






































Type of the Paper (Article


 

  

 

 
Cluj Vet J 2024, vol 29, issue 1 http://clujveterinaryjournal.ro 

Review  

Comprehensive Evaluation of Direct Methods for Failure of 
Passive Transfer Diagnosis in Neonatal Calves 
Dragoș Adrian Popescu 1, Florin Petrișor Posastiuc 1, 2, *, Nicolae Tiberiu Constantin 1, 3, Crina Raluca Andrei 1,3, 
Florina Marian4 and Mario Darius Codreanu 1    

1 Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Ro-
mania  

2 Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium  
3 Research and Development Institute for Bovine Balotești, Balotești, Romania  
4 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 

Romania 
*Correspondence: florin.posastiuc@ugent.be 

Abstract: Failure of Passive Transfer (FPT) is a critical concern in neonatal calves, marked by inadequate maternal im-
munoglobulin transmission due to the synepitheliochorial placenta. This leads to heightened disease prevalence, sig-
nificantly impacting calf health and imposing substantial economic burdens. The prevalence of FPT varies globally, 
with studies reporting incidence rates ranging from 13% to 41%, one of the explanations for such high variability being 
the use of different methods and non-adapted thresholds. Efficient diagnostic methodologies are essential to address 
this challenge, with ongoing exploration of alternative approaches beyond the established gold standard of radial im-
munodiffusion (RID). This review critically evaluates the available direct methods for FPT assessment gathering the 
most recent data on both established methods such as enzyme-linked immunosorbent assay (ELISA) and innovative 
methodologies such as immunoturbidimetry, split trehalase IgG assay (STIGA), ionization techniques (IT), mass spec-
trometry (MS), proteomics, and infrared spectroscopy (IS) with attenuated infrared spectroscopy (ATR). Exploring be-
yond conventional practices is vital for enhancing diagnostic accuracy and addressing the complexities of FPT in calf 
health management. 

Keywords: failure of passive transfer; radial immunodiffusion; calf management; immunoglobulins, total serum protein 
 

 

1. Introduction 
The synepitheliochorial placenta in cows, with its capacity to separate mater-

nal and fetal blood supplies, effectively inhibits the in-utero transmission of pro-
tective immunoglobulins [1]. Consequently, calves are born with insufficient im-
munity, making them highly dependent on colostrum intake [2].  

The colostrum incorporates blood serum components [3], such as immuno-
globulins, mainly IgG [4]. The secretion is transitory, and the antibody levels 
gradually decline over time, reaching very low levels 24 hours after birth [5]. An-
tibodies from the colostrum are absorbed by epithelial cells in the small intestine, 
particularly in the jejunum and ileum, via pinocytosis [1]. Afterward, immuno-
globulins are transferred to the main lymphatic tissue components, entering sys-
temic circulation via the thoracic duct [1]. Unfortunately, the pinocytotic mecha-
nism fully ceases the transfer of large molecules after the first 24 hours [2]. Beyond 

Received: 19 January 2024 

Accepted: 11 February 2024 

Published: 9 April 2024 

DOI:10.52331/bkynge51 

 

 

 

Copyright: © 2024 by the authors. 

Submitted for possible open access 

publication under the terms and 

conditions of the Creative Commons 

Attribution (CC BY) license 

(http://creativecommons.org/licenses

/by/4.0/). 

https://www.frontiersin.org/people/u/2595100


Cluj Vet J 2024, vol 29, issue 1 27 of 37 
 

this point, additional intake of colostral immunoglobulins can offer local intestinal protection but 
does not contribute to the calf's overall systemic protection [2]. 

The neonatal calf's inability to absorb sufficient colostral immunoglobulins during the initial 
hours of life leads to a failure in passive transfer (FPT) [6]. FPT was associated with high economic 
losses [6] and increased disease prevalence [7, 8]. Dairy calves experiencing FPT were twice as 
likely to develop diarrhea and necessitate antibiotic therapy before weaning [9-12]. Various thresh-
olds for serum IgG were contemplated based on the animal category. In the case of the mentioned 
dairy calves, the designated cut-off value for FPT was established at 10 g/L, with lower levels cor-
relating to elevated rates of mortality and morbidity [2, 13]. The situation appears to be more 
alarming in beef cattle, with the established threshold set at 24 g/L [14]. Beef calves with serum 
IgG levels below 24 g/L may be 1.6 times more susceptible to death compared to those with higher 
IgG values [14]. Other studies report even more concerning findings, considering a mortality rate 
2.7 times higher before weaning, under the same circumstances [15]. An alternative approach is 
represented by the measurement of serum total protein concentrations (STPC), considering a 
threshold of 52 g/dL as equivalent to IgG levels inferior to 10 g/L [16]. These results motivated a 
series of researchers to orient their work to different treatment and prophylactic alternatives, such 
as the use of hyperimmune plasma [17], along with non-enriched blood products [18].  

The prevalence of FPT was intensively studied in the last decade different prevalence rates 
being recorded, ranging from 13% [19] to 33% [20] and even 46.5% [21].  

However, these results may be method-dependent, and several direct or indirect techniques 
for FPT assessment being scrutinized such as: Brix refractometry (BR), gamma-glutamyl transfer-
ase activity, zinc sulfate turbidity test, enzyme-linked immunosorbent assay (ELISA), radial im-
munodiffusion (RID), electrophoresis, capillary electrophoresis (CE), turbidimetric immunoas-
says, Split trehalase IgG assay (STIGA), Ionization techniques (IT) and mass spectrometry (MS), 
Infrared spectroscopy (IS) with Transmitted & Attenuated Infrared Spectroscopy (ATR) and pro-
teomics. The suitability of various methods is still a topic of discussion, with some techniques 
proving more fitting for on-site calf applications but potentially lacking in sensitivity. The aim of 
this paper is to gather the most recent information on the direct methods available for FPT evalu-
ation, as indirect methods have been recently reviewed [22]. 

 
2. Direct methods for FPT evaluation 
 
2.1 Radial immunodiffusion (RID) – golden standard for FPT assessment 
Currently, the reference test for IgG determination is RID but even this method has several 

drawbacks being impractical for clinical use. This belief is backed up by the relatively high cost, 
the need for skilled operators, and a specifically equipped laboratory [23]. In addition, this method 
implies working on low-volume assays [23], the reagents and required antibodies having limited 
durability [24]. Results are usually obtained in 18 or 24 hours after admitting the samples to work, 
the measurements applied to the precipitation zones being time-consuming [25, 26]. In addition, 
if the coefficient of variation between the samples exceeds 10%, carrying out the test at least twice 
may be recommended [27, 28]. 

However, the method described by Mancini et al. (1965) with its variations has been 
extensively applied for different purposes in immunoglobulin quantitative studies [29, 30]. 

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Cluj Vet J 2024, vol 29, issue 1 28 of 37 
 

In contemporary literature, the use of RID is acknowledged as a valuable approach for 
assessing IgG levels in bovines. Findings indicate that, in certain instances, results obtained 
through RID may diverge from those obtained through alternative methods such as ELISA or 
electrophoresis [29] [31-33]. Gathering four different analytical methods of assessing FPT (ELISA, 
electrophoresis, and refractometry), a German research group used RID as gold standard, with 
results from the other procedures being referred to the latter [32]. Even if the same authors 
considered that the results from the other techniques are highly correlated to the standard, they 
stated that a true quantitative comparison between RID and ELISA or electrophoresis is 
inadequate. Moreover, the demand to adapt the thresholds for every assay depending on the 
sample type, serum or plasma, has been pinpointed. 

Beyond several studies comparing other methods to the RID IgG results in order to assess the 
applicability of different techniques [31-34], there is also data available regarding the comparison 
between commercial RID kits [35]. Thus, it has been ratified that even different available kits may 
be subject to variability. Accordingly, it was considered that a three internal standard kit 
formulation may reveal with higher precision IgG levels compared to a single RID kit [35]. 

 
2.2. Immunoturbidimetry  
Turbidimetric immunoassays have been recently developed and proposed for different 

applications such as acute phase protein determination in swine [36] and IgG serum evaluation in 
bovines [37]. Published research has indicated the potential for rapid analysis, with absorbance 
readings through spectrophotometry yielding results within 10 minutes [37]. This became 
achievable after adapting the original method [38] to the use of a portable laboratory instrument 
for in-farm applications [37]. The suitability of the method was backed up by analytical survey. 
Based on regression analysis, the automated immunoassay results closely paralleled those 
obtained by RID, with a correlation coefficient of 0.988,and a coefficient of determination value 
between immunoturbidimetry and RID of 0.98 [37].  

Point-of-care assays based on turbidimetry have proven good sensitivity and specificity [39]. 
Based on these premises other authors compared bovine specific turbidimetric assays to several 
techniques for FPT evaluation [40]. However, the turbidimetric immunoassays were deemed to be 
only fairly correlated to RID, and a large bias was determined between the two [40]. 

Further studies have analyzed the use of turbidimetry-based calf-side tests [41, 42]. The high 
efficiency of the above-mentioned for FPT diagnosis in calves is considered relatively sensitive 
and suitable for initial screening [42]. Moreover, to reduce processing time requirements, further 
validation of whole blood tests, through semiquantitative IgG determinations were proposed [41]. 
Unfortunately, the method did not prove to be sufficiently accurate for immune status assessments, 
exhibiting a 77% sensitivity and 44% specificity compared to RID [41].  

  
2.3. Split trehalase IgG assay (STIGA) 
A novel detection platform based on a glycolytic enzyme, trehalase respectively, was recently 

proposed [43].  
Further research has proven the possibilities of measuring IgG levels in more than one species, 

through the use of three bacterial immunoglobulin binding proteins (protein G, protein A, and 



Cluj Vet J 2024, vol 29, issue 1 29 of 37 
 

protein L) [44]. Thus, the use of this method has been ratified in a large number of species, with 
activation being detected in almost all tested situations, except for birds [44].  

In bovines, the results are spectacular, at an absorbance optical density of 0.2 sensitivity rate 
reaching 77.8% and specificity 98.2% [27]. Moreover, a blinded trial has been carried out, with high 
correlation values being observed when compared to RID for both colostrum and serum [27]. In 
this manner, the authors managed to obtain 100% sensitivity and 94.7% specificity, for FPT 
diagnosis when using serum samples [27]. 

The advantages of the method have been further depicted, highlighting its high efficiency in 
the direct detection of IgG levels through a single-step analysis [27]. In addition, STIGA provides 
swift results, requiring only 90 minutes as opposed to longer time intervals needed for RID (up to 
48 h) [27, 45]. From an economic perspective, STIGA is less expensive due to the lower production 
costs of the reagents compared to RID, less laborious, and suitable for automatization [27]. The 
latter statement was backed up by the user-friendly format that could be optimized for field testing, 
STIGAFIELD ®, which rendered the same high correlation between its results and the ones obtained 
by RID [27]. 

  
2.4. Enzyme-linked immunosorbent assay (ELISA) 
ELISA is broadly used for different means regarding veterinary practice. Among its diverse 

applications, this technique has been utilized extensively to determine IgG levels in colostrum, 
plasma, and sera [31, 33] [46-49]. 

Additionally, as a direct, laboratory-based method [49], ELISA proved to be a good alternative 
to RID in terms of costs [33]. Comparative studies have shown that the level of agreement between 
the two stated methods is considered to be high (94%), this assay being suitable for both screening 
and diagnosis of FPT. Another study gathering 206 Holstein-Friesian calves, compared ELISA to 
other methods while using different sample types. The Pearson correlation coefficients between 
the latter and RID remained at a high level (0.9) when serum was analyzed [32]. Also, a high 
correlation has been established between CE and ELISA (0.89), with the same authors proposing 
method-dependent cut-off values (5.4 mg IgG/mL for ELISA and 6.9 mg IgG/mL for CE) [32]. In 
contrast to these results, other authors were able to display only a relatively weak correlation 
between ELISA and RID (r= 0.59, P < 0.01), when using plasma samples [33], their results being 
closer to the ones obtained through immunoturbidimetry, also consistently lower than the RID 
values [50]. However, not only one statement rendering the comparison between such methods 
(RID vs ELISA) as being inadvisable, has been formulated [31, 33]. The main reason for the 
hindmost statement refers to the poor agreement between the assays [31]. 

A rapid test based on a competitive immunoassay was also compared to ELISA using 277 
samples obtained from calves housed in 16 farms [51]. The quick method and ELISA were highly 
significantly correlated but the strength of the correlation was only moderate (rs = –0.36) [51]. 
Moreover, the fast diagnosis kit revealed questionable sensitivity and specificity rates, reaching 
61.1% and 58.7%, respectively [51]. 

Even though ELISA is a time-consuming method, it is still broadly used when laboratory 
infrastructure is available. However, current data analysis does not consider it to have high 
potential for point-of-care or calf-side applications.  

 



Cluj Vet J 2024, vol 29, issue 1 30 of 37 
 

2.5. Ionization techniques (IT) and mass spectrometry (MS)  
IT such as solid or liquid matrix-assisted laser desorption/ionization (MALDI) are considered 

core techniques for molecular analysis through MS [52]. 
Solid MALDI MS profiling has been applied in many areas [53]. Thus, these attempts were 

carried out with variable success rates because of many reasons, including the limited analytical 
power [53]. Generally, liquid MALDI has additional advantages compared to the solid alternative 
such as greater sample homogeneity, greater ion beam stability, and lower sample consumption 
[54]. This kind of advanced diagnosis has been used for the detection of dairy cow mastitis, 
proving 98.5% accuracy and 100% specificity [53].  

Liquid MALDI MS is relatively affordable and highly efficient in terms of operating times as 
it has been previously shown [55] while dealing with sheep and goat milk protein and lipid 
profiling. Therefore, the latter study has highlighted a new potential alternative for colostrum 
discrimination via IgG detection. MALDI MS was compared to the presently employed methods, 
ELISA and BR, and was deemed globally superior [55]. 

Even if current literature does not state the use of these techniques for FPT evaluation in calves, 
it has been previously hypothesized that these advanced methods would be at a higher value in 
comparison with current alternatives [23]. Thus, research activity concerning these applications 
should be considered.  

 
2.6. Proteomics  
Fluid and tissue sample characterization through proteomic studies have been recently 

discussed, along with their implications in veterinary practice [56-59].  
Numerous proteins have been individualized through proteomics, during colostrum or milk 

analysis. One group identified 212 compounds, 208 being also quantified [59]. The same study 
highlighted the link between the decrease in protease inhibitors and the subsequent drop in im-
munoglobulins, suggesting that certain enzymes may have the ability to protect IgG from proteo-
lytic degradation [59]. Recently, IgG identification was reported in a proteomic study regarding 
colostrum compounds time-related variability [60]. Thus, using a peptide-centric LC–MS/MS, 212 
individual proteic sequences have been isolated, >50 % of the colostrum protein content being 
represented by IgG [60].  

Due to still absent information regarding serum compounds analysis in calves for FPT 
diagnosis, proteomics is still not established for this purpose. However, the results previously 
reported, particularly those concerning milk or colostrum examination, provide a foundation for 
a potential new tool that warrants further investigation.  

 
2.7. Infrared spectroscopy (IS) and transmitted & attenuated infrared spectroscopy (ATR) 
 
By courtesy of modern analytical chemistry, another tool has been proposed for different 

qualitative and quantitative measurements. IS has proved various applications being able to carry 
out multianalyte analysis [61, 62]. For IgG levels assessment IS has been trialed in humans [63], 
equines [64], and bovines [65, 66].  

One group tested ATR and IR for the measurement of bovine serum IgG concentration, thus 
FPT diagnosis [67]. The authors have stated that both methods are suitable for the proposed 



Cluj Vet J 2024, vol 29, issue 1 31 of 37 
 

applications, based on the sensitivity, specificity, and predictive values obtained.  Moreover, the 
use of different sample types was appraised, with optimal cut-off values leading to high levels of 
agreement (88.1%) between results derived from testing serum and plasma by transmitted IR 
spectroscopy [21].  

Transferability into field conditions was also scrutinized, and a portable ATR-IR spectrometer 
rendered suitable for use in alpacas [68]. Furthermore, the other rapid methods described [42] in 
correlation with the previously sketched portable ATR spectrometers [61, 62] have suggested their 
potential for calf-side applications. 

 
2.8. Electrophoresis and capillary electrophoresis (CE) 
Electrophoretic methods can be used to measure immunoglobulin concentrations in serum 

thanks to the final fraction migration [69]. The latter statement refers to IgG which is the least 
negatively charged molecule that will determine a slow migration through the electric field [23]. 
Therefore, it is considered that IgG electrophoretic analysis can be carried out with higher accuracy 
(89%) [29]. Moreover, a study regarding serum protein profiling has highlighted the utility of elec-
trophoretic findings in general practice situations such as neonatal calf diarrhea [70]. Nevertheless, 
the studies on ruminants have identified variables that could have affected the outcomes, includ-
ing the animal's age and physiological condition as well as sample pre-analytical preparations in 
the lab [71-73].  

For FPT assessment in calves, electrophoretic techniques have been applied and current data 
suggests adequate accuracy when compared to the gold standard (RID) [74]. 

Electrophoresis was successfully applied for IgG level determination in serum samples after 
colostrum supplementation in calves [75]. More data is available in equines, and a good agreement 
between RID and electrophoresis when determining IgG levels in foals being highlighted [76, 77].  

Thus, laboratory-based, electrophoresis is an interesting alternative to RID being a faster 
method, which still relies on specific cut-off values [32].  

CE is a superior technique that has been used in human medicine for serum protein recogni-
tion [78, 79]. Additionally, CE is more suitable for monoclonal components detection clinical im-
plications of these features being already depicted in related scientific works [79]. 

Fully automated [79] and cost-efficient [80], CE has been also used for veterinary research 
purposes regarding FPT. The method was applied in lambs, being considered reliable for serum 
IgG determination, due to its accordance with the gold standard RID [81]. CE was applied to sam-
ples from 216 clinically healthy Holstein Friesian calves, the results proving the high reliability of 
the method for FPT diagnosis when a cut-off value of 6.9 mg IgG/mL is considered [32].  

3. Conclusions 
The heightened awareness of a significantly high prevalence of FPT in neonatal calves accen-

tuates the imperative for reliable diagnostic methodologies. This underscores the need for accurate 
FPT assessment, given the economic implications and increased disease susceptibility that further 
emphasize the urgency of precise diagnostic tools. While RID serves as the established gold stand-
ard, persistent practical limitations necessitate the exploration of alternative approaches. Emerg-
ing technologies like immunoturbidimetry, and innovative methodologies such as the STIGA, MS, 

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Cluj Vet J 2024, vol 29, issue 1 32 of 37 
 

and proteomics show promise but require thorough validation for the establishment of practical, 
efficient modalities to ensure optimal calf health and productivity. 

Author Contributions: writing—original draft preparation, D.P., F.P.P., N.T.C, C.R.A.; writing—review and editing, 
D.P., F.P.P., N.T.C, F.M.; supervision, M.D.C.; All authors have read and agreed to the published version of the manu-
script. 

Funding: This research received no external funding. 

Conflicts of Interest: The authors declare no conflict of interest. 

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