Patra et al | Article 8016

J Pure Appl Microbiol. 2023. doi: 10.22207/JPAM.17.3.27

Received: 05 August 2022 | Accepted: 17 July 2023

P-ISSN: 0973-7510; E-ISSN: 2581-690X

RESEARCH ARTICLE OPEN ACCESS

Evaluation of an In-house Indirect ELISA for Differential

Detection of IgM and IgG anti-Brucella Antibodies in
Human Brucellosis

Sudipta Patra, Muneera M. Sahib, G. Shanmugam, Somy Skariah,

Samer Shamshad, Nagalingam Mohandoss, Bibek R. Shome and
Rajeswari Shome*

Indian Council for Agricultural Research -National Institute of Veterinary Epidemiology and Disease Informatics,
Bengaluru, Karnataka, India.

Abstract
Brucellosis caused by various species of the genus Brucella is one of the most important zoonotic
diseases of global importance with veterinary, public health, and economic concerns. The study
aimed to standardize IgM and IgG-based iELISA to detect anti-Brucella antibodies for serodiagnosis
of acute and chronic human brucellosis. The test was standardized using 1:320 dilution of smooth
lipopolysaccharide (sLPS) antigen from B. abortus S99 strain, 1:80 serum dilution, 1:4000 anti-human
IgM and IgG conjugates, respectively for both IgM and IgG iELISA. The cut-off using 50 each brucellosis
positive and negative human sera panel samples was set at ≥ 42 for both IgM and IgG iELISA. A total
of 700 human sera samples were evaluated (137 veterinary doctors, 157 artificial inseminators, and
406 veterinary assistants). Overall, the study detected 8.3%, 8.1%, 8%, and 6.1% positivity by in-house
IgG iELISA, RBPT, IgM iELISA, and SAT tests, respectively. Considering commercial iELISA kit as a gold
standard, the sensitivities of IgM and IgG iELISA were 90% and 97.9%, respectively, whereas, specificities
were >99%. The study established >98% specificity and >90% sensitivity for differential detection of
immunoglobulin classes in the standardized iELISA. The developed assay outperformed the other
evaluated tests with a shorter assay time and can be implemented in both endemic and non-endemic
regions for surveillance and diagnosis of human brucellosis.
Keywords: Brucellosis, Diagnosis, Human, Indirect ELISA

*Correspondence: rajeswarishome@gmail.com
Citation: Patra S, Sahib MM, Shanmugam G, et al. Evaluation of asn In-house Indirect ELISA for Differential Detection of IgM and
IgG anti-Brucella Antibodies in Human Brucellosis. J Pure Appl Microbiol. Published online 01 September 2023. doi: 10.22207/
JPAM.17.3.27
© The Author(s) 2023. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which
permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons license, and indicate if changes were made.

Journal of Pure and Applied Microbiology 1 www.microbiologyjournal.org

 Patra et al | J Pure Appl Microbiol. 2023. https://doi.org/10.22207/JPAM.17.3.27

INTRODUCTION of protective and long-lasting humoral immunity.

Therefore, serological tests are preferred diagnostic
Brucellosis caused by various species of tests worldwide. Currently available and widely
the genus Brucella is one of the most important used serological tests are Rose Bengal Plate Test
zoonotic diseases of global importance with (RBPT), Standard Agglutination Test (SAT), and
veterinary, public health, and economic concerns. 2-Mercaptoethanol (2-ME). However, these tests
Brucella spp. majorly infects a wide range of rely on whole-cell antigens, which reduces their
domestic and wild animals.1 Humans are incidental specificity (Sp) and makes them less suitable for
hosts and often get infected through direct or definitive diagnosis.7,8 Due to these limitations,
indirect contact with diseased animals and/ or there is a growing interest in developing more
their products. Brucellosis in animals is generally specific and sensitive diagnostic assays.
a disease of the reproductive system causing Enzyme-linked immunosorbent assay
abortion, retention of placenta, infertility, orchitis, (ELISA) is a widely accepted method for diagnosis
epididymitis, reduction of milk production, and and screening of various infectious diseases.9 A
rarely arthritis. Human brucellosis is a multi-system previous study reported lower sensitivity (60%)
disease with debilitating morbidities.2 Although it and specificity (84%) for IgM and IgG iELISA,
is an age-old disease, still possess a great threat respectively. 10 The other studies have reported
to most of the endemic countries in the world.3 a higher Se of 92.3% - 100% for IgG-IgM iELISA
Almost all the species of Brucella but exhibited low Sp of only 55% - 71.3%.11,12
are involved in causing brucellosis in animals; The results of these ELISAs lack good combined
however, only B. melitensis, B. suis, B. abortus, Se and Sp for the detection of both IgM and IgG
and B. canis and recently discovered B. inopinata antibodies.13
can infect humans.2 B. melitensis is the most Hence, the present work aimed to
common causative agent of human brucellosis evaluate the diagnostic utility of an indirect
and most virulent of them all.4 Brucella spp., ELISA (iELISA) for the detection of both IgM and
being a facultative intracellular pathogen, can IgG anti-Brucella antibodies against smooth
survive inside the host macrophages, multiply lipopolysaccharide (sLPS) antigen in human serum
and cause long-lasting chronic infection. Acute, samples.
sub-acute, and chronic brucellosis in humans
requires prolonged treatment with a minimum of MATERIALS AND METHODS
two to three-drug regimens for complete recovery.
Although the rate of mortality is <2%, delayed Ethics statement
diagnosis, and inadequate treatment not only The study was approved by Institutional
cause a severely debilitating and disabling illness Ethics Committee, ICAR- NIVEDI, Bangalore, India
but also have major socio-economic ramifications.5 (Project ID: IXX10708) and samples were collected
Therefore, an accurate and early diagnosis of after obtaining written consent from all the
human brucellosis is inevitable. participants.
Diagnosis of brucellosis has always
been a dilemma due to its non-specific clinical Study design, location, and population
presentations, cross-reacting serological tests, This cross-sectional study was conducted
and time-consuming culture methods. Isolation at ICAR-NIVEDI in Bangalore from June to
of Brucella spp. from clinical specimens gives the December 2021. Seven hundred human sera
confirmatory diagnosis of brucellosis. However, samples collected from risk personnel (veterinary
culturing Brucella spp. requires biosafety doctors and artificial inseminators) were included
containment facilities and trained laboratory in this study.
personnel. Consequently, less contagious
serological and molecular tests are generally Extraction of sLPS antigen
preferred for diagnosis and sero-surveillance.6 A The smooth lipopolysaccharide (sLPS)
major component of the adaptive host immune antigen was extracted from phenol-killed B.
response to Brucella infection is the generation abortus S99 cell pellet using the hot phenol water
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 Patra et al | J Pure Appl Microbiol. 2023. https://doi.org/10.22207/JPAM.17.3.27

extraction method as described previously. 14 and secondary antibodies was reduced to 30

Initially, 1 gm wet weight of cells was suspended minutes to shorten the assay time.
in 3.4 ml of distilled water and the suspension
was heated to 66°C. After reaching the desired Sample analysis/ evaluation of iELISA
temperature, 3.8 ml of pre-heated 90% phenol To analyze the diagnostic Se and Sp
(v/v) was added to the suspension at 66°C. The of iELISA, a total of 700 human sera samples
mixture was stirred continuously for 15 minutes [veterinary doctors-161, artificial inseminators-187
and subjected to centrifugation at 10,000 rpm and animal handlers- 406] were included in the
for 15 minutes at 4°C. After centrifugation, the study. All these samples were tested by RBPT, SAT,
brownish layer of phenol at the bottom of the 2-ME, commercial Brucella IgM, and IgG iELISA
tube was aspirated and the remaining debris was (NovaLisa, NovaTec, Germany) and compared with
filtered out using Whatman filter paper No. 1. A in-house IgM and IgG iELISA. RBPT and SAT were
10ml suspension of ice-cold methanol containing performed as described previously by Alton et al.,
0.1ml of methanol saturated with sodium 1988.15 Both RBPT and SAT antigens were obtained
acetate was used to precipitate the sLPS antigen from the Institute of Animal Health and Veterinary
and incubated at 4°C for 2 h. The precipitate Biologicals, Bengaluru, India. The commercial
was dissolved in 1.6ml of distilled water and IgM and IgG iELISA were performed following the
incubated for 18 h at 4°C with constant stirring. manufacturer’s instructions. The optical density
After centrifugation at 10,000 rpm for 10 min, (OD) taken at 450nm was converted into percent
the supernatant was used for further purification. positivity (PP) values with reference to the OD of
Protein contaminants were removed by treating the positive control serum.16 Confirmed human
the supernatant with 0.16gm of trichloroacetic sera positive by RBPT, with 2ME titer of 1:1280
acid (TCA) and finally, 3.2 ml supernatant was and SAT titer of 1:2560, were used as positive
dialyzed against distilled water. sera controls. To establish diagnostic confirmation,
paired sera samples in 20 suspected individuals
Standardization of in-house anti-human IgM and were collected 3-4 weeks apart and repeated
IgG-based iELISA testing by all the serological tests to demonstrate
Both the assays were carried out rising antibody titers
following the previously described protocol with
few modifications.15 Checkerboard titrations were Statistical analysis
performed to optimize the concentration of sLPS Statistical analysis was performed using
antigen, dilutions of primary human antibodies, SPSS software, version 22 (IBM, India). The cut-
and anti-human IgM and IgG conjugates (Sigma- off, Se, Sp, area under the curve (AUC), accuracy,
Aldrich, St. Louis, MO, USA). Using these optimal positive predictive value, negative predictive
dilutions of antigen, serum, and conjugates, the value and kappa statistics were determined using
analytical sensitivity (ASe) and analytical specificity ROC curve analysis and Epitool software (https://
(Asp) of both IgM and IgG iELISA were determined. epitools.ausvet.com.au/).17
Different dilutions of two reference positive serum
samples and 8 reference/ hyperimmune sera of RESULTS
cross-reactive Gram-negative bacteria comprising
E. coli O157, Salmonella (Poly O), six serovars of Y. Optimization of test parameters for in-house IgM
enterocolitica (O1 & O2, O3, O5, O8, O9), and V. and IgG iELISA
cholera (Poly Hikojima, Inaba, Ogawa) were used to Checkerboard titration revealed 1:320,
determine the ASe and Asp, respectively. The ROC 1:80, and 1:4000 as optimal dilutions for sLPS
curve analysis was performed to derive a cut-off antigen, serum, and conjugates, respectively for
of percentage positivity (PP) values for both IgM both IgM and IgG iELISA. A Se of both IgM and
and IgG iELISA using panel samples comprising 50 IgG iELISA were up to 1:320 dilutions using two
each brucellosis positive and negative human sera representative positive samples (Figure 1A).
samples. The incubation time with both primary Evaluation of Asp for both IgM and IgG iELISA

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showed non-reactivity with hyperimmune sera and a cut-off value of ≥42 PP with Se of 98% and
against various cross-reactive Gram-negative Sp of 99.9% for IgG iELISA (Figure 1C).
bacteria (Figure 1B). The ROC curve analysis of 50
each panel samples showed a cut-off value of ≥42 Evaluation of in-house iELISA
PP with Se of 98% and Sp of 100% for IgM iELISA Of 700 samples tested, 8.3%, 8.1%, 8%,
and 6.1% were positive by in-house IgG iELISA,

Figure 1. Determination of analytical parameters of in-house IgM and IgG iELISA. (A) ASe was estimated using two
representative positive samples (S1: IgM positive sample, S2: IgG positive sample) at different dilutions. (B) ASe was
estimated with hyperimmune sera against various cross-reactive Gram-negative bacteria. (C) Determination of cut-off
using ROC curve analysis of IgM and IgG iELISA drawn using 50 each brucellosis positive and negative human sera
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 Patra et al | J Pure Appl Microbiol. 2023. https://doi.org/10.22207/JPAM.17.3.27

RBPT, in-house IgM iELISA, and SAT respectively SAT (significant titer ≥ 1:160) as a gold standard
(Table 1). A total of 57 (8.1%) individuals were test, the Se and Sp of IgM iELISA were 92.8%
reported as positive and suggested brucellosis and 98.9% and the Se and Sp of IgG iELISA were
treatment. Due to insufficient serum samples, 90.7% and 98.8%, respectively. A very good kappa
we could perform 2-ME only for 311 samples. Of agreement for IgM and IgG iELISA vs. RBPT and IgM
these, 7 (2.3%) showed significant SAT and 2-ME and IgG iELISA vs. SAT (Table 2) was recorded. An
titers among the participants. All 700 samples interactive dot plot diagram of IgM iELISA showed
tested with the commercial IgM and IgG iELISA kit that at the cut-off value of >41.7 PP, the majority of
showed 95.8% and 94.4% agreements between the positive samples were above the cut-off (Figure
in-house and commercial IgG and IgM iELISAs, 2A). Similarly, for IgG iELISA, most of the positive
respectively. Considering RBPT as a gold standard samples were above the cut-off >40.3 (Figure 2B).
test, the Se and Sp of IgM iELISA were 91.1% and From 20 paired sera samples tested, seven samples
99.2% and the Se and Sp of IgG iELISA were 92.86% showed rising titers.
and 99.2%, respectively. Similarly, considering

Figure 2. Interactive dot plot of IgM (A) and IgG (B) iELISA drawn using both positive and negative samples, showing
the distribution of PP values among the positive and negative participants
Table 1. Comparative evaluation of in-house IgM and IgG iELISA with RBPT, SAT, 2-ME, and commercial IgM and
IgG ELISA

Risk Total RBPT SAT 2-ME IgM iELISA IgG iELISA

groups positives positives positives
In-house Commercial In-house Commercial
kit kit kit kit
positives positives positives positives

Veterinary 137 (19.6) 11 (8) 9 (6.6) 5 (3.6) 10 (7.3) 11 (8) 14 (10.2) 13 (9.5)
doctors
Artificial 157 (22.4) 18 (11.5) 11 (7) 5 (3.6) 18 (11.5) 18 (11.5) 16 (10.2) 15 (9.6)
inseminators
Veterinary 406 (58) 28(6.9) 23 (5.7) NT 28 (6.9) 28 (6.9) 28 (6.9) 27 (6.6)
assistants
Total 700 57 (8.1) 43 (6.1) 7 (2.3) 56 (8) 57 (8.1) 58 (8.3) 55 (7.8)

*Due to insufficient sample 2-ME was performed for 311 samples only; NT: Not tested; values in parenthesis represents
percentages; ≥1:160 titer in SAT and ≥1:80 titer in 2-ME SAT were considered as significant titers

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DISCUSSION overlaps with various other infectious or non-

infectious diseases making brucellosis diagnosis
In India, a significant part of the population challenging. Hence, a rapid, simple, inexpensive,
involved in livestock-associated activities are always and consistent diagnostic test is required for
at risk of contracting the disease.18 The diverse brucellosis diagnosis. Isolation of Brucella spp.
clinical symptoms presented by human brucellosis from human specimens is indisputable proof for

Table 2. Performance characteristics of in-house IgM and IgG iELISA at cut-off 42 (PP value) considering three
serological tests as gold standard.

Gold standard 1. Gold standard 2. Gold standard 3.

RBPT SAT Commercial ELISA
IgM iELISA IgG iELISA IgM iELISA IgG iELISA IgM ELISA IgG ELISA

Se (95% CI) 91.1 92.86 92.8 90.7 90 97.9

(80.4-97) (82.7-98) (80.5-98.5) (77.8-94.7) (76.3-97.2) (88.7-99.9)
Sp (95% CI) 99.2 99.2 98.9 98.7 99.4 99.5
(98.2-99.7) (98.2-99.7) (97.8-99.5) (97.6-99.5) (98.4-99.8) (98.6-99.9)
PPV (95% CI) 86.06 86.3 81.8 79.4 88.2 91.5
(71.9-93.7) (72.4-93.8) (68.2-90.5) (65.8-88.6) (73.7-95.2) (77.8-97.1)
NPV (95% CI) 99.53 99.6 99.6 99.5 99.5 99.9
(98.9-99.8) (99-99.8) (98.8-99.8) (98.7-99.8) (98.7-99.8) (99.2-99.9)
Accuracy 98.8 98.9 98.6 98.3 98.9 99.4
(97.7-99.5) (97.8-99.5) (97.4-99.4) (97.1-99.2) (97.8-99.5) (98.5-99.8)
Kappa (95% CI) 0.9 0.91 0.88 0.86 0.89 0.95
(0.84-0.96) (0.86-0.97) (0.81-0.95) (0.78-0.94) (0.82-0.97) (0.91-0.99)
Positive 117 120 86 73 142 206
likelihood ratio (48.8-281.9) (49.8-287.2) (40.8-179.9) (36.6-147.02) (53.24-379.7) (66.5-637.1)
Negative 0.09 0.072 0.072 0.094 0.1 0.02
likelihood ratio (0.04-0.21) (0.03-0.19) (0.02-0.21) (0.04-0.24) (0.04-0.25) (0-0.15)

*CI: confidence interval; PPV: positive predictive value; NPV: negative predictive value

Table 3. Overall comparison of in-house and commercial brucellosis diagnosis. However, blood cultures
Brucella IgM and IgG iELISA results considering RBPT have been reported to be positive for 38% to
as a reference test 90% of patients with brucellosis and chances of
successfully isolating the organism decrease as
In-house (95% CI) Commercial (95% CI)
the disease progresses.19,20 As a result, detection
Se 93.3 (83.8 – 98.2) 91.7 (81.6 – 97.2) of anti-Brucella antibodies using serological
Sp 99.4 (98.4 – 99.8) 99.1 (97.9 - 99.7) tests is preferred for routine diagnosis. Since the
PPV 88.7 (74.8 – 95.5) 83.8 (69.9 - 92) discovery of the very first test, several serological
NPV 99.6 (99.1 - 99.8) 99.6 (98.9 - 99.8) tests have been developed and improved for the
Accuracy 99.1 (98.1 - 99.6) 98.7 (97.6 – 99.4) diagnosis of human brucellosis.21 In this study,
Kappa 0.93 (0.87 – 0.97) 0.9 (0.84 – 0.95) we have described the diagnostic performance
Positive 150 (56.4 -399.5) 98.5 (44.3 – 219.3) of in-house IgM and IgG iELISA and compared it
likelihood with conventional serological techniques (RBPT,
ratio
SAT, and 2-ME) and commercial ELISA kits. As
Negative 0.07 (0.03 - 0.17) 0.08 (0.04 - 0.19)
likelihood per the results, artificial inseminators (11%) had
ratio the highest seropositivity, followed by veterinary
doctors (10%) and assistants (7%). Similarly, higher
*Se: sensitivity; Sp: specificity; CI: confidence interval; PPV: seroprevalence was reported in animal handlers,
positive predictive value; NPV: negative predictive value
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artificial inseminators, farmers, and veterinary with similar clinical manifestations.10,29,30 In this
doctors from various parts of the country.22,23 study, 8.3% and 8% of the screened individuals
RBPT and SAT are the two inexpensive were positive by in-house IgG and IgM iELISA,
and valuable laboratory tests for preliminary respectively. However, five participants showed
diagnosis of brucellosis and studies have reported false-positive results in IgM iELISA, whereas other
a high level of concordance between SAT and tests were negative which could be due to cross-
RBPT results.24 In this study, among the screened reacting antibodies. Repeat sampling after 3-4
veterinary health care individuals, 8.1%, 6.1%, weeks confirmed false positivity. Therefore, the
and 2.3% were positive by RBPT, SAT, and 2-ME, presence of only IgM antibodies should never be
respectively. The negative or insignificant titers considered as conclusive for acute infections. In
in SAT/2-ME tests in the considerable number of such cases, detection of IgG antibodies and/ or
participants (n-13) showed positive serological rise in antibody titer should be considered for
tests in RBPT, IgM, and IgG iELISA. This noticeable definitive diagnosis. According to our paired serum
finding of this study implies poor sensitivity of sample analysis, we observed various conditions
conventional agglutination tests for diagnosis. which indicated the usefulness of paired serum
Largely, the false-negative SAT and 2-ME results samples for definitive diagnosis of brucellosis
could be due to the presence of ‘blocking or among suspected individuals. Also, anti-Brucella
incomplete antibodies’ belonging to IgG and IgA antibodies persist for a long even after successful
classes.25 treatment and recovery.
Several serological techniques have In comparison, in-house IgG iELISA has
been widely studied for human brucellosis which detected 8.3% positivity whereas the commercial
includes complement fixation tests (warm and IgG ELISA kit detected only 7.8% positivity. Overall,
cold), Coomb’s test, radio-immunoassay (RIA), and comparison between the combined efficiency of
fluorescent polarization assay. These tests have IgM and IgG in-house and commercial ELISA kits
good Se, Sp, and effective for diagnosis of both showed that in-house assays have higher Se and
acute and chronic brucellosis. However, iELISA is Sp indicating the effectiveness for serodiagnosis of
preferred over these traditional tests due to its human brucellosis (Table 3). In the present study,
simplified test procedures, time efficiency, and we observed that 7% of the screened individuals
elimination of hazardous materials.13,26 were positive by RBPT, in-house IgM, and IgG
A p a r t f ro m s L P S , s e ve ra l o t h e r iELISA whereas, SAT and 2-ME detected only
immunodominant antigens of Brucella spp. have 6.1% and 2.3%, respectively. In routine diagnostic
been used for diagnostic purposes.27 The sLPS is laboratories with limited resources, performing
present in all classical species of Brucella except B. SAT and 2-ME for every sample is difficult because
ovis and B. canis and therefore, sLPS is well suited of their non-robust and time-consuming. In such
for the detection of anti-Brucella antibodies.28 cases, robust, specific, and sensitive IgM and IgG
Moreover, because of structural similarity and iELISA can be used as alternative assays for rapid
higher immunogenicity, purified sLPS antigen has and reliable disease diagnosis.
the potential to detect a wide range of Brucella
biovars.6 CONCLUSION
The presence of anti-Brucella IgM
antibodies are generally indicative of acute In Conclusion, in-house iELISA
brucellosis and IgM antibodies start appearing outperformed the commercial ELISA kits and
seven days of the disease onset, reaching highest other conventional serological tests. The current
level within 1 to 3 months after infection. Whereas brucellosis control program in India allows
IgG antibodies appear just about after 3 weeks prevention by vaccination and country-wide
and reach a peak 6 – 8 weeks after the onset of surveillance of the disease in animals. However,
the disease. Few of the previous studies have the most important measures for the prevention
reported a Sp of 100% for IgM ELISA and the of human brucellosis would be to educate artificial
possibility of over-diagnosis were highlighted in inseminators, animal handlers, and farmers,
areas with high burden of other infectious diseases about the disease and to conduct routine testing
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