the symptoms of malaria include fever and anemia , and most of the deaths are caused by the parasite plasmodium falciparum .
the merozoite form of the parasite invades red cells , grows to form ring- , trophozoite- and schizont stages , and after rupture of the infected red cell new merozoites are released that are ready to enter uninfected red cells .
merozoite invasion is a process that takes only a few minutes , but it involves several complex receptor - ligand interactions . initial attachment of the merozoite is mediated by merozoite surface proteins such as msp1 and msp2 , and is followed by reorientation of the merozoite where apical membrane antigen 1 ( ama1 ) is of importance [ 4 , 5 ] .
other ligands such as erythrocyte - binding antigens ( ebas ) , for example , eba140
, eba175 , and eba181 and p. falciparum reticulocyte - binding homologues ( pfrhs ) , including pfrh1 , pfrh2 , pfrh4 , and pfrh5 have also shown to be involved in the invasion process [ 69 ] , even though the exact function of each antigen is not known .
genetic polymorphisms exist for many of the above - mentioned ligands , and based on some genes like msp2 , parasites can be grouped into two major allelic types : 3d7 and fc27 .
serine repeat antigens ( seras ) are proteases that take part in forming a protein complex that is associated with the merozoite surface [ 1012 ] , and entry into the red blood cell is finally completed by an actin - myosin motor movement [ 13 , 14 ] .
individuals who live in malaria endemic areas eventually develop immunity , but only slowly and after repeated exposure [ 15 , 16 ] .
women have a greater risk of succumbing to malaria , and the fetus is also at risk .
it is known that antibodies are important in the defence against malaria , and it has been shown that passive transfer of antibodies from immune donors to individuals with p. falciparum infection reduces parasitemia and clears clinical symptoms [ 1821 ]
. however , exactly which specific antibodies are protective against future disease are not yet defined , and how they should be measured is even less clear .
this information is urgently needed to be able to develop a functioning vaccine , something that has so far failed .
we will here discuss the pros and cons for different methods available such as elisa , invasion inhibition assays ( iias ) , antibody - dependent cellular inhibition ( adci ) , and affinity , and we compare it to measurements of antibodies in other diseases and how the overall evaluation of immunity or vaccine status of malaria could possibly be improved .
when antibodies directed against different p. falciparum antigens have been measured , elisa has usually been the method of choice . in this static method
, proteins are coated to a plate and levels of antibodies in plasma from patients with ( or without ) malaria can be estimated .
when recombinant proteins are used , only antibodies directed against specific antigens are analyzed . in real life ,
the antigen of choice is located together with several other antigens , for example , on the merozoite surface , and there might be interaction or competition between binding of different antibodies , something that is not accounted for in an elisa .
it has , for example , been shown for msp1 that there are blocking antibodies that can compete with the binding of cleavage - inhibiting antibodies for epitopes on the merozoite [ 22 , 23 ] , and there are also other studies that have demonstrated that mixing of different antibodies can influence the outcome of the assay .
this kind of studies indicates that we should more look at using assays where the function of antibodies is studied , but the reason for why elisas are continued to be used to such a major extent is probably that they are very easy , fast , and robust to perform compared to functional assays .
when recombinant proteins are applied in elisas , the result might depend on which part of an antigen that is selected for use in the assay .
for msp1 , it has been shown that antibodies against msp1 - 19 were associated with some protection , while antibodies against msp1 block-1 were not . however , even when the same subdomain has been used such as in studies of eba175 , contradictory results have been achieved for whether there was a protective effect of antibodies or not [ 26 , 27 ] .
when red cells burst due to egress of merozoites , a lot of  debris  will be left in the blood stream that needs to be removed , and many of the antibodies might help in doing this but this does not mean that the antibodies will protect from future disease .
if only elisas are used , it is difficult to discern which antibodies are functionally important . in general
, higher levels of antibodies are found in elisas in older individuals in endemic areas , while lower levels of antibodies are seen in younger individuals in the same areas .
this was recently shown for the ebas for example   and it has been shown earlier for other antigens as well [ 7 , 26 , 27 , 29 , 30 ] . however , even though an individual has high levels of antibodies , they can still develop malaria , and an individual with relatively low levels of antibodies can be fully protected from clinical and severe malaria [ 25 , 3152 ] . in vaccine trials , antibodies measured by elisa
have been shown to often be short - lived , and most patients will still get malaria in spite of presence of antigen - specific antibodies . from a population perspective , elisas can be used to make an overall estimation of how much exposure there has been to malaria , but for each individual it is not possible to make an exact determination of the immune status .
the only thing that can for sure be concluded from a positive response in elisa is that the individual has at some stage during his / her lifetime been exposed to malaria . when elisas against different antigens
are combined , more information can possibly be acquired about the level of immunity in investigations of the breadth of antibodies , but the question of whether it is just a measure of exposure will still remain . a way of improving the elisas would be to more often use standardized controls , allowing for the measurement of exact amounts of specific antibodies instead of titers . in conclusion , elisas are easy and robust to perform , and they can clearly give us information about whether or not an individual has ever been exposed to malaria . with combinations of different antigens and standardization of the assays , more information can possibly be provided .
however , elisas do not tell us anything about the function of the antibodies , and on an individual level , elisas will not give us complete information about immunity .
one assay that has been used to try and better determine the function of antibodies in plasma is growth inhibition assay or invasion inhibition assay ( iia ) .
antibodies directed against merozoite antigens are thought to function by directly inhibiting invasion of new red cells , which will then stop further multiplication of parasites , or through adci . by adding immune plasma , which contains antibodies to growing parasites ,
the inhibitory function of the antibodies can be evaluated in comparison to parasites where no plasma has been added .
the downside of iias compared to elisas is that they are much more labor intensive , but on the other hand all proteins are expressed in their native environment and many both known and unknown potential interacting factors are included in the assay .
there have been several studies that have shown invasion inhibitory activity of antibodies from human plasma , both when total igg has been used and when malaria - antigen - specific fractions have been used [ 22 , 5458 ] . some studies
have shown increasing invasion inhibition with age   while others have shown more invasion inhibition in children [ 56 , 60 ] .
this kind of contradictory results might be explained by different functions in the antibodies repertoire being important during development of immunity , compared to when immunity is already established , but it might also mean that the assay is not yet fully optimized to show who is immune or not .
one attempt to improve the iia is by adding monocytes ( adci ) . when adci has been employed , some antigens like msp3 and glurp [ 61 , 62 ] have shown an inhibitory effect only when monocytes were included in the iia .
however , with adci there seems to be a major variability in the assay that can be seen from day to day even using the same donor of monocytes , making it difficult to standardize the assay .
if this assay could be improved and standardized , it might add very valuable information about different antibodies .
another attempt to try and improve iia is to use knockout lines of parasites . here
, a single antigen can be studied in its natural environment and with the correctly folded protein , and a comparison can be made between the wild - type and the knockout parasites .
this has been used , for example , for the ebas , where it was shown that antibodies against eba175 was responsible for a major part of the inhibitory activity in some individual plasma samples , while other samples seemed not to have any functional antibodies against this protein at all .
this kind of results is important for selection of potential vaccine candidates , especially for ruling out those antibodies that have no effect .
some antigens have been difficult to knock out , in which case other reagents might have to be added to the assay such as blocking agents , to find out which antibodies are causing the inhibitory effect . in conclusion , iias are labor intensive but can provide important information especially for comparisons between knockout and wild - type parasites , where the function of a single potential vaccine candidate antigen can be evaluated .
another way of looking at antibodies against merozoite antigens is to study the affinity / avidity of antibodies .
some studies have tried to use elisa with added nh4scn to evaluate affinity , but the results for this have been inconsistent [ 64 , 65 ] . a new way of investigating affinity for vaccine trials has opened up with methods like surface plasmon resonance ( spr ) [ 66 , 67 ] , where affinity of antibodies can be estimated under flow , something that ought to be more similar to the physiological situation compared to static assays . with this method , association and dissociation of antibodies binding to their target antigens
this method has before mainly been used with monoclonal antibodies in malaria research , but it has recently been applied also to polyclonal antibodies .
it has , for example , been shown that affinity of antibodies against ama1 increased with age , and the presence of high affinity antibodies in plasma against msp2 - 3d7 was associated with protection against malaria . in this study , most plasma samples showed a relatively rapid on - rate , indicating that whether the concentration of antibodies in the samples is high or low , the antibodies will still bind quite fast to their antigens .
this is important for considering whether an antibody will function in inhibiting merozoite invasion or not , since invasion is a process that takes only a couple of minutes
. the dissociation rate ( which is concentration independent ) might therefore be more important for whether an antibody will function or not . in the referred study ,
monoclonal antibodies were also used and it could be seen that some bound with so low dissociation rates , that a value for the dissociation rate could not be obtained .
however , investigations of affinity of antibodies in malaria are yet a very new field which needs a lot more studies to be able to make firm conclusions , and standardized protocols needs to be in place to facilitate interpretation of the results . in other infectious diseases , such as bacterial diseases [ 69 , 70 ] , toxoplasma , or hiv ,
studies of affinity of polyclonal antibodies in malaria is a new field that could add a lot of information both about how immunity is formed and for vaccine trials , and more work in this area is needed .
when different assays are used to evaluate antibodies against malaria , the methods often show results that do not correlate with each other . for example
, spr has been shown to correlate with elisa for ama1 , which binds with relatively high affinity , but not for msp2 , which binds with lower affinity . when iia has been compared to elisa
, some people have shown correlations while others have not seen any correlations [ 36 , 73 ] .
this is probably because iia is a functional assay , while elisa only estimates the levels of antibodies .
an example of the lack of major correlations between methods ( iia and elisa ) is shown in figure 1 .
this illustrates the difficulties in estimating immunity against malaria . for vaccine trials , one has to be careful with interpretation of results , as the results could vary a lot depending on which method is used . in other fields such as hiv ,
international consortia have established common standards to be used in immunological assays , and this might be applicable for elisas , but more challenging for the functional methods yet available in malaria .
none of the methods described here are good enough on their own to give a complete picture of an individual 's immune status .
elisas are good at giving us information about whether any immune response at all is mounted against a potential vaccine candidate , and from a population perspective when many samples need to be analyzed , elisas are easy to perform . however
, if we want to know something about the function of a specific antibody , iia with the usage of knockout and wild - type parasites should be the way forward . even though these assays are more cumbersome to perform , they will add valuable information .
affinity of antibodies has so far been very scarcely studied in malaria research compared to many other diseases , and expansion of this field should add important information both for knowledge about immunity and for vaccine trials .
more studies are needed that employ different methods together in the same patient cohorts to get a more full picture of which functions of antibodies are important during different stages of development of immunity against malaria . in conclusion , which method should be used depends on what we want to know about the antibodies . if we only want to know whether antibodies are formed or not , elisas can be used , but if we want to know something about the function of the antibodies , more elaborate assays such as iia have to be applied . to get the full picture about immunity status in an individual , the methods available have to be developed
more and probably combined to a bigger extent , but with new methods such as those available for affinity measurements there is hope that this situation can be improved .
 immunity against malaria develops slowly and only after repeated exposure to the parasite . 
 many of those that die of the disease are children under five years of age . 
 antibodies are an important part of immunity , but which antibodies that are protective and how these should be measured are still unclear . 
 we discuss the pros and cons of elisa , invasion inhibition assays / adci , and measurement of affinity of antibodies and what can be done to improve these assays , thereby increasing the knowledge about the immune status of an individual , and to perform better evaluation of vaccine trials . 
1. Introduction
2. ELISA
3. Growth Inhibition Assay/Invasion Inhibition Assay
4. Affinity
5. Comparing Different Ways of Measuring Antibodies
6. Which Method Should We Use to Measure Antibodies against Malaria?