Background: It has been observed that dwellers in malaria endemic areas, who are continuously exposed to malaria parasitaemia, over time develop some level of immunity against severe forms of the disease. The interaction between the pro- and anti-inflammatory cytokines is known to play key roles in the immune response to infectious diseases. The pathogenesis of malaria parasitaemia, including its progression to symptomatic manifestation, also seems to be strongly related to the interplay between these cytokines. 

Aim: The study showed correlations in between the plasma levels of Interferon-gamma (IFN-γ), Interleukin-six (IL-6), and Transforming Growth Factor-beta (TGF-β); and the correlation between Age and the plasma levels of the cytokines in children aged between 6 and 60 months, when infected with malaria parasitaemia.

Method: The study was a cross-sectional study involving children with uncomplicated malaria. In the study, malaria parasitaemia was confirmed by microscopy, using Giemsa-stain. The enzyme-linked immunosorbent assay (ELISA) method was used to evaluate the plasma levels of IFN-γ, IL-6 and TGF-β in the subjects. 

Results: The median plasma IFN-γ, IL-6 and TGF-β levels in participants with malaria parasitaemia were 225.15 pg/mL, 123.31 pg/mL and 2091.02 pg/mL, respectively. The relationship between plasma IFN-γ and IL-6, in the children with malaria parasitaemia showed a statistically significant positive correlation (r = 0.793; p < 0.001). 

Conclusion: The findings in this work showed that malaria parasitaemia in under-five years’ children is associated with a significant positive correlation in the plasma levels of IFN-γ and IL-6, and a significant negative correlation between IFN-γ and TGF-β.

Malaria is an infectious disease caused by caused by species of Plasmodium (Ghosh and Stumhofer, 2021). In 2021, the global estimation of the malaria incidence was 247 million cases in 84 countries, which are mostly from the Sub-Saharan Africa. Four countries in the sub-Saharan Africa, with Nigeria inclusive, accounted for 48% of malaria cases globally 1. In the year 2021, across Africa mortality from malaria, was estimated at 593,000; under-five children comprising of 76% of the mortality. Thus, making the disease to be ranked the largest single component of disease burden in Africa by the WHO 1.

Species of Plasmodium are the major aetiological factor for malaria in humans and are transmitted by infected female Anopheles mosquitoes, which are widely distributed throughout Africa, Asia, and Latin America. Six species of Plasmodium implicated in human malaria are: Plasmodium falciparum, P. vivax, P. ovale curtis, P. ovale wallikeri, P. malariae, and P. knowlesi 2. In the sub-Saharan African, P. falciparum is the most prevalent cause of malaria, and is mostly implicated in severe forms of malaria, which is responsible for most malaria fatalities globally 3.

In the sub-Saharan African countries, where malaria is endemic, the disease is associated with high rate of transmission, and under-five years’ children appear to bear the brunt of severe forms of the disease with their attendant complications. These complications come with a very high morbidity and mortality in this age group 4. Nevertheless, in the non-endemic regions, where the exposure to bites from infected female Anopheles mosquitoes is relatively low, these is associated low level of acquired immunity to malaria evenly spread across all age groups in the populace. The morbidity and mortality also appear to be dependent on the extent of exposure to bites from infected female Anopheles mosquitoes and not age groups 5,6.

Mediators of inflammation have been shown to be very important in explaining the pathogenesis of malaria, and other febrile illnesses 7,8,9. Tumour Necrosis Factor-alpha (TNF-α) appear to be helpful in stimulating effector cells and also offers protective immune response in the disease by induction of fever, which is helpful in creating unfavorable environment for the parasite’s development. However, elevated TNF-α and some other pro-inflammatory cytokines, have also been observed to be associated with severe forms of malaria, and are noted to be key mediators in determining the severity of the disease 8,9. When the activities of these pro-inflammatory cytokines are not well modulated by anti-inflammatory cytokines, the pathologic consequences are observed to be out of proportion, thus resulting in severe form of the disease. This shows that the anti-inflammatory cytokines also play some critical roles in determining the clinical course of malaria, and is also important the protection against severe forms of malaria, especially, in children 10,11,12. 

As the quest for a durable malaria vaccine is still ongoing, to ameliorate or eliminate the morbidity and mortality often associated with P. falciparum infection, among the under-five, a more critical review of the interplay between these cytokines in malaria endemic zones could be of help in taming this childhood-killer-disease, in the sub-Saharan Africa and other malaria endemic regions of the world.

This study was designed to underscore any correlation between the plasma levels of Interferon-gamma (IFN-γ) and Interleukin-6 (IL-6), which are pro-inflammatory cytokines; Transforming Growth Factor-beta (TGF-β), an anti-inflammatory cytokine; and the ages groups of under-five Nigerian children, infected with P. falciparum. This is in view of this aiding in the designing of the long awaited, durable and potent malaria vaccine.

Study Area: The study was carried out at the Children Out-Patient Clinic (ChOPC) of Federal Teaching Hospital Abakaliki (FETHA) with Children on Clinic visitation and with pupils of Redeemer’s International School Abakaliki (RISA). Both facilities are located in Abakaliki Local Government Area of Ebonyi State, South-Eastern Nigeria. Ebonyi State shares a border with Benue State to the north, Enugu State to the west, Imo and Abia States to the south, and Cross River State to the east. 

Federal Teaching Hospital Abakaliki, being a tertiary health facility, provides specialized tertiary health care services, with Children Out-Patient Clinic has between 100-160 children in attendance, on daily basis.

Study Participants: Children aged between six and sixty months, who presented at the Children Out-Patient Clinic (ChOPC) of the hospital, and apparently healthy-looking pupils from Redeemer’s International School Abakaliki (RISA), were enrolled, with consent from their caregivers, after detailed information on what the study was all about. 

Study Design: The study was a cross-sectional descriptive study.

Inclusion Criteria

All children, aged between 6 and 60 months, with febrile illness and any healthy-looking child within the age bracket (as controls).

Exclusion Criteria

Severely malnourished child/ Grade III malnutrition, using Gomez’s Classification i.e 60% or less of Weight for Age (WFA) 13; Child with confirmed immunosuppressive disease, like: HIV/AIDS, Malignancy, Measles, Tuberculosis, cough of more than 2 weeks, etc; and Child with burns.

Ethical Consideration

Ethical Approval for the research was obtained from the Research Ethics Committee of the Federal Teaching Hospital, Abakiliki (FETHA). Only the children whose parent/guardian gave their consent were enrolled into the study. Confidentiality was maintained throughout the study. Caregivers were privy to the results of the investigations carried out on their wards, if they wished. As a form of incentive, children who tested positive to malaria infection were given free-malaria-treatment with Artemisinin-based Combined Therapy (ACT), following the dose prescription by the resident doctor at the Clinic, whereas, those that tested negative were given haematinic syrup. 

Sample Collection: The Nurses at the Children Outpatient Clinic of FETHA assisted in taking the biophysical profiles of the children, during the registration. A total of 2ml of blood sample was collected from each enrollee in the survey, via venepuncture, and was promptly transferred into the sample bottle which contains Ethylene diamine-tetracetic acid (EDTA). Each batch of samples collected, was taken to the Research laboratory of FETHA for processing, within 20minutes of their collection from the participants. 

Laboratory Procedures: In the laboratory, for each specimen, blood films (thick and thin films) for malaria parasite microscopy were quickly made on the glass slides, for malaria parasite identification. The remaining sample is centrifuged for 15 minutes, and the supernatant (plasma) separated into a plain specimen bottle, and then frozen at -200C. The frozen plasma specimens were transported, under cold chain maintenancee, to Ohize Medical Center, Benin City, Edo State in Nigeria, where the cytokine analysis took place, using the commercial standard Enzyme-Linked Immunosorbent Assay (ELISA) kits.

The Malaria Parasite Microscopy

Making a Thin Blood Film (for intracellular parasite identification): A drop of blood sample (using capillary tube) was placed on each properly identified dry glass slide. The blood drop was immediately spread using a smooth-edged slide-spreader and allowed to air-dry. Afterwards, the slide was placed horizontally on a staining rack and a drop of methanol applied. This was allowed for 2minutes to fix the thin blood film.

Making a Thick Blood Film (for specie identification): Twenty-five microlitre of blood sample (using automatic pipette) was applied on properly identified clean slide. The blood sample was gently spread to make the thick smear, which evenly covered an area of about 15x15mm on each slide. With the slide in a horizontal position, it was allowed too thoroughly air-dry, 

Staining of the slides: The slides (both the thick and thin blood films) were faced upwards, supported on two rods in a staining trough, flooded with 10% Giemsa-stain solution and allowed for 10minutes. Afterwards, clean water was used to flush the stain from the slides. The back of the slides was wiped clean and placed in a draining rack to air-dry, before viewing under the microscope at x100 (oil immersion).

Statistical Analyses

The data collected were analyzed using Statistical Package for Social Sciences (SPSS) version 23. The study population was partitioned into five (5) age groups: 6 to 11months, 12 to 23months, 24 to 35months, 36 to 47months, and 48 to 59months. It was also partitioned according to sex. The malaria infected and the uninfected (control) populations were also, separately, partitioned according to age and sex. The median plasma levels of INF-γ, IL-6 and TGF-β for the infected and uninfected groups were determined. Analyses for correlations between plasma levels of INF-γ, IL-6 and TGF-β and Age in the infected population were done using the Spearman’s Correlation Test. Positive Spearman’s correlation coefficients, (+ r-values) were taken as positive correlation, and negative Spearman’s correlation coefficients, (- r-values) were taken as negative correlation. Probability value (p-value) of less than 0.05 was considered to be statistically significant.

Sample Characteristics: A total of eighty-nine (89) children, aged between 6months and 59months were enrolled in the survey. Analyses for age distribution showed 10 were within 6 to 11 months age group, 18 were within 12 to 23 months, 27 were within 36 and 47 months, and 17 were between 48 and 59 months (Table 1). For the sex distribution, 42 were females, while 47 were males; with female to male ratio of 1:1.1 (Figure 1).

Malaria Parasitaemia: Presence of malaria parasite was noticed in 50 out of the 89 samples collected from the study population, as shown in Table 1. The 39 children without malaria parasitaemia were used as control group in the study. The age distributions of the infected and uninfected groups are both illustrated in Figure 1. The age group of 24-35 months had the highest number of children (16 children) with malaria parasitaemia, whereas, children aged between 6-11 months had the least (5 children). For the uninfected population (control group), the age group of 24-35 months, also had the highest number of children (11 children), whereas, children aged between 48-59 months had the least (3 children).

The Sex Distribution of the Infected and Uninfected Population 

The males appeared to be more infected with the malaria parasites than the female population; with the total of 30 out of the 50 infected participants being males. The proportion of the affected males was 30 out of the 47 male participants (i.e 63.8% of the male participants), unlike the females in which out of the 39 participants, 20 had malaria parasitaemia, (i.e 51.2% of the female participants). These are illustrated in Figure 1.

Table 1:  Age Distribution of All the Participants, showing the Infected and Uninfected Population

Figure 1: Bar-chart Showing Sex Distribution of the Participants

Description of the Cytokines Levels

For the population with malaria parasitaemia, the median levels for INF-γ, IL-6 and TGF-β were 225.15 (110.84-549.15) pg/mL, 123.36 (53.55-168.25) pg/mL, 2091.02 (1182.76-3685.17) pg/mL. For the population not infected with malaria, the mean levels for INF-γ, IL-6 and TGF-β, were 178.86 (107.61-272.70) pg/mL, 65.78 (37.60-180.71) pg/mL, and 4813.74 (2013.51-6414.23) pg/mL. These are shown in Table 2.

Table 2: The Cytokines Levels in the Infected and Uninfected Population

Spearman’s Correlations between the Cytokines’ levels and Age in Infected Population

Among the population with malaria parasitaemia, the Spearman’s Correlation test between the plasma levels of IFN-γ, IL-6 and TGF-β and the age variations, showed a significant positive correlation between plasma levels IFN-γ and IL-6 (Spearman’s correlation coefficient, rho, = +0.561, p <0 xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed>

Table 3: Spearman’s Correlations between the Cytokines’ levels and Age in Infected Population

* = Spearman’s correlation coefficient (rho)

** = p-value 

Spearman’s Correlations between the Cytokines’ levels and Age in Uninfected Population

Among the population not infected with malaria parasitaemia, the Spearman’s Correlation test between the plasma levels of IFN-γ, IL-6 and TGF-β and the age variations, showed a significant positive correlation between plasma levels IFN-γ and IL-6 (rho= +0.546, p <0 xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed xss=removed>

Table 4: Spearman’s Correlations between the Cytokines’ levels and Age in Uninfected Population

* = Spearman’s correlation coefficient (rho)

** = p-value 

Correlation of plasma IFN-γ and plasma IL-6

There was a very significant positive correlation between the plasma levels of IFN-γ and IL-6 in the population with malaria parasitaemia, in this index study. This was in keeping with the study conducted among children, aged five to fourteen years, who resided in a region of Papua New Guinea, a malaria endemic zone, where red blood cells that were infected with P. falciparum, were applied to isolated peripheral blood mononuclear cells. Production of pro-inflammatory cytokines, including IFN-γ and IL-6, by the mononuclear cells were assayed and their key sources investigated. The study showed that increased P. falciparum-induced IL-6 and IFN-γ levels were associated with an increase in incidence of P. falciparum clinical episodes 14. This was also, supported by the study of the interplay between IFN-γ and IL-6 signaling during polymorphonuclear cell migration in acute inflammation, where it was noted that the murine IL-6 got elevated on induction of IFN-γ. The neutrophil infiltration was reported to be controlled by the IFN-γ, which also modulates IL-6 signaling through its soluble receptor (sIL-6R) to promote their apoptosis and clearance 15.

Correlation of plasma IFN-γ and plasma TGF-β 

There was significant negative correlation between IFN-γ and TGF-β among the population infected which was in support of the findings in by de Jong et al, in a Controlled Human Malaria Infection (CMMI) conducted in volunteer subjects. When the subjects were infected with malaria parasite, there was a significant decrease in serum TGF-β when compared to the baseline, and a concomitant increase in the concentration of assayed pro-inflammatory indicators, like IFN-γ, IL-6, D-dimer and von Willebrand. However, those that showed sustained increase in the TGF-β, showed less severe clinical symptoms and the lesser markers of inflammation 16. This could be as a result of the immune modulating effect of the TGF-β, released by the T-regulatory cells, which regulates the activities of the immune mediators, thus ameliorating the clinical symptoms which are resultant effects of the activities of the pro-inflammatory cytokines, in a bid to rid the body of the parasite 9,12. The findings by Omar and Riley, also showed that TGF-β activities, in murine malaria, are quite important in maintaining the balance between control and clearance of infectious organisms and at the same time in the prevention of immune-mediated pathology. Thus, regulation of host-parasite interaction and mitigating the pathogenesis or worsening of the disease process 17. In a study done by Wenisch et al, in Bangkok, Thailand, patients with acute P. falciparum malaria were observed to have decreased levels of serum TGF-β, when compared to the healthy control group, prior to treatment. However, there was subsequent increase in the levels after initiation of treatment, and attainment of normal range on day 21 after the treatment commencement. They also noticed a significant inverse correlation of TGF-β and a pro-inflammatory cytokine, Tumour Necrosis Factor-alpha. This shows that with clearance of the pathogen which induces inflammatory response, the TGF-β production is increased to modulate the pro-inflammatory activity, with restoration of homeostasis 18. 

Correlation of plasma IL-6 and plasma TGF-β

There was a negative correlation between IL-6 and TGF-β, though not statistically significant, unlike some studies where the relationship between TGF-β and other pro-inflammatory cytokines like, IFN-γ and TNF-α, were observed to be have significant negative correlation with IL-6 9,12,18. This could be as a result of using a population that comprised of children with uncomplicated malaria parasitaemia, including those with asymptomatic parasitaemia, in a bid to avoid confounding factors like sepsis, which is often associated with severe/complicated malaria due to the attendant immunosuppression. In a study by Wu et al, in investigating the correlation between malaria and immune cells, TGF-β was found to be negatively correlated with the pro-inflammatory cytokines- IFN-γ and IL-6, in individuals with severe forms of malaria. They also noted that low TGF-β in a host prevents the host from being able to manage the inflammation that Plasmodium causes, resulting in pathological situation that leaves the host vulnerable to fatal infection 19. In other words, with the decrease in the level of the anti-inflammatory cytokine, TGF-β, in malaria parasitaemia, there is attendant reduction in its immune-modulatory activities, causing both IFN-γ and IL-6 activities to get exaggerated, thus worsening the clinical outcome of the disease process 20. Keswani et al, in their study on association between TGF-β and IL-6 in mouse model during Plasmodium berghei ANKA (PbA) infection, showed that there was worse clinical manifestation with treatment with anti-TGF-β, whereas, there was delayed immunopathology process with treatment with IL-6. Showing that worse disease progression was also associated with down-regulation of TGF-β and up-regulation of IL-6 in splenic cells during the PbA infection 21.

Correlation of Age and plasma IFN-γ

In this study, there was a positive correlation between the ages of the children and their levels of plasma IFN-γ, though not significant. The study by Weigering et al, showed age-associated increase in plasma levels of IFN-γ, in healthy children aged between zero to eighteen years, when their peripheral blood mononuclear cells or cord blood mononuclear cells were stimulated, in vitro, with phorbol-12-myristate-13-acetate or ionomycin 22. This is as due to immune immaturity of the very young, on account of their lower proportion of IFN-γ-producing CD4-positive and CD8-positive cells, when compared to the adults with aged microenvironment for lymphocyte maturation 23,24.

Correlation of Age and plasma IL-6 

There was negative correlation between the age of the children infected with malaria parasitaemia and their levels of plasma IL-6, although the relationship was not statistically significant. However, IL-6, being a key mediator of inflammatory processes, is known to increase with age, and this is often as a result of some physical changes associated with advance in age, particularly those related to chronic inflammatory diseases, which are not common in the very young age group 25.

Correlation of Age and plasma TGF-β 

A negative correlation was also observed between the age and plasma level of TGF-β, in children infected with malaria parasitaemia, although the relationship was not statistically significant. However, the relationship which was also a negative correlation was found to be very significant in the control group, which comprised of apparently healthy children, without malaria parasitaemia. A similar finding was also noted, in apparently healthy children, in the work done by Stark et al, while studying the role of TGF-β on Marfan syndrome 26.

Summary

In this study, children of age 24-35 months accounted for the highest number of children with malaria parasitaemia, whereas, children aged between six to eleven months had the least. The study also showed a higher incidence of malaria parasitaemia in the males than the females. A very significant positive correlation between the plasma levels of IFN-γ and IL-6 in the population with malaria parasitaemia was noted. Likewise, a significant correlation between IFN-γ and TGF-β was observed. Age related differences in the levels of the cytokines in the children with malaria parasitaemia were noted, however, none was statistically significant.

The host-plasmodium interaction in human results in complex cellular immune responses, which results in diverse interactions between the pro-inflammatory and the anti-inflammatory cytokines and other components of immune response. The interplay between these cytokines (both pro-inflammatory and the anti-inflammatory cytokines) play a critical role in determining the severity of the disease. The findings in this work, showed that, not with-standing the malaria endemicity in Nigeria, under-five years Nigerian children infected with Plasmodium falciparum exhibit statistically significant positive correlation in the plasma levels of IFN-γ and IL-6, and a negative correlation between IFN-γ and TGF-β in those with malaria parasitaemia. 

Limitations

-Not determining the Parasite density.

-Not determining the submicroscopic infections using PCR. 

Recommendations

From the results obtained the following recommendations are made:

1.These findings can be of help in the development of effective malaria vaccine.

2.Further research of this sort determining the inflammatory cytokines levels in other common inflammatory diseases, among these age groups (6-60 months) and correlating the levels to those with complicated and those with uncomplicated malaria.

3.A similar but more robust research involving the young adults, who are seemingly not prone to severe forms of P. falciparum malaria. 

Author Contributions

EOE: Wrote the first draft

EMC: Critical revision of the work

EO: Guidance with regards to handling the study population

ACO: Collection/Processing of the blood samples

UGC: Collection/Processing of the blood samples

ONA: Collection/Processing of the blood samples

OSO: Making and viewing of the slides for MP microscopy

EOM: Editing of the work

EAN: Collection/Processing of the blood samples

NFO: Collection/Processing of the blood samples

Acknowledgement:

We are grateful to Dr. Nnachi Oluomachi, the Head of department of Haematology, Alex Ekwueme Federal University Teaching Hospital, Abakaliki for the provision of a conducive environment in the Research laboratory, where the initial processing/storage of the samples took place before being transported to another laboratory for the ELISA analyses.

Conflict Of Interest

The authors declare that the research was conducted without any form of commercial of financial relationship that could portend a conflict of interest.

Appendix

The cytokines analyses by enzyme-linked immunosorbent assay (elisa)

1. Transforming growth factor-beta (tgf-β) 

Manufacturer

Bioassay Technology Laboratory, 

1713 Junjiang International Bldg. 218Ningguo Rd. 

Yangpu Dist. Shanghai, 

China. Cat.No E30511Hu

Specification

Standard Curve Range: 10pg/mL-400pg/L

Sensitivity: 5.11pg/mL

Intra-Assay: CV < 8>

Inter-Assay: CV<10>

Storage: 2-8OC

The principle 

The ELISA kit used the Sandwish-ELISA principle. The TGF-β was added to the wells pre-coated with TGF-β monoclonal antibody. After incubation a biotin-conjugated anti-human TGF-β antibody was added and bound to human TGF-β. After incubation unbound biotin-conjugated anti-human TGF-β antibody was washed away during a washing step. Streptavidin-Horseradish Peroxidase (HRP) was added and bound to the biotin-conjugated anti-human TGF-β antibody. After incubation unbound Streptavidin-HRP was washed away during a washing step.  Substrate solution was then added and colour developed in proportion to the amount of human TGF-β. The reaction was terminated by addition of acid stop solution and absorbance was measured at 450nm. 

Reagent preparation

All reagents were brought to room temperature. Then, 120µL of the standard (4800ng/L) was reconstituted with 120µL of standard diluent to generate a 2400ng/L standard stock solution.  This was allowed sit for 15 minutes with gentle agitation prior to making dilutions. Duplicate standard points were prepared by serially diluting the standard stock solution (2400ng/L) 1:2 with standard diluent to produce 1200ng/L, 600ng/L, 300ng/L, 150ng/L, solutions. The standard diluent served as the zero standard (0ng/L). 

Assay procedure

All the reagents were brought to room temperature. Then, 50µL of the standard was added to standard well. Afterwards, 40µL sample was added to sample wells and then 10µL anti-TGF-β into antibody wells. Then 50µL streptavidin-HRP was added to sample wells and standard wells (not blank control well). This was mixed well and before the plate was covered with a sealer, and then incubated for 60 minutes at 37oC.

Afterwards, the sealer was removed and the plate washed 5 times with wash buffer. The plate was afterwards, blotted onto paper towel. 50µL substrate solution A was added to each well and then 50µL of substrate solution B added, subsequently. The plate was covered with a new sealer and incubated for another 10 minutes at 37oC in the dark.

Then, 50µL of Stop Solution was added to each well, and the blue colour changed into yellow immediately. The optical density (OD) values of each well were determined, using a microplate reader set to 450 nm within 30 minutes 

3.9.2.2. interleukin-6 (il-6)

Manufacturer

Elabscience Biotechnology Inc. 

Building B18, 2nd Phase of Biomedical Park, 

#858 Gaoxin Road, Donghu Hi-Tech Development Area, 

Wuhan, Hubei, Central China.

Specification

Sensitivity: 4.69 pg/mL

Detection Range: 7.81-500 pg/mL

Specificity: this kit recorgnizes natural and some recombinant

Standard Curve Range: linearly dependent coefficient >0.9900

Repeatability: <10>

Storage: 2-8OC

Test principle

The ELISA kit also used the Sandwish-ELISA principle. The micro-ELISA plate provided in the kit had been pre-coated with an antibody specific to Human IL-6. Standards or samples were added to the micro-ELISA plate and combined with the specific antibody. Then a biotinylated detection antibody specific for Human IL-6 and Avidin-Horseradish Peroxidase (HRP) conjugate were added to each micro plate well and incubated. Free components were washed away. The substrate solution was added to each well. The enzyme-substrate reaction was terminated by the addition of stop solution. The optical density (OD) was measured spectrophotometrically at wavelength of 450±2nm. The OD value is proportional to the concentration of Human IL-6. The concentration of Human IL-6 in each sample was calculated by comparing the OD of the sample to the standard curve. 

Reagents preparation

All reagents and samples were brought to room temperature before use. The concentrated wash buffer was diluted 25 times to working solution. The reference standard was then diluted to different concentrations. The Concentrated HRP Conjugated was diluted 100x to working solution. This was done 15 minutes earlier before reconstitution of the wash buffer. The Microplate reader was pre-heated while reconstituting the Concentrated HRP Conjugated after.

Assay procedure

After all the reagents were brought to room temperature, 100 µL of standard and sample were added to corresponding wells and incubated for 90 minutes at 37oC. Afterwards, the liquid was removed, and 100 µL of Biotinylated Detected Ab added to the wells and incubated for 1 hour at 37oC. The content was removed and washed for 3 times, before adding 100 µL HRP Conjugate, and was incubated for 30 minutes at 37oC. Again, wells were emptied of the liquid and washed for 5 times, before adding 90 µL of Substrate Reagent, and then incubated for 15 minutes at 37oC. Afterwards, 50 µL of Stop Solution was added to the wells, and the OD value determine at 450nm immediately. The results were now calculated.

3.9.2.3 Interferon-Gamma (Ifn-Γ)

Manufacturer

Elabscience Biotechnology Inc. Building B18, 2nd Phase of Biomedical Park, #858 Gaoxin Road, Donghu Hi-Tech Development Area, Wuhan, Hubei, Central China.

Specification

Sensitivity: 9.38 pg/mL

Detection Range: 15.63-1000 pg/mL

Specificity: This kit recorgnizes Human IFN-γ in samples. No significant cross-reactivity or interference between Human IFN-γ and analogues was observed.

Standard Curve Range: linearly dependent coefficient >0.9900

Repeatability: Coefficient of variation is <10>

Storage: 2-8OC

Test principle

IFN-γ ELISA kits also used Sandwich-ELISA principle. The micro-ELISA plate provided in the kit was already pre-coated with an antibody specific to Human IFN-γ. Standards or samples were added to the micro-ELISA plate and combined with the specific antibody. Then a biotinylated detection antibody specific for Human IFN-γ and Avidin-Horseradish Peroxidase (HRP) conjugate were added to each micro plate well and incubated. Free components were washed away. The substrate was added to each well. The enzyme-substrate reaction was terminated by the addition of stop solution. The optical density (OD) was measured spectrophotometrically at wavelength of 450±2nm. The OD value is proportional to the concentration of Human IFN-γ. The concentration of Human IFN-γ in each sample was calculated by comparing the OD of the sample to the standard curve. 

Reagent preparation

The kit was brought out from the refrigerator 20 minutes in advance to bring all reagents to room temperature before use. The concentrated wash buffer was diluted to 25 times. The Reference Standard was diluted to different concentrations, and the Biotinylated Detection Ab diluted to 100 times. The 100x Concentrated HRP Conjugated was diluted to 100 times working solution 15 minutes earlier before step before dilution of concentrated wash buffer was completed. The Microplate reader was then pre-heated.

Assay procedure

After all the reagents were brought to room temperature, 100 µL of standard and sample were added to corresponding wells and incubated for 90 minutes at 37oC. Afterwards, the liquid was removed, and 100 µL of Biotinylated Detected Ab added to the wells and incubated for 1 hour at 37oC. The content was removed and washed for 3 times, before adding 100 µL HRP Conjugate, and was incubated for 30 minutes at 37oC. Again, wells were emptied of the liquid and washed for 5 times, before adding 90 µL of Substrate Reagent, and then incubated for 15 minutes at 37oC. Afterwards, 50 µL of Stop Solution was added to the wells, and the OD value determine at 450nm immediately. The results were now calculated.