thyroglobulin in serum. Standard curves in varying dilutions of human serum were similar to standard curves obtained in serum-free medium, thus demonstrating no significant cross-reactivity with any serum proteins other than thyroglobulin. Levels of serum thyroglobulin detected by ELISA correlated significantly (r = 0.93, P less than 0.001) with those from a standardized and well-characterized radioimmunoassay. The coefficients of variation within and between ELISA assays were 3.9 and 7.1% respectively. Thyroglobulin was detectable in 87% of 54 normal subjects who had no history of thyroid or autoimmune disease, the mean (+/- S.D.) for this group being 15 +/- 6.6 pmol/l with a range of 1.51-53 pmol/l. Using this assay, levels of thyroglobulin were shown to be significantly (P less than 0.002) increased in patients with untreated hyperthyroid Graves' disease compared with normal subjects.     

粗提抗原检测炭疽血清抗体ELISA方法的初步评价

目的 对使用粗提抗原检测炭疽血清抗体的酶联免疫吸附试验(ELISA)方法进行初步评价.方法 用间接ELISA方法检测人群血清(健康人血清42份、炭疽病人血清42份)特异性抗体,用阳性血清对照绘制标准曲线,按照标准曲线计算出每份血清标本的抗体相对含量,所得结果与重组致死因子(rLF)方法的检测结果进行比较.结果 病人组血清抗体相对含量中位数为1.19,健康人组血清抗体相对含量中位数为0.24,两组比较差异有统计学意义(uc=7.643,P＜0.05).粗提抗原检测与rLF检测结果并不完全对应,但两种方法显示出较高的一致性.结论 粗提抗原检测炭疽血清抗体的方法能区分大部分的病人和健康人,有一定的应用潜力,可用在炭疽疾病监测工作中.     

Combined use of an antigen and antibody detection enzyme-linked immunosorbent assay for cysticercosis as tools in an epidemiological study of epilepsy in Burundi

OBJECTIVE: To evaluate the benefits of the detection of both circulating antibodies (Ab) and antigens (Ag) for the diagnosis of cysticercosis in people with epilepsy. Neurocysticercosis is a cause of neurological diseases world-wide, especially epilepsy. The clinical symptoms of neurocysticercosis are non-specific and diagnosis is often difficult. METHODS: Serum samples were collected from subjects in a matched case-control study for epilepsy in the Kiremba area, Burundi, between March and April 2001 (epileptic cases=303; controls without epilepsy=606). The enzyme-linked immunosorbent assay (ELISA) was used for the detection of antibodies (Ab-ELISA) and circulating Ag (Ag-ELISA). RESULTS: The Ab-ELISA revealed 58.7% positivity in epilepsy cases and 31.4% in healthy controls; and Ag-ELISA revealed 38.3% positivity in epilepsy cases and 20.0% in controls. The matched odds ratios were 3.6 (95% CI: 2.5-4.9) for Ab-ELISA, and 2.9 (95% CI: 2.1-4.3) for Ag-ELISA. CONCLUSION: Both Ag- and Ab-ELISA detected a significantly higher number of seropositives among people with epilepsy than among controls. The risk of epilepsy was high in cases with a positive Ag-ELISA, although less important than in cases with positivity for Ab-ELISA. Dead or degenerating cysticerci appear to be more frequently associated with epilepsy than living cysts. The high number of people with circulating Ag of Taenia solium suggests that the study area is a focus of active transmission of the parasite.     

The feasibility of a Dot enzyme-linked immunosorbent assay (Dot-ELISA) for the diagnosis of Plasmodium falciparum antigens and antibodies

An enzyme-linked immunosorbent assay for rapid detection of malarial antibodies and antigens was developed. Plasmodium falciparum antigen preparation, obtained from sonicated cultures of the parasite at a parasitemia of 10%-15%, was applied to cellulose filter discs in volumes of 0.1 microliter in 96-well microtiter plates. Antibodies were detected by successive incubations with: bovine serum albumin for blocking, tested serum at different dilutions, peroxidase-conjugated antihuman IgG, and the precipitable substrate 4-chloro-1-naphtol. Positive reactions appeared as blue dots on a white background which are easily read by eye. Pools of sera from patients with recent disease or from individuals with a history of malaria, contained antibodies detectable up to a dilution of 1:64,000. Negative results were obtained when normal RBC were used for dotting the filters. Normal sera showed no reaction at any antigen concentration. P. falciparum antigens were detected by their ability to inhibit the binding of antibody to the filters. RBC infected with P. falciparum in vitro can be detected at a level of 0.001% parasitemia. This report presents the feasibility of an assay for detecting malarial antibodies and antigens in blood samples which is easily applicable to field conditions.     

Monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Plasmodium malariae sporozoites in mosquitoes

A monoclonal antibody specific for a repeated epitope of the circumsporozoite protein of Plasmodium malariae sporozoites has been used to develop a two-site, single antibody-based enzyme-linked immunosorbent assay that can detect P. malariae sporozoites in mosquitoes. The assay uses a purified monoclonal antibody produced against sporozoites of the Uganda I/CDC strain of P. malariae to capture the antigen and the same monoclonal antibody labeled with horseradish peroxidase as the detector. Sporozoites have been detected in laboratory-infected mosquitoes stored at room temperature in the presence of a desiccant for as long as 18 months. The detection limit of the assay is approximately 50 P. malariae sporozoites per test well. Cross-reaction has not been observed with mosquitoes infected with P. falciparum, P. vivax, or P. ovale sporozoites.     

Circumsporozoite antibody as a serologic marker of Plasmodium falciparum transmission.

In a longitudinal study of a malaria-endemic village in southeastern Thailand, circumsporozoite (CS) antibody to sporozoites of Plasmodium falciparum was measured by an enzyme-linked immunosorbent assay to determine its usefulness as a seroepidemiologic marker of malaria transmission. The CS anti-(NANP)n antibody level and prevalence during a 25-month period paralleled the pattern of seasonal transmission consistent with conventional parasitologic and entomologic measurements. The prevalence and level of antibody decreased during the non-transmission wet season, and increased over a 1-2-month transition period between the end of monsoon rains and the onset of dry conditions, an interval of maximum vector activity. Antibody increased with age in the population. The prevalence of antibody to the asexual blood stage as measured by conventional indirect fluorescent antibody assay did not coincide with changes in transmission and was sustained throughout the study period. Thus, CS antibody appeared to reflect the relative population exposure to mosquito inoculation of P. falciparum sporozoites and provided a useful measure of malaria transmission dynamics.     

Simultaneous use of Plasmodium falciparum crude antigen and red blood cell control antigen in the enzyme-linked immunosorbent assay for malaria

An enzyme-linked immunosorbent assay for malaria, based upon duplicate testing of serum samples on both a crude Plasmodium falciparum antigen and a red blood cell control antigen, is evaluated. Results were analyzed using the Student's t-test for identification of positive serum samples (t greater than or equal to 2.92, P less than or equal to 0.05) and for calculation of the mean difference in absorbance values (delta ABS) obtained between the P. falciparum wells and the control wells. Cross-evaluation with the IFA test for P. falciparum antibodies gave 89.6% concording positive or negative results. Among discrepant sera 8.35% were ELISA+/IFA- and 2.05% ELISA-/IFA+. In addition, delta ABS values in ELISA were highly correlated to titers obtained in immunofluorescence (r = 0.80, P less than 0.001). The results confirm the high degree of species-specificity of the ELISA using P. falciparum crude antigen. The necessity of the simultaneous use of red blood cell control antigen with a crude plasmodial antigen is demonstrated by comparing the presented results with those obtained on the P. falciparum antigen only.     

A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay.

We report a double-site enzyme-linked lactate dehydrogenase immunodetection assay (DELI), a highly sensitive antigen-capture enzyme-linked immunosorbent assay, which proved to be more sensitive for the detection of Plasmodium falciparum than thick blood smears, as sensitive as the polymerase chain reaction, and probably more reliable. This technique can help to detect infra-microscopic parasitemias (one parasite in 10(6)-10(8) red blood cells) from biological samples, and being quantitative, provide a fast substitute to thick smears for epidemiologic purposes. The technique can also be used to measure the in vitro drug sensitivity of P. falciparum with greater ease, much greater speed, and simpler equipment than that required for the isotopic microtest. Results obtained with four antimalarial drugs upon 16 strains closely paralleled those obtained by the isotopic assay (R = 0.95). In contrast with the latter, much lower parasite densities could be tested in the DELI assay (as low as 0.005%), thereby extending the number of isolates that can be investigated. The ease of implementation and low cost of the DELI-microtest may contribute to a revived interest in using in vitro methods to survey resistance to antimalarial drugs, so as to better predict future in vivo drug failures and provide public health recommendations.     

Immunodiagnosis of human fascioliasis by enzyme-linked immunosorbent assay using excretory-secretory products

In sera from patients with fascioliasis the enzyme-linked immunosorbent assay (ELISA) was used to detect antibody using excretory-secretory products (ES) from Fasciola hepatica adult worms. The specificity of ES-ELISA (with OD values greater than 0.38) allowed the differentiation among fascioliasis, schistosomiasis, clonorchiasis, and other human parasite infections.     

Detection of antibodies in human sera to the repeating epitope of the circumsporozoite protein of Plasmodium falciparum using the synthetic peptide (NANP)3 in an enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibody in human sera to a synthetic peptide, Asn-Ala-Asn-Pro (NANP)3, derived from the repeating amino acid sequence found in the surface circumsporozoite protein of Plasmodium falciparum sporozoites. One hundred four sera from U.S. residents were used to determine a cut-off value for reactivity. Test sera were considered reactive when the absorbance was greater than that at the 95th percentile of the control sera. Sera from 112 Kenyans living in an area of holoendemic malaria transmission were tested. Of the total number of sera, 65% had detectable antibody to (NANP)3. The percentage of reactive sera increased from 41% in sera from children under 4 years of age to 85% in sera from adults 20 to 39 years of age. The high exposure to malaria parasites of the Kenyans was reflected in indirect fluorescent antibody assay titers to blood stage P. falciparum parasites. All of the Kenyan sera had antibody present at titers greater than 1:256.